Results for “Microbial” — Page 44

NASA NTRSDate not supplied

The BioMole Facility: Advancement of In Situ Microbiome Analysis for the International Space Station

Characterization of the International Space Station (ISS) microbiome has been enabled by sample return and Earth-based analysis. As human exploration pushes beyond low-Earth orbit, microbial-related crew health, planetary protection, and space research requires in situ capabilities. Steps toward reducing Earth-dependence for complex sample analysis began in 2016 with the amplification of DNA within the miniPCR thermal cycler and DNA sequencing with the MinION sequencer onboard the ISS; for both, samples were prepared on Earth. In 2017, these platforms synergistically enabled the in-situ identification of unknown bacteria collected and cultured from ISS surfaces, thereby shifting the paradigm that microbial cultures had to be returned to Earth. The following year, a culture-independent, swab-to-sequencer method further advanced spaceflight microbiology, demonstrating that culturing could be excluded and provided enhanced insight into the bacterial profile of ISS surfaces. Based on the success of these payloads in confirming the ability to meet crew health identification requirements and the benefits accompanying a culture-independent method, the BioMole Facility was established by the medical operations Crew Health Care Systems team. BioMole is the set of hardware, consumables, and procedures required to support sample preparation and nanopore sequencing onboard the ISS. BioMole goals include expanding sample sources, comparing data to previous methods, demonstrating onboard data analytics, and validating new hardware. To date, comparative surface analysis, molecular- and culture-based, has been completed. Additionally, the demonstration of a sample-to-answer process was achieved when BioMole data was processed onboard using the IBM Open Data and AI Edge software platform installed on the ISS-residing Spaceborne Computer-2. The taxonomic profiles generated from the edge analysis were as expected and paralleled that of the downlinked processed data. Future BioMole efforts involve microbial profiling of the ISS water system, ISS validation of the MinION Mk1C, and an expansion to a research facility available to investigators.

Sarah L. Castro-Wallace↗

NASA NTRSDate not supplied

Method Development for In Situ Microbiome Profiling of the Water Recovery System’s Wastewater Tank Onboard the International Space Station

A distinctive microbial community has inhabited the wastewater tank within the International Space Station Water Recovery System (WRS) for over 14 years and experienced the stressors associated with the microgravity environment. The WRS generates potable water for the crew from urine distillate, humidity condensate, Sabatier product water, and the occasional off-loading of ground-supplied water. The reservoir for these products, the wastewater tank, does not have a means of microbial control. While samples are occasionally collected for analysis, the time between sample collection and the return to Earth, as well as the lack of preservation, results in a skewed depiction of the microbiome. Routinely observed from these returned samples are high counts (105 – 106 colony forming units per mL) and two prevailing genera, Burkholderia and Ralstonia. The wastewater tank likely contains a more diverse microbiome, as a higher diversity of bacteria and fungus has been noted upstream and downstream of the tank. To characterize the microbial profile of the tank, analysis needs to occur at the time of sample collection. Toward this goal, a method for in situ analysis based on nanopore sequencing was developed. The filter-to-sequencer method evolved from previous work that has been validated onboard the ISS (BEST payload and the BioMole Crew Health Care Systems Facility). The method, including filtration, DNA extraction, purification, amplification, library preparation, and nanopore sequencing will be described. Additionally, data collected with this method from both ISS and terrestrial samples will be detailed. The consumables needed to support in situ analysis of the tank are set to the launch to the ISS in the spring of 2023. This investigation will allow for the first accurate characterization of the microbiome of the tank providing insight for crew health, planetary protection, and has the potential to enable engineering controls for future space station water systems.

Sarah Stahl-Rommel↗

NASA NTRSDate not supplied

To Biocide or not to Biocide? Exploring the "No Biocide" Option in Spacecraft Potable Water Systems

Residual biocide has been used to protect against microbial growth in spacecraft potable water systems since the Gemini program. Iodine, the biocide currently used on the International Space Station (ISS) has a long history of use but presents a few challenges for exploration: it must be removed using a consumable absorbent before crew consumption and is known to lose its biocidal concentration due to interactions with wetted components, especially during periods of dormancy. Biocidal silver is being considered for exploration missions as it is safe for crew consumption, but it too has challenges with proper material design to maintain sufficient disinfection residuals. An idea proposed by some in the Environmental Control and Life Support System (ECLSS) community has been that the biocide does not provide a true barrier against microbial growth, and therefore, perhaps the challenges of maintaining a residual biocide in the system could be best overcome by removing the biocide altogether. In this argument, they point to the regenerative water treatment system on the ISS which produces high purity water that theoretically meets the microbiological requirements before the iodine is added. Exploration water processing systems are expected to have similar water processing capabilities. This paper will discuss the role that residual biocide may play in controlling microbial growth and presents perspectives on the potential risks associated with removing it from exploration water systems.

biocide↗

NASA NTRS2022

2022 Annual Report: Culture-Based Environmental Microbiology Monitoring of Crop-based Space Food Systems (Veggie Monitoring)

Crewmembers live and work in a closed environment that is monitored to ensure their health and safety. Quarterly monitoring of the microorganisms in the International Space Station (ISS) environment supports crew safety and contributes to a large set of environmental microbial data from the air, surface, and water samples that are collected. This study leverages quarterly operational Environmental Health System (EHS) sampling by collecting additional microbial samples from the surface of the station’s Veggie plant production system. Longer exploration missions may require spaceflight-based systems for growth of plants, and this investigation is expected to provide additional data to help establish requirements to protect these systems, plants, and crew, mitigating adverse microbial exposure.

Tanner Hamilton↗

NASA NTRS2022

Microgreens Root Zone/Shoot Zone Partitioned Planting Box

To enable sustainable food production in future human exploration missions, plant growth is being studied by the Space Crop Production Team at KSC. Microgreens are good candidates for food supplements and contain specific nutrients that are lacking in the prepackaged diet, including vitamin C and vitamin K. Because they are densely sown, typical growth methods do not allow the ability to distinguish between the levels of evaporation from the rooting substrate and transpiration from the leaves. With larger plants, the root and shoot zone can be separated to distinguish these fluxes and accurate transpiration measurements of plant canopies are feasible. Furthermore, separation of the root and shoot zone may also be beneficial when harvesting microgreens in microgravity as it may reduce microbial contamination of the edible biomass by the roots, which have high microbial loads. Using a root and shoot separator box when harvesting may help with microbial contamination, but these tiny plants are challenging to handle in microgravity, so harvest management remains an open question. The innovation proposed here is a microgreen root/shoot partitioned planting box, which offers a solution to these challenges with accurate gas exchange measurements and a safe microgreen harvest in low gravity environments. Being able to measure transpiration of a microgreen canopy will be important for modeling plant growth in reduced gravity environments, so the first objective was to develop a planting unit with a seal between the root zone and the shoot zone. With an unsuitable harvesting technique, freshly harvested microgreens may add debris to the cabin, so the second objective was to test different harvesting techniques and management approaches associated with this innovation. These two objectives were pursued in parallel since many goals were the same: develop a planting unit that 1) separates the shoots from the roots, 2) allows acceptable germination rate, and 3) allows for seedlings to emerge and develop. What differed was the need to have a seal, which was only applicable for our gas exchange goal, and the need to have an embedded harvesting mechanism and bagging method which only applied to our harvesting goal. Testing of the various harvesting mechanisms and bagging methods was performed during a series of parabolic flights. All parabolic flight procedures took place inside a rented secondary containment chamber (e.g., glovebox) that was developed by the University of Louisville specifically for experiments involving fluids and other materials that may become airborne during reduced gravity flight. Three different harvesting methods and two different bagging collection methods were tested for microgreens. A third bagging method was initially tested but found to be unsuitable. Human factors were also taken into consideration, to identify which harvest and bagging collection methods would be easiest to use with favorable results in microgravity. Three parabolic flight tests were performed in total, one in November 2021 and two in December 2021.

Gioia Massa↗

NASA NTRSDate not supplied

To Biocide or not to Biocide? Exploring the "No Biocide" Option in Spacecraft Potable Water Systems

Residual biocide has been used to protect against microbial growth in spacecraft potable water systems since the Gemini program. Iodine, the biocide currently used on the International Space Station (ISS) has a long history of use but presents a few challenges for exploration: it must be removed using a consumable absorbent before crew consumption and is known to lose its biocidal concentration due to interactions with wetted components, especially during periods of dormancy. Biocidal silver is being considered for exploration missions as it is safe for crew consumption, but it too has challenges with proper material design to maintain sufficient disinfection residuals. An idea proposed by some in the Environmental Control and Life Support System (ECLSS) community has been that the biocide does not provide a true barrier against microbial growth, and therefore, perhaps the challenges of maintaining a residual biocide in the system could be best overcome by removing the biocide altogether. In this argument, they point to the regenerative water treatment system on the ISS which produces high purity water that theoretically meets the microbiological requirements before the iodine is added. Exploration water processing systems are expected to have similar water processing capabilities. This paper will discuss the role that residual biocide may play in controlling microbial growth and presents perspectives on the potential risks associated with removing it from exploration water systems.

biocide↗

NASA NTRSDate not supplied

Verification Testing and Veg-05 Tomato Crop Production on the International Space Station

Production of fresh, nutritious, and tasty produce for astronauts during spaceflight may provide health-promoting, bioavailable nutrients and enhance the dietary experience as we move into longer-duration missions. Growing and caring for plants may also reduce the psychological stresses associated with spaceflight and enhance connections to Earth. Requirements to consistently grow a diversity of crops under spaceflight environmental conditions remain poorly defined. The VEG-05 experiment is part of a series of experiments with pick-and-eat salad crops to better define best practices for crop production and handling in space. VEG-05 and predecessor experiments VEG-04A and VEG-04B, use the Veggie vegetable production facilities on the International Space Station to grow salad crops under different spectral compositions. In VEG-04A and B, mizuna mustard was cultivated with two different red: blue light treatments, and in VEG-05 we are cultivating ‘Red Robin’ dwarf cherry tomatoes under the same light spectra. Light can impact the growth habit, yield, nutritional composition, microbial levels, and even flavor attributes within crops, and our team will assess these characteristics for this crop during VEG-05. Prior to launch and installation on ISS in late 2022, both a science verification test (SVT), and an experiment verification test (EVT) were conducted at Kennedy Space Center in ISS Environment Simulator Chambers. Science verification testing, and a previous fertilizer test, grew plants in both plant pillows and PONDS (Passive Orbital Nutrient Delivery System) units and tested two different fertilizer treatments in both sets of hardware, with each test under only one of the light conditions. Because of challenges validating the PONDS hardware on ISS, and with good crop production in plant pillows, the EVT moved forward using only plant pillows with the highest fertilizer composition tested during SVT, and two Veggie units were utilized. One Veggie had light settings consisting of equal levels of red: blue light (150 µmol/m2/s for each color) plus green light (30 µmol/m2/s) while the second Veggie had a 90:10 ratio of red: blue light (270 µmol/m2/s red and 30 µmol/m2/s blue) plus green, so each unit provided 330 µmol/m2/s of photosynthetically active radiation to the tomato crops on average. Our original plan, based on prior ground testing, was to grow the crop for 104 days and harvest at 80, 90 and 104 days after initiation. For SVT, under the equal red: blue light treatment, fruit ripening in plant pillows was delayed and fruit were not ripe by day 80, so actual harvest days were days 90, 97, and 104. For EVT we saw fruit ripening earlier, especially in the high red treatment, and so we harvested at days 83, 90, and 99 days after initiation. In SVT we had mostly daily watering, and this led to excess water in plant pillows, which leaked out. This excess water also caused fungus to grow on one leaf and a couple of plant stems. To reduce this excess moisture, we throttled back the watering for EVT, and used the root mat reservoir more frequently. This led to watering only every other day, reducing crew time needed for plant care, however, two wilting events occurred during this EVT, at days 51 and 75. Plants recovered from these wilting events, but these events may have influenced the rate of fruit ripening and flower formation. Regardless, more than 10 fruit were produced from each plant on average, with the high red treatment producing slightly heavier fruit. Microbial testing from fruit during SVT indicated that fruit were safe for consumption with microbial levels below detection limits. VEG-05 flight and ground operations are expected to run between December 2022 and March 2023. This research was co-funded by the Human Research Program and Space Biology (MTL#1075) in the ILSRA 2015 NRA call.

Gioia D. Massa↗

NASA NTRSDate not supplied

To Biocide or Not to Biocide? Exploring the "No Biocide" Option in Spacecraft Potable Water Systems

Residual biocide has been used to protect against microbial growth in spacecraft potable water systems since the Gemini program. Iodine, the biocide currently used on the International Space Station (ISS) has a long history of use but presents a few challenges for exploration: it must be removed using a consumable absorbent before crew consumption and is known to lose its biocidal concentration due to interactions with wetted components, especially during periods of dormancy. Biocidal silver is being considered for exploration missions as it is safe for crew consumption, but it too has challenges with proper material design to maintain sufficient disinfection residuals. An idea proposed by some in the Environmental Control and Life Support System (ECLSS) community has been that the biocide does not provide a true barrier against microbial growth, and therefore, perhaps the challenges of maintaining a residual biocide in the system could be best overcome by removing the biocide altogether. In this argument, they point to the regenerative water treatment system on the ISS which produces high purity water that theoretically meets the microbiological requirements before the iodine is added. Exploration water processing systems are expected to have similar water processing capabilities. This paper will discuss the role that residual biocide may play in controlling microbial growth and presents perspectives on the potential risks associated with removing it from exploration water systems.

biocide↗

NASA NTRSDate not supplied

To Biocide or Not to Biocide? Exploring the "No Biocide" Option in Spacecraft Potable Water Systems

Residual biocide has been used to protect against microbial growth in spacecraft potable water systems since the Gemini program. Iodine, the biocide currently used on the International Space Station (ISS) has a long history of use but presents a few challenges for exploration: it must be removed using a consumable absorbent before crew consumption and is known to lose its biocidal concentration due to interactions with wetted components, especially during periods of dormancy. Biocidal silver is being considered for exploration missions as it is safe for crew consumption, but it too has challenges with proper material design to maintain sufficient disinfection residuals. An idea proposed by some in the Environmental Control and Life Support System (ECLSS) community has been that the biocide does not provide a true barrier against microbial growth, and therefore, perhaps the challenges of maintaining a residual biocide in the system could be best overcome by removing the biocide altogether. In this argument, they point to the regenerative water treatment system on the ISS which produces high purity water that theoretically meets the microbiological requirements before the iodine is added. Exploration water processing systems are expected to have similar water processing capabilities. This paper will discuss the role that residual biocide may play in controlling microbial growth and presents perspectives on the potential risks associated with removing it from exploration water systems.

biocide↗

NASA NTRSDate not supplied

Analysis of Bioburden Associated with Nonmetallic Spacecraft Materials

Planetary Protection (PP) requirements help to ensure the integrity of life-detection science conducted on solar system bodies, particularly those with liquid water. Such requirements are often met by a combination of cleaning and verification steps to reduce microbial contamination (i.e. bioburden) to an acceptable limit. There are only two cleaning methods approved by the National and Space Administration (NASA), vapor hydrogen peroxide (VHP) and heat microbial reduction (HMR), the latter of which is the most commonly employed. However, HMR can be harsh and produce its own concerns about material integrity, like aging or increasing hardness. When bioburden reduction methods cannot be employed, the project must rely on assumed NASA specification values of bioburden which can cause compliance issues with strict PP bioburden limitations. This work aimed to more accurately define the risk of contamination of a target body by providing empirical data associated with raw materials commonly used in solid rocket motors for lander missions. In this study, we pulverized nonmetallic materials using a cryogenic grinder, then analyzed the resulting substrate for microbial colony forming units (CFU). We found that many nonmetallic materials do not harbor large bioburden when compared with the NASA specification values, though a range existed depending on the material, treatment, and handling. The results from this work provide quantitative data to help reduce concerns of contamination, while also providing a foundation for follow up studies into additional sterilization methods and molecular identification of contaminating microbes.

Planetary Protection↗

NASA NTRSDate not supplied

AMMPER: a user-friendly agent-based model that recapitulates simple metabolic responses of yeast to deep-space radiation

For humans venturing to deep space, radiation exposure poses a major health risk. Fundamental research into the biological effects of space radiation are essential for enabling exploration, and the first experimental organisms we send to deep space will be microbial. Yet there are many ways in which microorganisms are likely to experience the effects of high-energy particle radiation (such as Galactic Cosmic Rays) differently from multicellular animals, partly due to the simple fact that microbes are small and unicellular-- less likely to get hit in the first place, and less likely to communicate damage between cells. Computational modeling can aid in designing experiments and predicting the biological effects of radiation, but thus far particle radiation models have not focused on microbes. Here we present the latest developments in AMMPER, the Agent-based Model for Microbial Populations Exposed to Radiation. Originally written in 2021, AMMPER is a Python-based model that incorporates radiation track data from NASA's RITRACKS software and simulates the growth, damage, and death of yeast cells in 3D. It is now freely available as an open-source package on NASA's GitHub repository. Recent improvements include the ability to simulate the dynamics of alamarBlue, a color-changing redox dye commonly used to track metabolic activity in microbial spaceflight experiments. We demonstrate that a simple blue-pink-clear transition model is able to recapitulate key features observed in empirical data from ground studies. AMMPER also includes a new graphical user interface and introductory tutorial to facilitate ease of use by a wider audience. AMMPER can help us to understand how spatially heterogeneous particle radiation damage at the single-cell level can translate to growth differences at the population level, ultimately allowing us to better interpret experiments using microbes as model organisms and how well their results apply to humans.

yeast↗

NASA NTRSDate not supplied

ISS External Microorganisms: Collecting Planetary Protection Samples During Extravehicular Activity

Introduction: We have designed1, built, and tested a sampling kit (Fig. 1) to aseptically collect microbiological samples from exterior surfaces on the ISS (International Space Station). The kit was flown to ISS as part of the NG-19 commercial cargo mission in August of 2023. Astronauts will use the kit to collect samples from six exterior surfaces on ISS. These samples will be frozen at -80°C after collection and returned to the ground for next generation DNA sequencing. The results of this experiment will help inform planetary protection requirements for crewed missions. We hypothesize that there are detectable microbial communities outside ISS and that these communities originate from inside ISS. Current life support systems do not include any components for reducing microbial leakage. Gases are vented from inside ISS without filtration and there are currently no protocols in place to minimize bioburden on space suit exteriors prior to use. It is important to quantify the bioburden on current vehicles so that achievable limits can be set for crewed missions to astrobiologically relevant locations like Mars. Figure 1: The sampling kit contains eight swab canisters in total (six on the top and two on the bottom). A resuable end effector (1) is used to remove and reinstall the swabs. The kit also contains a handrail (2), tether loop (3), and, bayonet probes(4) to allow easy manipulation during EVA. Methods: We have designed a kit to carry 8 commercially available foam swabs into and out of vacuum without compromising the swabs’ sterility. The specially designed swab canisters contain a 0.2 µm Teflon filter that allows the canister to accommodate pressure changes without introducing unwanted contaminants. The kit meets existing EVA (Extravehicular Activity) safety requirements and has been used by human test subjects in the neutral buoyance lab and at vacuum in test chambers at the Johnson Space Center. In the early part of 2024, astronauts will use this sampling kit to swab six surfaces on the exterior of ISS. We will collect samples from: the airlock vestibule, the interior surface of the airlock thermal cover, an exterior handrail, a vent connected to the CO2 removal system, and a vent connected to the payload vacuum system inside ISS. Approximately 300 cm2 will be swabbed at each location. A seventh swab will be exposed to the vacuum of space without touching any surfaces as a blank. The eighth swab will remain sealed as a process control. After the EVA the swab kit will be frozen at -80°C and returned to earth at the earliest possible opportunity. The samples will remain frozen until they are thawed for next generation DNA sequencing on earth. We will use amplicon sequencing to identify any bacteria, archaea or fungi present in the samples. If there is enough DNA present, we will use shotgun metagenomic sequencing to further characterize the microbial ecology outside ISS. Preliminary Results: Results from ground-based testing demonstrate that the swab kit is capable of cycling in and out of vacuum without contaminating the swabs. Test subjects, wearing flight-like EVA gloves could remove swabs from the canister and sample discrete locations without inadvertently touching any other surfaces. Several types of bacteria and fungi were collected during these ground tests and survived up to 6 hours at vacuum. This includes non-spore forming bacterial like Staphyloccus capitis that are not traditionally considered extremophiles. Shotgun metagenomic sequencing of the ground-test samples revealed bacteria associated with human skin and airways on the exterior of space suits used during these tests 2 In addition to these results we will present preliminary results from our space-flight samples. We will also present lessons learned from attempting to collect microbiological samples during an EVA and describe how our results will affect planetary protection requirements for future crewed missions.

Aaron B. Regberg↗

NASA NTRS2024

2023 Cleanroom Monitoring for OSIRIS-REx

OSIRIS-REx is an asteroid sample return mission that delivered asteroid regolith from Bennu to Earth on 9/24/2023 [1]. Cleanroom monitoring of OSIRIS-REx Curation facilities has been ongoing since 2021 when construction of the OSIRIS-REx lab at Johnson Space Center (JSC) was completed [2]. The monitoring continues inside of the OSIRIS-REx lab in Houston. Monitoring was also conducted on a temporary cleanroom constructed inside a hangar at the Utah Test and Training Range (UTTR) to initially receive the Sample Return Capsule (SRC) upon its landing there. The following reports on cleanroom monitoring that has taken place in the months leading up to as well as after OSIRIS-REx return. Monitoring techniques used include particle counts, deployment of Si wafer witness plates (Balazs, Inc.) and Al foil witness plates, gas sampling (Balazs, Inc.) and microbiological monitoring. We collected particle counts at both the lab at JSC as well as the temporary cleanroom at UTTR. Particle counts were taken monthly from six locations in the lab at JSC. Particle counts were taken from seven locations at UTTR in July, August, and September. The UTTR cleanroom particle counts were taken daily in the five days leading to OSIRIS-REx return as well as consistently the day of return (every 2 minutes and 15 seconds 7:21 am- 6:27 pm) from one location inside the cleanroom. The particle counts at UTTR consistently stayed well within the ranges of its required ISO 7 equivalent classification (352,000 maximum particles/ m3 ≥ 0.5 μm). Most analysis showed that the particle counts were far below ISO 7 requirements (highest measured count was 61,484 particles/ m3 ≥ 0.5 um). but the elevated instances correlated to exterior hangar doors being open. Particle counts were reduced when keeping exterior hangar doors closed. The particle counts at JSC have stayed consistently within their ISO 5 equivalent classification (3,520 maximum particles/ m3 ≥ 0.5 um). In most instances, the counts are much better except in one instance where the count measured 13,381 particles/ m3 ≥ 0.5 um. The high spike in particle counts in the JSC lab correlated with the air handlers being shut down temporarily the previous day. The particle counts taken in the same location quickly reduced and measured 0 when taken a month later with the air handlers on. We deployed 8-inch semiconductor polished Si wafer witness plates for 24-hour periods inside of the OSIRIS-REx Curation lab at JSC (in July, August, September, and November 2023) and the cleanroom at UTTR (July and September 2023) to test for possible organic and inorganic contaminants. We also deployed these wafers inside of the OSIRIS-REx Touch-and-Go sample Acquisition Mechanism (TAGSAM) glovebox (where Bennu sample processing occurs) and desiccator (Bennu sample storage) at JSC in August 2023. These samples were all sent to Balazs for analysis via Thermal Desorption Gas Chromatography Mass Spectroscopy (TD-GC-MS) to quantify organic compounds and vapor phase decomposition inductively coupled plasma mass spectrometry (VPD-ICP-MS) to quantify inorganic contaminants. We collected air samples using an adsorbent tube connected to a pump for six hours (100 mL/minute) inside of both the JSC lab and the UTTR cleanroom. These samples were taken at JSC in July, August, September, November, and December and at UTTR in July and September. Additionally, a sample was taken just outside of the TAGSAM glovebox airlock door in July. These samples were sent to Balazs for analysis of volatile organics in air. Overall, the OSIRIS-REx lab at JSC and cleanroom at UTTR yielded very low organic and inorganic contaminants (similar results to Genesis lab at JSC which is ISO 4 equivalent) except for boron, which is attributed to the borosilicate glass in the fan filter units. We collected monthly surface and air samples inside the JSC lab to monitor potential microbial contamination. We report the recovery rate for each sampling event, which is defined as the number of samples exhibiting bacterial or fungal growth divided by the total number of samples collected. Since the lab was commissioned in 2021 the median recovery rate is 29%. This recovery rate is consistent with other ISO 5 equivalent labs used to curate astromaterials collections. Increases in recovery rate correspond to construction and/or extra activity in the lab. However, increases in recovery rate do not reliably correlate to increases in particle counts, which highlights the need for a dedicated microbial monitoring program for biologically sensitive collections. We sampled the temporary clean room at UTTR in July, August, and September of 2023 to monitor potential microbial contamination. The recovery rate decreased from 80% in August to 29% in September. However, in all instances, the diversity of bacteria and fungi was higher in the UTTR cleanroom than in the JSC lab. We routinely collected more than twenty different organisms from the UTTR cleanroom. In the JSC lab the median diversity is 2 organisms. Based on these results we hypothesize that microbes from UTTR could be transported back to JSC with the Bennu samples. To mitigate this risk, in September of 2023, we implemented additional cleaning procedures in the JSC lab to reduce the bioburden on surfaces that could come into direct contact with hardware used to process Bennu samples. Since adopting these additional cleaning measures, the median recovery rate has decreased to 14%. We did not observe an increase in fungal or bacterial diversity in the lab in the October sampling. We will continue to monitor this trend closely for the next several months. Supported by NASA under Award NNH09ZDA007O and Contract NNM10AA11C.

Rachel Comstock Funk↗

NASA NTRS2023

Viral Enumeration Using Cost-Effective Wet-Mount Epifluorescence Microscopy for Aquatic Ecosystems and Modern Microbialites

Enumeration is a fundamental measure of community ecology in which viruses represent the most numerous biological identities. Epifluorescence microscopy (EFM) has been the gold standard method for environmental viral enumeration for over 25 years. Currently, standard EFM methods using the Anodisc filters are no longer cost-effective (>$15 per slide) and have yet to be applied to modern microbialites. Microbialites are microbially driven benthic organosedimentary deposits that have been present for most of Earth’s history. We present a cost-effective method for environ mental viral enumeration from aquatic samples, microbial mats, and exopolymeric substances (EPSs) within modern microbialites using EFM. Our integrated approach, which includes filtration, differential centrifugation, chloroform treatment, glutaralde hyde fixation, benzonase nuclease treatment, probe sonication (EPS and mat only), SYBR Gold staining, wet mounting, and imaging, provides a robust method for modern microbialites and aquatic samples. Viral abundances of modern microbialites and aquatic samples collected from Fayetteville Green Lake (FGL) and Great Salt Lake (GSL) did not differ across ecosystems by sample type. EPS and microbial mat samples had an order of magnitude higher viral-like particle abundance when compared to water regardless of the ecosystem (107 vs 106). Viral enumeration allows for estimates of total viral numbers and weights. The entire weight of all the viruses in FGL and GSL are ~598 g and ~2.2 kg, respectively. Further development of EFM methods and software is needed for viral enumeration. Our method provides a robust and cost-effective (~$0.75 per sample) viral enumeration within modern microbialites and aquatic ecosystems.

Madeline Bellanger↗

NASA NTRSDate not supplied

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

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

Planetary Protection; Lasers; Sterilization; Bacil↗

NASA NTRSDate not supplied

ISS External Microorganisms: Collecting Planetary Protection Samples During Extravehicular Activity

Introduction: We have designed1, built, and tested a sampling kit (Fig. 1) to aseptically collect microbiological samples from exterior surfaces on the ISS (International Space Station). The kit was flown to ISS as part of the NG-19 commercial cargo mission in August of 2023. Astronauts will use the kit to collect samples from six exterior surfaces on ISS. These samples will be frozen at -80°C after collection and returned to the ground for next generation DNA sequencing. The results of this experiment will help inform planetary protection requirements for crewed missions. We hypothesize that there are detectable microbial communities outside ISS and that these communities originate from inside ISS. Current life support systems do not include any components for reducing microbial leakage. Gases are vented from inside ISS without filtration and there are currently no protocols in place to minimize bioburden on space suit exteriors prior to use. It is important to quantify the bioburden on current vehicles so that achievable limits can be set for crewed missions to astrobiologically relevant locations like Mars. Figure 1: The sampling kit contains eight swab canisters in total (six on the top and two on the bottom). A resuable end effector (1) is used to remove and reinstall the swabs. The kit also contains a handrail (2), tether loop (3), and, bayonet probes(4) to allow easy manipulation during EVA. Methods: We have designed a kit to carry 8 commercially available foam swabs into and out of vacuum without compromising the swabs’ sterility. The specially designed swab canisters contain a 0.2 µm Teflon filter that allows the canister to accommodate pressure changes without introducing unwanted contaminants. The kit meets existing EVA (Extravehicular Activity) safety requirements and has been used by human test subjects in the neutral buoyance lab and at vacuum in test chambers at the Johnson Space Center. In the early part of 2024, astronauts will use this sampling kit to swab six surfaces on the exterior of ISS. We will collect samples from: the airlock vestibule, the interior surface of the airlock thermal cover, an exterior handrail, a vent connected to the CO2 removal system, and a vent connected to the payload vacuum system inside ISS. Approximately 300 cm2 will be swabbed at each location. A seventh swab will be exposed to the vacuum of space without touching any surfaces as a blank. The eighth swab will remain sealed as a process control. After the EVA the swab kit will be frozen at -80°C and returned to earth at the earliest possible opportunity. The samples will remain frozen until they are thawed for next generation DNA sequencing on earth. We will use amplicon sequencing to identify any bacteria, archaea or fungi present in the samples. If there is enough DNA present, we will use shotgun metagenomic sequencing to further characterize the microbial ecology outside ISS. Preliminary Results: Results from ground-based testing demonstrate that the swab kit is capable of cycling in and out of vacuum without contaminating the swabs. Test subjects, wearing flight-like EVA gloves could remove swabs from the canister and sample discrete locations without inadvertently touching any other surfaces. Several types of bacteria and fungi were collected during these ground tests and survived up to 6 hours at vacuum. This includes non-spore forming bacterial like Staphyloccus capitis that are not traditionally considered extremophiles. Shotgun metagenomic sequencing of the ground-test samples revealed bacteria associated with human skin and airways on the exterior of space suits used during these tests 2 In addition to these results we will present preliminary results from our space-flight samples. We will also present lessons learned from attempting to collect microbiological samples during an EVA and describe how our results will affect planetary protection requirements for future crewed missions.

Aaron B. Regberg↗

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BioNutrients-3: Precision Fermentation, Pasteurization, and Pathogen Detection - Towards Safe Fermentation and Production of Nutrients in Space

The BioNutrients (BN) project is developing a microbial manufacturing approach to supplement the NASA food system to address known nutrient degradation associated with long-term storage. BN uses synthetic biology to deliver high-value nutrients and therapeutics through genetic engineering of microbes and production of fermented food products like yogurt and kefir, in a fully dehydrated system. On-demand production of nutrients for human consumption requires rigorous safety protocols to ensure contaminants are not introduced during the fermentation process. The third iteration of the BN flight project, BN-3, further develops the BN project by investigating strategies for pathogen detection and pasteurization in microgravity. BN-3 will test the limits of pathogen detection using whole genome sequencing, standard microbial assays and the NASA Ames E-Nose, a volatile carbon nano tube-based sensor array, to detect unwanted microbes. BN-3 also expands on the number of nutrients produced in a single bioreactor to enhance efficiency of the system by combining production of B vitamins in conjunction with the carotenoids, beta-carotene and zeaxanthin. Serial production of food products through yogurt passaging, new updates to the fluorinated ethylene propylene bags to allow crew access via a straw, as well as use of a food safe pH indicator dye to indicate readiness of the food product, will further enhance the useability of this system. This presentation will provide status of the BN-3 flight project with the aim of advancing in-space biomanufacturing for on-demand microbially based food production for future space exploration.

Biomanufacturing↗

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

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

BioNutrients↗