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NASA NTRS2003

Energy Requirements of Hydrogen-utilizing Microbes: A Boundary Condition for Subsurface Life

Microbial ecosystems based on the energy supplied by water-rock chemistry carry particular significance in the context of geo- and astrobiology. With no direct dependence on solar energy, lithotrophic microbes could conceivably penetrate a planetary crust to a depth limited only by temperature or pressure constraints (several kilometers or more). The deep lithospheric habitat is thereby potentially much greater in volume than its surface counterpart, and in addition offers a stable refuge against inhospitable surface conditions related to climatic or atmospheric evolution (e.g., Mars) or even high-energy impacts (e.g., early in Earth's history). The possibilities for a deep microbial biosphere are, however, greatly constrained by life s need to obtain energy at a certain minimum rate (the maintenance energy requirement) and of a certain minimum magnitude (the energy quantum requirement). The mere existence of these requirements implies that a significant fraction of the chemical free energy available in the subsurface environment cannot be exploited by life. Similar limits may also apply to the usefulness of light energy at very low intensities or long wavelengths. Quantification of these minimum energy requirements in terrestrial microbial ecosystems will help to establish a criterion of energetic habitability that can significantly constrain the prospects for life in Earth's subsurface, or on other bodies in the solar system. Our early work has focused on quantifying the biological energy quantum requirement for methanogenic archaea, as representatives of a plausible subsurface metabolism, in anoxic sediments (where energy availability is among the most limiting factors in microbial population growth). In both field and laboratory experiments utilizing these sediments, methanogens retain a remarkably consistent free energy intake, in the face of fluctuating environmental conditions that affect energy availability. The energy yields apparently required by methanogens in these sediment systems for sustained metabolism are about half that previously thought necessary. Lowered energy requirements would imply that a correspondingly greater proportion of the planetary subsurface could represent viable habitat for microorganisms.

Hoehler, Tori M.↗

NASA NTRS2003

ATP as a biomarker of viable microorganisms in clean-room facilities

A new firefly luciferase bioluminescence assay method that differentiates free extracellular ATP (dead cells, etc.) from intracellular ATP (viable microbes) was used to determine the viable microbial cleanliness of various clean-room facilities. For comparison, samples were taken from both clean-rooms, where the air was filtered to remove particles >0.5 microm, and ordinary rooms with unfiltered air. The intracellular ATP was determined after enzymatically degrading the sample's free ATP. Also for comparison, cultivable microbial populations were counted on nutrient-rich trypticase soy agar (TSA) plates. Both the cultivable and ATP-based determinations indicate that the microbial burden was lower in clean-room facilities than in ordinary rooms. However, there was no direct correlation between the two sets of measurements because the two assays measured very different populations. A large fraction of the samples yielded no colony formers on TSA, but were positive for intracellular ATP. Subsequently, genomic DNA was isolated directly from selected samples and 16S rDNA fragments were cloned and sequenced, identifying nearest neighbors, many of which are known to be noncultivable in the media employed. It was concluded that viable microbial contamination can be reliably monitored by measurement of intracellular ATP, and that this method may be considered superior to cultivable colony counts due to its speed and its ability to report the presence of viable but noncultivable organisms. When the detection of nonviable microbes is of interest, the ATP assay can be supplemented with DNA analysis.

NASA Discipline Environmental Health↗

NASA NTRS1999

Post-Viking microbiology: new approaches, new data, new insights

In the 20 years since the Viking experiments, major advances have been made in the areas of microbial systematics, microbial metabolism, microbial survival capacity, and the definition of environments on earth, suggesting that life is more versatile and tenacious than was previously appreciated. Almost all niches on earth which have available energy, and which are compatible with the chemistry of carbon-carbon bonds, are known to be inhabited by bacteria. The oldest known bacteria on earth apparently evolved soon after the formation of the planet, and are heat loving, hydrogen and/or sulfur metabolizing forms. Among the two microbial domains (kingdoms) is a great deal of metabolic diversity, with members of these forms being able to grow on almost any known energy source, organic or inorganic, and to utilize an impressive array of electron acceptors for anaerobic respiration. Both hydrothermal environments and the deep subsurface environments have been shown to support large populations of bacteria, growing on energy supplied by geothermal energy, thus isolating these ecosystems from the rest of the global biogeochemical cycles. This knowledge, coupled with new insights into the history of the solar system, allow one to speculate on possible evolution and survival of life forms on Mars.

Review↗

NASA NTRS2005

Influence of Hydroponically Grown Hoyt Soybeans and Radiation Encountered on Mars Missions on the Yield and Quality of Soymilk and Tofu

Soybeans were chosen for hmar and planetary missions due to their nutritive value and ability to produce oil and protein for further food applications. However, soybeans must be processed into foods prior to crew consumption. Wilson et al. (2003) raised questions about (1) the influence of radiation (on germination and functional properties) that the soybeans would be exposed to during bulk storage for a Mars mission, and (2) the impact of using hydroponically grown versus field grown soybeans on the yield and quality of soyfoods. The influence of radiation can be broken down into two components: (A) affect of surface pasteurization to ensure the astronauts safety from food-borne illnesses (a Hazard Analysis Critical Control Point), and (B) affect of the amount of radiation the soybeans receive during a Mars mission. Decreases in the amount of natural antioxidants and free radical formation and oxidation induced changes in the soybean (lipid, protein, etc.) will influence the nutritional value, texture, quality, and safety of soyfoods made from them. The objectives of this project are to (1) evaluate the influence of gamma and electron beam radiation on bulk soybeans (HACCP, CCP) on the microbial load, germination, ease of processing, and quality of soymilk and tofu; (2) provide scale up and mass balance data for Advanced Life Support subsystems including Biomass, Solid Waste Processing, and Water Recovery Systems; and (3) to compare Hoyt field grown to hydroponically grown Hoyt soybeans for soymilk and tofu production. The soybean cultivar Hoyt, a small standing, high protein cultivar that could grow hydroponically in the AIMS facility on Mars) was evaluated for the production of soymilk and tofu. The quality and yield of the soymilk and tofu from hydroponic Hoyt, was compared to Vinton 81 (a soyfood industry standard), field Hoyt, IA 2032LS (lipoxygenase-free), and Proto (high protein and antioxidant potential). Soymilk and tofu were produced using the Japanese method. The soymilk was coagulated with calcium sulfate dihydrate. Soybeans and tofu were evaluated using chemical, microbial, and instrumental sensory methods. The surface radiation of whole dry soybeans using electron beam or gamma rays at 10 or 30 kGy did provide microbial safety for the astronauts. However, these doses caused oxidative changes that resulted in tofu with rancid aroma, darkening of the tofu, lower tofu yields, more solid waste, and loss of the ability of the seeds to germinate. While lower doses may reduce these problems, we lose the ability to insure microbial safety (cross-contamination) of bulk soybeans for the astronauts. Counter measures could include vacuum packaging, radiating under freezing conditions. A No Effect Dose for food quality, below 10 kGy needs to be determined. Better estimates of the radiation that the food will be exposed to need to determined and shared. Appropriate shielding for the food as well as the astronauts needs to be developed. The Hoyt soybean did not provide a high yielding, high quality tofu. A new small scale system for evaluating soybeans was developed using 50 g quantities of soybeans.

Wilson, Lester A.↗

NASA NTRS2005

Assessment of Microbiologically Influenced Corrosion Potential in the International Space Station Internal Active Thermal Control System Heat Exchanger Materials: A 6-Momths Study

The fluid in the Internal Active Thermal Control System (IATCS) of the International Space Station (ISS) is water based. The fluid in the ISS Laboratory Module and Node 1 initially contained a mix of water, phosphate (corrosion control), borate (pH buffer), and silver sulfate (Ag2SO4) (microbial control) at a pH of 9.5+/-0.5. Over time, the chemistry of the fluid changed. Fluid changes included a pH drop from 9.5 to 8.3 due to diffusion of carbon dioxide (CO2) through Teflon(reistered Trademark) (DuPont) hoses, increases in dissolved nickel (Ni) levels, deposition of silver (Ag) to metal surfaces, and precipitation of the phosphate (PO4) as nickel phosphate (NiPO4). The drop in pH and unavailability of a antimicrobial has provided an environment conducive to microbial growth. Microbial levels in the fluid have increased from >10 colony-forming units (CFUs)/100 ml to 10(exp 6) CFUs/100 ml. The heat exchangers in the IATCS loops are considered the weakest point in the loop because of the material thickness (=7 mil). It is made of a Ni-based braze filler/CRES 347. Results of a preliminary test performed at Hamilton Sundstrand indicated the possibility of pitting on this material at locations where Ag deposits were found. Later, tests have confirmed that chemical corrosion of the materials is a concern for this system. Accumulation of micro-organisms on surfaces (biofilm) can also result in material degradation and can amplify the damage caused by the chemical corrosion, known as microbiologically influenced corrosion (MIC). This paper will discuss the results of a 6-mo test performed to characterize and quantify the damage from microbial accumulation on the surface of the ISS/ATCS heat exchanger materials. The test was designed to quantify the damage to the materials under worst-case conditions with and without micro-organisms present at pH 8.3 and 9.5.

Roman, Monsi C.↗

NASA NTRS2005

Mineralized Remains of Morphotypes of Filamentous Cyanobacteria in Carbonaceous Meteorites

The quest for conclusive evidence of microfossils in meteorites has been elusive. One difficulty arises from the fact than many abiotic or inorganic microstructures, mineral grains, and coating artifacts can mimic the smaller representatives of the microbial world that possess very simple morphologies (unicellular cocci or bacilli). However, there exist a wide variety of large, filamentous trichomic prokaryotic microorganisms (cyanobacteria and sulfur bacteria) with sufficiently well known and complex morphologies that they can be recognized and are known to be of unquestionable biogenic origin. The taphonomic modes of fossilization and their of their life habits and processes frequently result in distinctive chemical biosignatures associated with carbonization, silicification, calcification, phosphatization and metal-binding properties of their cell-walls, trichomes, sheaths and extracellular polymeric substances (EPS). Strong differences of mineral concentrations in closely associated and visibly differentiated cellular microstructures provide strong evidence of biogenicity. This evidence is further enhanced by the detection of recognizable and distinct microstructures (e.g., uniseriate or multiseriate filaments, trichomes, sheaths, cells of proper sizes and size distributions) and growth characteristics (e.g., basal or apical cells, true or false branching of trichomes, tapered or uniform filaments, robust or thin sheaths) and reproductive and nitrogen fixation habits (e.g., baeocytes, hormogonia, akinetes and heterocysts), Microfossils of cyanobacteria and cyanobacterial mats and stromatolites have been recognized a described from many of the most ancient rocks on Earth. The crucial problem lies in developing valid protocols and methodologies for establishing that the putative microfossils are truly indigenous and not merely recent microbial contaminants. During the past several years, we have conducted Field Emission Scanning Electron Microscopy (FESEM) investigations of freshly fractured interior surfaces of carbonaceous meteorites, terrestrial rocks, and recent microbial extremophiles and filamentous cyanobacteria. These studies have resulted in the detection in a several carbonaceous meteorites of the mineralized remains of a wide variety of complex filamentous trichomic microorganisms. These embedded forms are consistent in size and microstructure with well-preserved morphotypes of mat- forming filamentous trichomic cyanobacteria and the degraded remains of microfibrils of cyanobacterial sheaths. We present the results of comparative imaging studies and EDAX elemental analyses of recent cyanobacteria (e.g. Calothrix, Oscillatoria, and Lyngbya) that are similar in size, morphology and microstructure to morphotypes found embedded in meteorites. EDAX elemental studies reveal that forms found in carbonaceous meteorites often have highly carbonized sheaths in close association with permineralized filaments, trichomes and microbial cells. Ratios of critical bioelements (C:O, C:N, C:P, and C:S) reveal dramatic differences between microfossils in Earth rocks and meteorites and in filaments, trichomes, hormogonia, and cells of recent cyanobacteria.

Hoover, Richard B.↗

NASA NTRS2005

Microbiological Methodology in Astrobiology

Searching for life in astromaterials to be delivered from the future missions to extraterrestrial bodies is undoubtedly related to studies of the properties and signatures of living microbial cells and microfossils on Earth. As model terrestrial analogs of Martian polar subsurface layers are often regarded the Antarctic glacier and Earth permafrost habitats where alive microbial cells preserved viability for millennia years due to entering the anabiotic state. For the future findings of viable microorganisms in samples from extraterrestrial objects, it is important to use a combined methodology that includes classical microbiological methods, plating onto nutrient media, direct epifluorescence and electron microscopy examinations, detection of the elemental composition of cells, radiolabeling techniques, PCR and FISH methods. Of great importance is to ensure authenticity of microorganisms (if any in studied samples) and to standardize the protocols used to minimize a risk of external contamination. Although the convincing evidence of extraterrestrial microbial life will may come from the discovery of living cells in astromaterials, biomorphs and microfossils must also be regarded as a target in search of life evidence bearing in mind a scenario that alive microorganisms had not be preserved and underwent mineralization. Under the laboratory conditions, processes that accompanied fossilization of cyanobacteria were reconstructed, and artificially produced cyanobacterial stromatolites resembles by their morphological properties those found in natural Earth habitats. Regarding the vital importance of distinguishing between biogenic and abiogenic signatures and between living and fossil microorganisms in analyzed samples, it is worthwhile to use some previously developed approaches based on electron microscopy examinations and analysis of elemental composition of biomorphs in situ and comparison with the analogous data obtained for laboratory microbial cultures and fossilized microorganisms. This communication will be focused on the analysis of our experience in working with ancient microorganisms and fossils and discussion of some issues that are crucial for development of the program for future finding of extraterrestrial life and its evidence.

Abyzov, S. S.↗

NASA NTRS2007

Multispecies Biofilm Development on Space Station Heat Exhanger Core Material

Investigations of microbial contamination of the cooling system aboard the International Space Station (ISS) suggested that there may be a relationship between heat exchanger (HX) materials and the degree of microbial colonization and biofilm formation. Experiments were undertaken to test the hypothesis that biofilm formation is influenced by the type and previous exposure of HX surfaces. Acidovorax delafieldii, Comamonas acidovorans, Hydrogenophaga pseudoflava, Pseudomonas stutzeri, Sphingomonas paucimobilis, and Stenotrophomonas maltophilia, originally isolated from ISS cooling system fluid, were cultured on R2A agar and suspended separately in fresh filter-sterilized ISS cooling fluid, pH 8.3. Initial numbers in each suspension ranged from 10(exp 6)-10(exp 7) CFU/ml, and a mixture contained greater than 10(exp 7) CFU/ml. Coupons of ISS HX material, previously used on orbit (HXOO) or unused (HXUU), polycarbonate (PC) and 316L polished stainless steel (SS) were autoclaved, covered with multispecies suspension in sterile tubes and incubated in the dark at ambient (22-25 C). Original HX material contained greater than 90% Ni, 4.5% Si, and 3.2% B, with a borate buffer. For approximately 10 weeks, samples of fluid were plated on R2A agar, and surface colonization assessed by SYBR green or BacLight staining and microscopy. Suspension counts for the PC and SC samples remained steady at around 10(exp 7) CFU/ml. HXUU counts declined about 1 log in 21 d then remained steady, and HXOO counts declined 2 logs in 28 d, fluctuated and stabilized about 10(exp 3) CFU/ml from 47-54 d. Predominantly yellow S. paucimobilis predominated on plates from HXOO samples up to 26 d, then white or translucent colonies of other species appeared. All colony types were seen on plates from other samples throughout the trial. Epifluorescence microscopy indicated microbial growth on all surfaces by 21 d, followed by variable colonization. After 54 d, all but the HXOO samples had well-distributed live and dead cells; the HXOO samples had few cells and most were live by BacLight. The results suggest that HX materials themselves are inhibiting microbial growth on the surfaces. The HX exposed on orbit to cooling system fluid inhibited growth of some species originally isolated from the system, whereas the unused HX material had a moderate effect compared to no inhibition with PC or SS controls. It is possible that chemistry or microbiology of the ISS system increased deposition of inhibitory compounds on the HXOO coupon surfaces; these may inhibit inoculated species to differing degrees.

Pyle, B. H.↗

NASA NTRS2008

Deep-Sea Hydrothermal-Vent Sampler

An apparatus is being developed for sampling water for signs of microbial life in an ocean hydrothermal vent at a depth of as much as 6.5 km. Heretofore, evidence of microbial life in deep-sea hydrothermal vents has been elusive and difficult to validate. Because of the extreme conditions in these environments (high pressures and temperatures often in excess of 300 C), deep-sea hydrothermal- vent samplers must be robust. Because of the presumed low density of biomass of these environments, samplers must be capable of collecting water samples of significant volume. It is also essential to prevent contamination of samples by microbes entrained from surrounding waters. Prior to the development of the present apparatus, no sampling device was capable of satisfying these requirements. The apparatus (see figure) includes an intake equipped with a temperature probe, plus several other temperature probes located away from the intake. The readings from the temperature probes are utilized in conjunction with readings from flowmeters to determine the position of the intake relative to the hydrothermal plume and, thereby, to position the intake to sample directly from the plume. Because it is necessary to collect large samples of water in order to obtain sufficient microbial biomass but it is not practical to retain all the water from the samples, four filter arrays are used to concentrate the microbial biomass (which is assumed to consist of particles larger than 0.2 m) into smaller volumes. The apparatus can collect multiple samples per dive and is designed to process a total volume of 10 L of vent fluid, of which most passes through the filters, leaving a total possibly-microbe-containing sample volume of 200 mL remaining in filters. A rigid titanium nose at the intake is used for cooling the sample water before it enters a flexible inlet hose connected to a pump. As the water passes through the titanium nose, it must be cooled to a temperature that is above a mineral-precipitation temperature of 100 C but below the upper working temperature (230 C) of switching valves and tubes in the apparatus. The sample water then passes into a manifold tube, from whence the switching valves can direct the water through either a bypass tube or any one of the filter arrays, without contamination from a previous sample. Each filter array consists of series of filters having pore sizes decreasing in the direction of flow: 90-, 60-, 15-, and 7-micron prefilters and a large-surface-area 0.2-micron collection filter. All the filter taps are located between the intake and the bypass tube so that each time the bypass tube is used, the entire manifold tube is flushed as well.

Behar, Alberto E.↗

NASA NTRS2009

Surface, Water and Air Biocharacterization - A Comprehensive Characterization of Microorganisms and Allergens in Spacecraft Environment

A Comprehensive Characterization of Microorganisms and Allergens in Spacecraft (SWAB) will use advanced molecular techniques to comprehensively evaluate microbes on board the space station, including pathogens (organisms that may cause disease). It also will track changes in the microbial community as spacecraft visit the station and new station modules are added. This study will allow an assessment of the risk of microbes to the crew and the spacecraft. Research Summary: Previous microbial analysis of spacecraft only identify microorganisms that will grow in culture, omitting greater than 90% of all microorganisms including pathogens such as Legionella (the bacterium which causes Legionnaires' disease) and Cryptosporidium (a parasite common in contaminated water) The incidence of potent allergens, such as dust mites, has never been systematically studied in spacecraft environments and microbial toxins have not been previously monitored. This study will use modern molecular techniques to identify microorganisms and allergens. Direct sampling of the ISS allows identification of the microbial communities present, and determination of whether these change or mutate over time. SWAB complements the nominal ISS environmental monitoring by providing a comparison of analyses from current media-based and advanced molecular-based technologies.

Pierson, Duane L.↗

NASA NTRS2009

Human System Risk Management - Tools of our Trade

The risk of infectious disease to select individuals has historically been difficult to predict in either spaceflight or on Earth with health care efforts relying on broad-based prevention and post-infection treatment. Over the past 10 years, quantitative microbial risk assessment evaluations have evolved to formalize the assessment process and quantify the risk. This process of hazard identification, exposure assessment, dose-response assessment, and risk characterization has been applied by the water and food safety industries to address the public health impacts associated with the occurrence of and human exposure to pathogens in water and food for the development of preventive strategies for microbial disease. NASA is currently investigating the feasibility of using these techniques to better understand the risks to astronauts and refine their microbiological requirements. To assess these techniques, NASA began an evaluation of the potable water system on the International Space Station to determine how the microbial risk from water consumption during flight differed from terrestrial sources, such as municipal water systems. The ultimate goal of this work is to optimize microbial requirements which would minimize unnecessary cargo and use of crew time, while still protecting the health of the crew. Successful demonstration of this risk assessment framework with the water system holds the potential to maximize the use of available resources during spaceflight missions and facilitate investigations into the evaluation of other routes of infection, such as through the spaceflight foods system.

Ott, C. Mark↗

NASA NTRS2007

Benchtop Detection of Proteins

A process, and a benchtop-scale apparatus for implementing the process, have been developed to detect proteins associated with specific microbes in water. The process and apparatus may also be useful for detection of proteins in other, more complex liquids. There may be numerous potential applications, including monitoring lakes and streams for contamination, testing of blood and other bodily fluids in medical laboratories, and testing for microbial contamination of liquids in restaurants and industrial food-processing facilities. A sample can be prepared and analyzed by use of this process and apparatus within minutes, whereas an equivalent analysis performed by use of other processes and equipment can often take hours to days. The process begins with the conjugation of near-infrared-fluorescent dyes to antibodies that are specific to a particular protein. Initially, the research has focused on using near-infrared dyes to detect antigens or associated proteins in solution, which has proven successful vs. microbial cells, and streamlining the technique in use for surface protein detection on microbes would theoretically render similar results. However, it is noted that additional work is needed to transition protein-based techniques to microbial cell detection. Consequently, multiple such dye/antibody pairs could be prepared to enable detection of multiple selected microbial species, using a different dye for each species. When excited by near-infrared light of a suitable wavelength, each dye fluoresces at a unique longer wavelength that differs from those of the other dyes, enabling discrimination among the various species. In initial tests, the dye/antibody pairs are mixed into a solution suspected of containing the selected proteins, causing the binding of the dye/antibody pairs to such suspect proteins that may be present. The solution is then run through a microcentrifuge that includes a membrane that acts as a filter in that it retains the dye/antibody/protein complexes while allowing any remaining unbound dye/antibody pairs to flow away. The retained dye/antibody/protein complexes are transferred to a cuvette, wherein they are irradiated with light from a miniature near-infrared laser delivered via a fiber-optic cable. The resulting fluorescence from the dye(s) is measured by use of a miniature spectrometer, the output of which is digitized, then analyzed by laptop computer. The software running in the computer identifies the protein species by the wavelengths of their spectral peaks and determines the amounts of the proteins, and thus, one day, microbes of the various species from the intensities of the peaks. The abovementioned removal of the unbound dye/antibody pairs during centrifugation prevents false positive readings. The process proves successful in detecting proteins in solution and thus can now be employed for use in microbe detection.

Scardelletti, Maximilian C.↗

NASA NTRS2012

Preparation for the Proof of Concept Flight of the Veggie Plant Growth Chamber

Veggie is a small plant growth chamber designed and built by ORBITEC that will fly to the International Space Station on SpaceX-3, scheduled for the summer of 2013. Ultimately Veggie will be used for research, education and outreach, and crew recreation. We want to demonstrate the functionality of this hardware by testing a scenario that could allow the crew to grow and consume fresh vegetables. Veggie will be collapsed and transported flat in a cargo transfer bag, and deployed on orbit, where it will be installed in an EXPRESS rack. The chamber consists of three subsystems: an LED light cap, a transparent bellows, and a root mat reservoir assembly. The bellows and flexible support arms allow the distance between plants and light cap to be adjusted for different ages and types of plants. Researchers at Kennedy Space Center and ORBITEC have been working to develop the plant growth interfaces for the proof of concept flight. We have developed a rooting pillow, consisting of a small bag containing media, time release fertilizer, seeds, and a wicking surface to conduct water from the root mat reservoir. Prototype pillows have been tested and results have influenced the design of flight pillows, which will be modified for microgravity from flight-approved materials. Several studies have been conducted selecting species and comparing media types in analog systems. Water content seems to be the most important factor differentiating media types in these small growth volumes (100 mL). Media type also influenced microbial levels on plants. Since produce sanitizing agents are not currently approved for growing food crops on orbit, plants and media types having very low microbial levels are being selected. Lettuce, mizuna, and other salad greens typically have microbial counts less than 10(exp 4) colony forming units and thus are good candidates for spaceflight. As we approach flight verification testing, we will finalize species, media selection, harvesting, and microbial sampling procedures. Next steps include testing of Veggie flight and ground hardware and associated equipment. This research was funded by NASA.

Massa, Gioia↗

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 NTRS2014

Towards a General Equation for the Survival of Microbes Transferred between Solar System Bodies

It should be possible to construct a general equation describing the survival of microbes transferred between Solar System bodies. Such an equation will be useful for constraining the likelihood of transfer of viable organisms between bodies throughout the lifetime of the Solar System, and for refining Planetary Protection constraints placed on future missions. We will discuss the construction of such an equation, present a plan for definition of pertinent factors, and will describe what research will be necessary to quantify those factors. Description: We will examine the case of microbes transferred between Solar System bodies as residents in meteorite material ejected from one body (the "intial body") and deposited on another (the "target body"). Any microbes transferred in this fashion will experience four distinct phases between their initial state on the initial body, up to the point where they colonize the target body. Each of these phases features phenomena capable of reducing or exterminating the initial microbial population. They are: 1) Ejection: Material is ejected from the initial body, imparting shock followed by rapid desiccation and cooling. 2) Transport: Material travels through interplanetary space to the target body, exposing a hypothetical microbial population to extended desiccation, irradiation, and temperature extremes. 3) Infall: Material is deposited on the target body, diminishing the microbial population through shock, mass loss, and heating. 4) Adaptation: Any microbes which survive the previous three phases must then adapt to new chemophysical conditions of the target body. Differences in habitability between the initial and target bodies dominate this phase. A suitable general-form equation can be assembled from the above factors by defining the initial number of microbes in an ejected mass and applying multiplicitive factors based on the physical phenomena inherent to each phase. It should be possible to present the resulting equation in terms of initial ejection mass, ejection shock magnitude, transfer time, initial microbial load and/or other terms and generate graphs defining the number of surviving microbes. The general form of the equation is: x(sub f) = x(sub i) f(sub1) f(sub 2) f(sub 3) f(sub 4) Where x(sub f) is the final number of microbes to survive transfer, x(sub i) is the initial population prior to ejection, and f(sub 1-4) are mortality factors for the four phases described above. Among other considerations, f(sub 1) will vary with respect to impact shock magnitude and f(sub 2) will be time-dependent. Considerable research has been performed to date to quantify the survival rates of various microbes in response to portions of these four phases, both as vegetative cells and/or spores. Results indicate that many species tend to respond differently to the pertinent mortality factors, especially in the case of extremophiles. Therefore, a complete equation will include species-specific responses to the mortality factors.

Fries, M.↗

NASA NTRS2015

Dormancy and Recovery Testing for Biological Wastewater Processors

Bioreactors, such as aerated membrane type bioreactors have been proposed and studied for a number of years as an alternate approach for treating wastewater streams for space exploration. Several challenges remain before these types of bioreactors can be used in space settings, including transporting the bioreactors with their microbial communities to space, whether that be the International Space Station or beyond, or procedures for safing the systems and placing them into dormant state for later start-up. Little information is available on such operations as it is not common practice for terrestrial systems. This study explored several dormancy processes for established bioreactors to determine optimal storage and recovery conditions. Procedures focused on complete isolation of the microbial communities from an operational standpoint and observing the effects of: 1) storage temperature, and 2) storage with or without the reactor bulk fluid. The first consideration was tested from a microbial integrity and power consumption standpoint; both room temperature (25 C) and cold (4 C) storage conditions were studied. The second consideration was explored; again, for microbial integrity as well as plausible real-world scenarios of how terrestrially established bioreactors would be transported to microgravity and stored for periods of time between operations. Biofilms were stored without the reactor bulk fluid to simulate transport of established biofilms into microgravity, while biofilms stored with the reactor bulk fluid simulated the most simplistic storage condition to implement operations for extended periods of nonuse. Dormancy condition did not have an influence on recovery in initial studies with immature biofilms (48 days old), however, a lengthy recovery time was required (20+ days). Bioreactors with fully established biofilms (13 months) were able to recover from a 7-month dormancy period to steady state operation within 4 days (approximately 1 residence cycle). Results indicate a need for future testing on biofilm age and health and further exploration of dormancy length.

Dormancy↗

NASA NTRS2016

Risk of Adverse Health Effects Due to Host-Microorganism Interactions

Numerous spaceflight experiments have been conducted to investigate alterations in microbial responses resulting from culture during spaceflight and spaceflight-analogs. However, recent studies investigating spaceflight-associated alterations in microbial virulence have initiated the review and production of evidence to better understand the impact these alterations would have on the incidence of infectious disease during a spaceflight exploration mission. The preponderance of evidence indicates that alterations in microbial gene expression and phenotype (including virulence) are occurring; however, the clinical implications of such changes are still unclear. Greater knowledge is required including a better understanding of the mechanism behind unique spaceflight-associated microbial responses to determine how this environmental stimulus impacts various microorganisms, their diversity and concentration in the spacecraft and crew microbiome, their impact on the vehicle and crew, and their resistance to current mitigation and antibiotic regimens. This knowledge will enable us to determine requirements, guidelines, and processes for design and monitoring of the next generation vehicles.

Ott, C. Mark↗

NASA NTRS2019

Constraints on the Metabolic Activity of Microorganisms in Atacama Surface Soils Inferred from Refractory Biomarkers: Implications for Martian Habitability and Biomarker Detection

Dryness is one of the main environmental challenges to microbial survival. Understanding the threshold of microbial tolerance to extreme dryness is relevant to better constrain the environmental limits of life on Earth and critically evaluate long-term habitability models of Mars. Biomolecular proxies for microbial adaptation and growth were measured in Mars-like hyper-arid surface soils in the Atacama Desert that experience only a few millimeters of precipitation per decade, and in biologically active soils a few hundred kilometers away that experience two- to fivefold more precipitation. Diversity and abundance of lipids and other biomolecules decreased with increasing dryness. Cyclopropane fatty acids (CFAs), which are indicative of adaptive response to environmental stress and growth in bacteria, were only detected in the wetter surface soils. The ratio of trans to cis isomers of an unsaturated fatty acid, another bacterial stress indicator, decreased with increasingly dry conditions. Aspartic acid racemization ratios increased from 0.01 in the wetter soils to 0.1 in the driest soils, which is indicative of racemization rates comparable to de novo biosynthesis over long timescales (10,000 years). The content and integrity of stress proteins profiled by immunoassays were additional indicators that biomass in the driest soils is sin situ microbial growth in the driest surface soils of the Atacama, and any metabolic activity is likely to be basal for cellular repair and maintenance only. Our data add to a growing body of evidence that the driest Atacama surface soils represent a threshold for long-term habitability (i.e., growth and reproduction). These results place constraints on the potential for extant life on the surface of Mars, which is 100–1000 times drier than the driest regions in the Atacama.

Wilhelm, Mary Beth↗