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NASA NTRS2012

Gene Expression Measurement Module (GEMM) - A Fully Automated, Miniaturized Instrument for Measuring Gene Expression in Space

The capability to measure gene expression on board spacecraft opens the door to a large number of high-value experiments on the influence of the space environment on biological systems. For example, measurements of gene expression will help us to understand adaptation of terrestrial life to conditions beyond the planet of origin, identify deleterious effects of the space environment on a wide range of organisms from microbes to humans, develop effective countermeasures against these effects, and determine the metabolic bases of microbial pathogenicity and drug resistance. These and other applications hold significant potential for discoveries in space biology, biotechnology, and medicine. Supported by funding from the NASA Astrobiology Science and Technology Instrument Development Program, we are developing a fully automated, miniaturized, integrated fluidic system for small spacecraft capable of in-situ measurement of expression of several hundreds of microbial genes from multiple samples. The instrument will be capable of (1) lysing cell walls of bacteria sampled from cultures grown in space, (2) extracting and purifying RNA released from cells, (3) hybridizing the RNA on a microarray and (4) providing readout of the microarray signal, all in a single microfluidics cartridge. The device is suitable for deployment on nanosatellite platforms developed by NASA Ames' Small Spacecraft Division. To meet space and other technical constraints imposed by these platforms, a number of technical innovations are being implemented. The integration and end-to-end technological and biological validation of the instrument are carried out using as a model the photosynthetic bacterium Synechococcus elongatus, known for its remarkable metabolic diversity and resilience to adverse conditions. Each step in the measurement process-lysis, nucleic acid extraction, purification, and hybridization to an array-is assessed through comparison of the results obtained using the instrument with those from standard laboratory protocols. Once developed, the system can be used with minor modifications for multiple experiments on different platforms in space, including extension to higher organisms and microbial monitoring. A proposed version of GEMM that is capable of handling both microbial and tissue samples on the International Space Station will be briefly summarized.

Pohorille, Andrew↗

NASA NTRSDate not supplied

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

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

C M Ott↗

NASA NTRS2024

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

C.M. Ott↗

NASA NTRS2005

Effects of artificial defoliation of pines on the structure and physiology of the soil fungal community of a mixed pine-spruce forest

Loss of photosynthetic area can affect soil microbial communities by altering the availability of fixed carbon. We used denaturing gradient gel electrophoresis (DGGE) and Biolog filamentous-fungus plates to determine the effects of artificial defoliation of pines in a mixed pine-spruce forest on the composition of the fungal community in a forest soil. As measured by DGGE, two fungal species were affected significantly by the defoliation of pines (P < 0.001); the frequency of members of the ectomycorrhizal fungus genus Cenococcum decreased significantly, while the frequency of organisms of an unidentified soil fungus increased. The decrease in the amount of Cenococcum organisms may have occurred because of the formation of extensive hyphal networks by species of this genus, which require more of the carbon fixed by their host, or because this fungus is dependent upon quantitative differences in spruce root exudates. The defoliation of pines did not affect the overall composition of the soil fungal community or fungal-species richness (number of species per core). Biolog filamentous-fungus plate assays indicated a significant increase (P < 0.001) in the number of carbon substrates utilized by the soil fungi and the rate at which these substrates were used, which could indicate an increase in fungal-species richness. Thus, either small changes in the soil fungal community give rise to significant increases in physiological capabilities or PCR bias limits the reliability of the DGGE results. These data indicate that combined genetic and physiological assessments of the soil fungal community are needed to accurately assess the effect of disturbance on indigenous microbial systems.

Pinus/growth & development/physiology↗

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 NTRS1988

Test results on re-use of reclaimed shower water: Summary

A microgravity whole body shower (WBS) and waste water recovery systems (WWRS) were evaluated in three separate closed loop tests. Following a protocol similar to that anticipated for the U.S. Space Station, test subjects showered in a prototype whole body shower. The WWRS processes evaluated during the test series were phase change and reverse osmosis (RO). A preprototype Thermoelectric Integrated Hollow Fiber Membrane Evaporation Subsystem phase change process was used for the initial test with chemical pretreatment of the shower water waste input. The second and third tests concentrated on RO technologies. The second test evaluated a dynamic RO membrane consisting of zirconium oxide polyacrylic acid (ZOPA) membranes deposited on the interior diameter of 316L porous stainless steel tubes while the final test employed a thin semipermeable RO membrane deposited on the interior surface of polysulfone hollow fibers. All reclaimed water was post-treated for purity using ion exchange and granular activated carbon beds immediately followed by microbial control treatment using both heat and iodine. The test hardware, controls exercised for whole body showering, types of soaps evaluated, shower subject response to reclaimed water showering, and shower water collection and chemical pretreatment (if required) for microbial control are described. The WWRS recovered water performance and the effectiveness of the reclaimed water post-treatment techniques used for maintaining water purity and microorganism control are compared. Results on chemical and microbial impurity content of the water samples obtained from various locations in the shower water reuse system are summarized.

Verostko, C. E.↗

NASA NTRS2011

Parabolic Flight Evaluation of Bacterial Adhesion on Multiple Antimicrobial Surface Treatments

This report describes the development of a test method and the evaluation of the effectiveness of antimicrobial technologies in reduced gravity based on parabolic flight experiments. Microbial growth is a common occurrence on fully immersed wetted surfaces in spacecraft environmental control and life support systems despite the use of chemical and/or physical \disinfection. Many materials and surface treatments with antimicrobial properties are commercially available but none have been vetted for spaceflight applications. Herein a test method is explained that included ground and reduced gravity parabolic flight experiments with a standard microorganism recovered from spacecraft, Pseudomonas aeruginosa, added at a concentration of 1 x 10(exp 5) cells per milliliter (mL) onto challenge material coupon surfaces. Several experimental materials were observed to slightly reduce microbial attachment in reduced gravity flight experiments, but none were capable of eliminating all challenge bacteria. Lunar gravity had an increased antimicrobial effect in 28 out of 36 test coupons compared to microgravity when provided otherwise identical conditions for growth, suggesting trace .amounts of gravity may be required for maximum antimicrobial performance. Bacterial cells exposed to variable gravity had more than twice as ,much intracellular adenosine triphosphate (ATP) when compared to control cells exposed only to Earth gravity due to a short duration response to environmental stress. An ATP luminescence assay was the method most amenable to development of an in-flight microbial monitoring assay

Birmele, Michele↗

NASA NTRS2002

Rejuvenation of Spent Media via Supported Emulsion Liquid Membranes

The overall goal of this project is to maximize the reuseability of spent fermentation media. Supported emulsion liquid membrane separation, a highly efficient extraction technique, is used to remove inhibitory byproducts during fermentation; thus, improving the yield while reducing the need for fresh water. The key objectives of this study are: Develop an emulsion liquid membrane system targeting low molecular weight organic acids which has minimal toxicity on a variety of microbial systems; Conduct mass transfer studies to allow proper modeling and design of a supported emulsion liquid membrane system; Investigate the effect of gravity on emulsion coalescence within the membrane unit; Access the effect of water re-use on fermentation yields in a model microbial system; Develop a perfusion-type fermentor utilizing a supported emulsion liquid membrane system to control inhibitory fermentation byproducts; Work for the coming year will focus on the determination of toxicity of various solvents, selection of the emulsifying agents, as well as characterizing the mass transfer of hollow-fiber contactors.

Wiencek, John M.↗

NASA NTRSDate not supplied

Life Finds A Way, the Dynamics of E. Coli Evolution in Microgravity

Investigating the evolutionary dynamics of Escherichia coli in microgravity offers a unique opportunity to understand microbial adaptation to extreme environments. Here, we explored the effects of simulated microgravity (SµG) on gene expression and genome evolution of Escherichia coli REL606, a strain continuously evolved and documented terrestrially for 35 years. We used transcriptomic profiling over a 24-hour growth cycle to examine how short-term exposure to SµG under glucose-limiting and glucose-replete conditions may influence the genetic adaptations in microbial populations. Pathway analyses of differentially expressed genes suggest that SµG may alter cell membrane structure and function across all conditions, while changes to protein synthesis machinery were uniquely observed in glucose-replete samples. Furthermore, altered expression of several prophage genes across conditions in SµG samples and upregulation of general stress response factors hints at the potential for stress-induced mutagenesis in response to microgravity. We further investigated the impact of long-term exposure to SµG on genome evolution and observed a more rapid accumulation of base substitutions and deletions in SµG sample genomes across time. Specifically, mutations in the mraZ and elyC genes suggest a mechanism for increased production of peptidoglycan in the cell membrane. These findings offer insights into bacterial adaptations in long-term microgravity environments and pave the way for further detailed investigations.

Brittney Lozzi↗

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 NTRS2002

Rejuvenation of Spent Media via Supported Emulsion Liquid Membranes

The overall goal of this project was to maximize the reuseability of spent fermentation media. Supported emulsion liquid membrane separation, a highly efficient extraction technique, was used to remove inhibitory byproducts during fermentation; thus, improve the yield while reducing the need for fresh water. The key objectives of this study were: (1) Develop an emulsion liquid membrane system targeting low molecular weight organic acids which has minimal toxicity on a variety of microbial systems. (2) Conduct mass transfer studies to allow proper modeling and design of a supported emulsion liquid membrane system. (3) Investigate the effect of gravity on emulsion coalescence within the membrane unit. (4) Access the effect of water re-use on fermentation yields in a model microbial system. and (5) Develop a perfusion-type fermentor utilizing a supported emulsion liquid membrane system to control inhibitory fermentation byproducts (not completed due to lack of funds)

Wiencek, John M.↗

NASA NTRS1991

Waste treatment integration in space

The circumstances and criteria for space-based waste treatment bioregenerative life-support systems differ in many ways from those needed in terrestrial applications. In fact, the term "waste" may not even be appropriate in the context of nearly closed, cycling, ecosystems such as those under consideration. Because of these constraints there is a need for innovative approaches to the problem of "materials recycling". Hybrid physico-chemico-biological systems offer advantages over both strictly physico-chemico or biological approaches that would be beneficial to material recycling. To effectively emulate terrestrial cycling, the use of various microbial consortia ("assemblies of interdependent microbes") should be seriously considered for the biological components of such systems. This paper will examine the use of consortia in the context of a hybrid-system for materials recycling in space.

Waste Management/methods↗

NASA NTRS2001

Ecological physiology of Synechococcus sp. strain SH-94-5, a naturally occurring cyanobacterium deficient in nitrate assimilation

Synechococcus sp. strain SH-94-5 is a nitrate assimilation-deficient cyanobacterium which was isolated from an ammonium-replete hot spring in central Oregon. While this clone could grow on ammonium and some forms of organic nitrogen as sole nitrogen sources, it could not grow on either nitrate or nitrite, even under conditions favoring passive diffusion. It was determined that this clone does not express functional nitrate reductase or nitrite reductase and that the lack of activity of either enzyme is not due to inactivation of the cyanobacterial nitrogen control protein NtcA. A few other naturally occurring cyanobacterial strains are also nitrate assimilation deficient, and phylogenetic analyses indicated that the ability to utilize nitrate has been independently lost at least four times during the evolutionary history of the cyanobacteria. This phenotype is associated with the presence of environmental ammonium, a negative regulator of nitrate assimilation gene expression, which may indicate that natural selection to maintain functional copies of nitrate assimilation genes has been relaxed in these habitats. These results suggest how the evolutionary fates of conditionally expressed genes might differ between environments and thereby effect ecological divergence and biogeographical structure in the microbial world.

Nitrates/metabolism↗

NASA NTRS2004

Impact Processes in the Solar System

The three main topics of this program as described initially in our May 2003 proposal are: 1) Shock-induced damage and attenuation in planetary materials. 2 ) Shock-induced melting and phase changes. 3) Impact-induced volatilization and vapor speciation of planetary materials Topic 4 has been the subject of a continuing investigation since approximately 1990. On Topic 5, we have a paper in preparation and have submitted a proposal to Astrobiology. 4) Responses of planetary atmospheres to giant impact, 5) Effects of impact-induced shock waves on microbial life

Ahrens, Thomas J.↗

NASA NTRS2019

Lipid Decontamination Procedures for Life Detection Missions

Molecular biosignatures are key targets for current, proposed, and future life detection missions. With the high accuracy and low limit of detection (LOD) that new and future instruments will require, decontamination of life detection hardware is necessary to prevent false positives. Lipids are a molecular biosignature of interest, as they are ubiquitous to all life as we know it, can survive unaltered in the geologic record for longer than any other biomolecule (i.e. billions of years), and form through both biotic and abiotic processes. Lipids display origin-diagnostic molecular patterns that can reveal biotic or abiotic synthesis, so finding them and ascertaining their molecular features is important for potentially detecting evidence of life elsewhere. Traditional methods of decontamination, or contamination control (CC), primarily clean hardware through fabrication in sterile (cleanroom) environments, killing microbes, and removing/flushing contaminants off instrument and spacecraft components. However, research suggests that some standard cleaning methods are either unlikely to remove lipid contaminants or are incompatible with life detection instrument materials. To solve this problem, I propose to find, test, and verify a decontamination method that thoroughly cleans instruments by destroying lipid molecules, but is simultaneously compatible with major materials used in these instruments. I will study the effects of traditional CC methods (including Dry Heat Microbial Reduction and Vapor phase Hydrogen Peroxide) and experimental CC methods (Electron Beam Irradiation) on lipid molecules for application to life detection instrumentation. I will then develop a CC plan for a novel lipid detector (ExCALiBR, Extractor for Chemical Analysis of Lipid Biomarkers in Regolith) searching for lipids in either soil or icy world scenarios. This plan will uphold planetary protection regulation requirements and validate experimental analyses of in-situ life detection tests.

Decontamination↗

NASA NTRS1998

Long Term Effects of Poultry Litter on Soil Physical and Chemical Properties in Cotton Plots

Poultry litter and compost can alter the moisture holding capacity of a soil. These organic materials can also increase the nutrient status of a soil during the decomposition process by microbial actions. The objective of this study was to evaluate the effect of poultry litter and compost on the dielectric constant and moisture holding capacity of soil. The Delta-T theta-probe was used to measure volumetric soil water content and the apparent dielectric constant of the upper 6-cm of the soil profile. Soil texture, pH, and organic matter were also determined for each plot. Results of these analyses indicated that the pH of the soil ranged from 6.4 to 7.7 and the volumetric soil moisture content ranged from 0.06 to 0.18 cu m/cu m for the upper 6-cm of the soil profile. The effect of poultry litter and compost on soil properties resulted in an increase in the volumetric moisture content and dielectric constant of the soil due to the improvement of the soil structure.

Surrency, J.↗

NASA NTRS1975

Microbiological investigations

The crew microbiology program was conducted to evaluate lunar contamination, to detect potentially pathogenic microoganisms, to identify medically important microorganisms recovered from ill crewmen, to aid in diagnosis and treatment, and to collect microbiological data that would aid in elucidating the response of the crew microbial autoflora to the space flight environment and in evaluating the resultant effect on the crewmember. Microbiological sampling of selected sites in the command module was initiated in support of the quarantine program. During lunar quarantine missions, microbial screening was accomplished for all support personnel to be isolated with the returning crewman. Virology support for the Apollo project consisted of characterization of the viral and mycoplasma flora of the crewmembers and performance of viral serology for crewmembers, crew contacts, and key mission personnel. Procedures and results are discussed in detail.

James K. Ferguson↗

NASA NTRS1994

Suitability of Gray Water for Hydroponic Crop Production Following Biological and Physical Chemical and Biological Subsystems

The water present in waste streams from a human habitat must be recycled in Controlled Ecological Life Support Systems (CELSS) to limit resupply needs and attain self-sufficiency. Plants play an important role in providing food, regenerating air, and producing purified water via transpiration. However, we have shown that the surfactants present in hygiene waste water have acute toxic effects on plant growth (Bubenheim et al. 1994; Greene et al., 1994). These phytotoxic affects can be mitigated by allowing the microbial population on the root surface to degrade the surfactant, however, a significant suppression (several days) in crop performance is experienced prior to reaching sub-toxic surfactant levels and plant recovery. An effective alternative is to stabilize the microbial population responsible for degradation of the surfactant on an aerobic bioreactor and process the waste water prior to utilization in the hydroponic solution (Wisniewski and Bubenheim, 1993). A sensitive bioassay indicates that the surfactant phytotoxicity is suppressed by more than 90% within 5 hours of introduction of the gray water to the bioreactor; processing for more than 12 hours degrades more than 99% of the phytotoxin. Vapor Compression Distillation (VCD) is a physical / chemical method for water purification which employees sequential distillation steps to separate water from solids and to volatilize contaminants. The solids from the waste water are concentrated in a brine and the pure product water (70 - 90% of the total waste water volume depending on operating conditions) retains non of the phytotoxic effects. Results of the bioassay were used to guide evaluations of the suitability of recovered gray water following biological and VCD processing for hydroponic lettuce production in controlled environments. Lettuce crops were grown for 28 days with 100% of the input water supplied with recovered water from the biological processor or VCD. When compared with the growth of plants in control hydroponic solution containing pure deionized water, no growth difference could be measured resulting from any of the recovered water treatments. Both biological treatment and VCD offer alternative technology approaches to recovering water from waste streams appropriate for input into a crop production system. A high level of crop performance (food, air, and water production) can be maintained with either processor; selection decisions can be based on other factors regarding system integration.

Bubenheim, David L.↗