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At least 55 records · Page 3

Current Concepts and Future Directions of CELSS

Bioregenerative life support systems for use in space were studied. Concepts of such systems include the use of higher plants and/or microalgae as sources of food, potable water and oxygen, and as sinks for carbon dioxide and metabolic wastes. Recycling of materials within the system will require processing of food organism and crew wastes using microbiological and/or physical chemical techniques. The dynamics of material flow within the system will require monitoring, control, stabilization and maintenance imposed by computers. Studies included higher plant and algal physiology, environmental responses, and control; flight experiments for testing responses of organisms to weightlessness and increased radiation levels; and development of ground based facilities for the study of recycling within a bioregenerative life support system.

Macelroy, R. D.↗

Biomolecular Effects of Cold Plasma Exposure

The effects of cold plasma exposure on Deinococcus radiodurans, plasmid DNA and model proteins were assessed using microbiological, spectrometric and biochemical techniques. Exposure of D. radiodurans, an extremely radiation resistant microbe, to O2 plasma (less than or equal to 25 W, approx. 45 mTorr, 90 min) yielded a approx. 99.999 % sterilization and the sterilization rate was increased approx. 10-fold at 100 W and 500 mTorr. AFM images shows that the exposed cells are significantly deformed and possess 50-70 nm concavities. IR analysis indicates the chemical degradation of lipids, proteins and carotenoids of the cell wall and membrane. Intracellular damage was indicated by major absorbance loss at 1245, 1651 and 1538/cm corresponding to degradation of DNA and proteins, respectively. Biochemical experiments demonstrate that plasmas induce strand scissions and crosslinking of plasmid DNA, and reduction of enzyme activity; the degradation is power dependent with total sample loss occurring in 60 s at 200 W and 500 mTorr. Emission spectroscopy shows that D. radiodurans is volatilized into CO2, CO, N2 and H2O confirming the removal of biological matter from contaminated surfaces. The O2 plasma impacts several cellular components predominantly through chemical degradation by atomic oxygen. A CO2, plasma, however, was not effective at degrading D. radiodurans, revealing the importance of plasma composition, which has implications for planetary protection and the contamination of Mars.

Mogul, Rakesh↗

Impact of low-temperature plasmas on Deinococcus radiodurans and biomolecules

The effects of cold plasma on Deinococcus radiodurans, plasmid DNA, and model proteins were assessed using microbiological, spectrometric, and biochemical techniques. In low power O(2) plasma (approximately 25 W, approximately 45 mTorr, 90 min), D. radiodurans, a radiation-resistant bacterium, showed a 99.999% reduction in bioburden. In higher power O(2) plasma (100 W and 500 mTorr), the reduction rate increased about 10-fold and observation by atomic force microscopy showed significant damage to the cell. Damage to cellular lipids, proteins, and chromosome was indicated by losses of infrared spectroscopic peaks at 2930, 1651, 1538, and 1245 cm(-1), respectively. In vitro experiments show that O(2) plasmas induce DNA strand scissions and cross-linking as well as reduction of enzyme activity. The observed degradation and removal of biomolecules was power-dependent. Exposures to 200 W at 500 mTorr removed biomolecules to below detection limits in 60 s. Emission spectroscopy indicated that D. radiodurans cells were volatilized into CO(2), CO, N(2), and H(2)O, confirming that these plasmas were removing complex biological matter from surfaces. A CO(2) plasma was not as effective as the O(2) plasma, indicating the importance of plasma composition and the dominant role of chemical degradation. Together, these findings have implications for NASA planetary protection schemes and for the contamination of Mars.

Evaluation Studies↗

Community-level physiological profiling performed with an oxygen-sensitive fluorophore in a microtiter plate

Community-level physiological profiling based upon fluorometric detection of oxygen consumption was performed on hydroponic rhizosphere and salt marsh litter samples by using substrate levels as low as 50 ppm with incubation times between 5 and 24 h. The rate and extent of response were increased in samples acclimated to specific substrates and were reduced by limiting nitrogen availability in the wells.

NASA Discipline Life Support Systems↗

Microbiological sampling of spacecraft cabling, antennas, solar panels and thermal blankets

Sampling procedures and techniques described resulted from various flight project microbiological monitoring programs of unmanned planetary spacecraft. Concurrent with development of these procedures, compatibility evaluations were effected with the cognizant spacecraft subsystem engineers to assure that degradation factors would not be induced during the monitoring program. Of significance were those areas of the spacecraft configuration for which special handling precautions and/or nonstandard sample gathering techniques were evolved. These spacecraft component areas were: cabling, high gain antenna, solar panels, and thermal blankets. The compilation of these techniques provides a historical reference for both the qualification and quantification of sampling parameters as applied to the Mariner Spacecraft of the late 1960's and early 1970's.

Koukol, R. C.↗

Deep learning enhanced super-resolution x-ray fluorescence microscopy by a dual-branch network

X-ray fluorescence (XRF) microscopy is a powerful technique for quantifying the distribution of elements in complex materials, which makes it a crucial imaging technique across a wide range of disciplines in physical and biological sciences, including chemistry, materials science, microbiology, and geosciences. However, as a scanning microscopy technique, the spatial resolution of XRF imaging is inherently constrained by the x-ray probe profile and scanning step size. Here we propose a dual-branch machine learning (ML) model, which can extract scale-variant features and bypass abundant low-frequency information separately, to enhance the spatial resolution of the XRF images by mitigating the effects of blurring from the probe profile. The model is trained by simulated natural images, and a two-stage training strategy is used to overcome the domain gap between the natural images and experimental data. The tomography reconstruction from enhanced XRF projections shows an improvement in resolution by a scale factor of four and reveals distinct internal features invisible in low-resolution XRF within a battery sample. This study offers a promising approach for obtaining high-resolution XRF imaging from its low-resolution version, paving the way for future investigations in a broader range of disciplines and materials.

36 MATERIALS SCIENCE↗

Single-cell stable isotope probing in microbial ecology

Abstract Environmental and host-associated microbiomes are typically diverse assemblages of organisms performing myriad activities and engaging in a network of interactions that play out in spatially structured contexts. As the sum of these activities and interactions give rise to overall microbiome function, with important consequences for environmental processes and human health, elucidating specific microbial activities within complex communities is a pressing challenge. Single-cell stable isotope probing (SC-SIP) encompasses multiple techniques that typically utilize Raman microspectroscopy or nanoscale secondary ion mass spectrometry (NanoSIMS) to enable spatially resolved tracking of isotope tracers in cells, cellular components, and metabolites. SC-SIP techniques are uniquely suited for illuminating single-cell activities in microbial communities and for testing hypotheses about cellular functions generated for example from meta-omics datasets. Here, we illustrate the insights enabled by SC-SIP techniques by reviewing selected applications in microbiology and offer a perspective on their potential for future research.

Alcolombri, Uria (ORCID:0000000335615091)↗

Microbiological concerns and methodological approaches related to bacterial water quality in spaceflight

A number of microbiological issues are of critical importance to crew health and system performance in spacecraft water systems. This presentation reviews an army of these concerns which include factors that influence water treatment and disinfection in spaceflight such as biofilm formation and the physiological responses of bacteria in clean water systems. Factors associated with spaceflight like aerosol formation under conditions of microgravity are also discussed within the context of airborne infections such as Legionellosis. Finally, a spectrum of analytical approaches is reviewed to provide an evaluation of methodological alternatives that have been suggested or used to detect microorganisms of interest in water systems. These range from classical approaches employing colony formation on specific microbiological growth media to direct (i.e. microscopic) and indirect (e.g. electrochemical) methods as well as the use of molecular approaches and gene probes. These techniques are critically evaluated for their potential utility in determining microbiological water quality through the detection of microorganisms under the influence of ambient environmental stress inherent in spaceflight water systems.

Pyle, Barry H.↗

Planned Environmental Microbiology Aspects of Future Lunar and Mars Missions

With the establishment of the Constellation Program, NASA has initiated efforts designed similar to the Apollo Program to return to the moon and subsequently travel to Mars. Early lunar sorties will take 4 crewmembers to the moon for 4 to 7 days. Later missions will increase in duration up to 6 months as a lunar habitat is constructed. These missions and vehicle designs are the forerunners of further missions destined for human exploration of Mars. Throughout the planning and design process, lessons learned from the International Space Station (ISS) and past programs will be implemented toward future exploration goals. The standards and requirements for these missions will vary depending on life support systems, mission duration, crew activities, and payloads. From a microbiological perspective, preventative measures will remain the primary techniques to mitigate microbial risk. Thus, most of the effort will focus on stringent preflight monitoring requirements and engineering controls designed into the vehicle, such as HEPA air filters. Due to volume constraints in the CEV, in-flight monitoring will be limited for short-duration missions to the measurement of biocide concentration for water potability. Once long-duration habitation begins on the lunar surface, a more extensive environmental monitoring plan will be initiated. However, limited in-flight volume constraints and the inability to return samples to Earth will increase the need for crew capabilities in determining the nature of contamination problems and method of remediation. In addition, limited shelf life of current monitoring hardware consumables and limited capabilities to dispose of biohazardous trash will drive flight hardware toward non-culture based methodologies, such as hardware that rapidly distinguishes biotic versus abiotic surface contamination. As missions progress to Mars, environmental systems will depend heavily on regeneration of air and water and biological waste remediation and regeneration systems, increasing the need for environmental monitoring. Almost complete crew autonomy will be needed for assessment and remediation of contamination problems. Cabin capacity will be limited; thus, current methods of microbial monitoring will be inadequate. Future methodology must limit consumables, and these consumables must have a shelf life of over three years. In summary, missions to the moon and Mars will require a practical design that prudently uses available resources to mitigate microbial risk to the crew.

Ott, C. Mark↗

Control of microbial contamination.

Two specific applications are discussed of microbial contamination control in planetary quarantine. Under the first concept, using the clean room to control environmental microorganisms, the objective is to reduce the microbial species and keep the numbers of microorganisms within an enclosure at a low level. The clean room concept is aimed at obtaining a product that has a controlled and reduced level of microbial contamination. Under the second concept, using the microbiological barrier to control microbial contamination of a specific product, the barrier techniques are designed to prevent the entry of any microorganisms into a sterile work area. Thus the assembly of space flight hardware within the confines of a microbiological barrier is aimed at obtaining a sterile product. In theory and practice, both approaches are shown to be applicable to the planetary quarantine program.

Mcdade, J. J.↗

Studying microbially induced corrosion on glass using ToF-SIMS

Microbially induced corrosion (MIC) is an emerging topic that has huge environmental impacts, such as long-term evaluation of microbial interactions with radioactive waste glass, environmental cleanup and disposal of radioactive material, and weathering effects of microbes. Time-of-flight secondary ion mass spectrometry (ToF-SIMS), a powerful mass spectral imaging technique with high surface sensitivity, mass resolution, and mass accuracy, can be used to study biofilm effects on different substrates. Understanding how to prepare biofilms on MIC susceptible substrates is critical for proper analysis via ToF-SIMS. We present here a step-by-step protocol for preparing bacterial biofilms for ToF-SIMS analysis, comparing three biofilm preparation techniques: no desalination, centrifugal spinning (CS), and water submersion (WS). Comparisons of two desalinating methods, CS and WS, show a decrease in the media peaks up to 99% using CS and 55% using WS, respectively. Proper desalination methods also can increase biological signals by over four times for fatty acids using WS, for example. ToF-SIMS spectral results show chemical compositional changes of the glass exposed in a Paenibacillus polymyxa SCE2 biofilm, indicating its capability to probe microbiologically induced corrosion of solid surfaces. This represents the proper desalination technique to use without significantly altering biofilm structure and substrate for ToF-SIMS analysis. ToF-SIMS spectral results showed chemical compositional changes of the glass exposed by a Paenibacillus bacterial biofilm over 3-month inoculation. Finally, possible MIC products include various phosphate phase molecules not observed in any control samples with the highest percent increases when experimental samples were compared with biofilm control samples.

36 MATERIALS SCIENCE↗

Recovery of hygiene water by multifiltration

A multifiltration hygiene water reclamation process that utilizes adsorption and particulate filtration techniques is described and evaluated. The applicability of the process is tested using a simulation of a 4-man subsystem operation for 240 days. It is proposed the process has a 10 year life, weighs 236 kg, and uses 88 kg of expendable filters and adsorption beds to process 8424 kg of water. The data reveal that the multifiltration is an efficient nonphase change technique for hygiene water recovery and that the chemical and microbiological purity of the product water is within the standards specified for the Space Station hygiene water.

Putnam, David F.↗

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↗