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NASA NTRSDate not supplied

The Need for Earth-Based Experiments to Inform Microbial Evolution on Planetary Surfaces

Introduction: Historically, the focus of planetary protection at NASA has been on unmanned, robotic missions. Such missions have paved the way for understanding how to implement planetary protection in a feasible and cost-sensitive way. However, with the introduction of crewed missions to Mars in the not-sodistant future, there is a need to better define and understand how to implement planetary protection under new circumstances, as well as understand the risk of contaminating Mars. One unavoidable fact is that microbes will go where humans go. Therefore, it is critical to understand how these microbes may (and will) impact our ability to conduct meaningful, reliable astrobiological science. Microorganisms have spent millions of years evolving to survive in extreme environments here on Earth. Already there are indications that microbes aboard the International Space Station evolve and adapt to life in low earth orbit. The microbes that are eventually taken to Mars with humans will also adapt, potentially causing harmful effects to crew and/or the planetary or astrobiological science conducted. Therefore, it is of critical interest that we evaluate and characterize the potential risks of microbial evolution on Mars. It is expected that microbes carried by humans will begin to evolve to new environments even before landing on Mars, during the several month cruise phase. Once landed, microbes will encounter different stressors within the crew habitats on Mars. During extravehicular activities, venting, or other release events, microbes will find their way out onto the Martian surface. The induced environments around crewed systems will create potentially-favorable conditions for microbes to continue evolving on Mars. Eventually, microbes may find their way beyond the close confines of the crewed area and continue evolving so as to fill new or distant niches on the Martian surface. It is challenging to replicate Martian environments here on Earth, making it nearly impossible to predict the evolutionary changes that microbes would undergo on Mars. But this work is critical. Serial passaging experiments performed by Richard Lenski on E. coli show the dramatic changes microbes can undergo even within a laboratory setting. Furthermore, experiments performed by Michael Baym also demonstrate the power of single mutations in microbial development of antibiotic resistance [3]. Long duration experiments should be performed on a suite of microbes exposed to environments likely to be experienced on the Martian surface. While simulating space environments can be challenging, facilities exist that can achieve individual and combinatorial environmental conditions to simulate space and planetary conditions. Such chambers should be employed for microbial studies. Currently, at the Marshall Space Flight Center, we have used various stressors like drying, vacuum, proton radiation, and ultraviolet light both separately and in combination, to evaluate the survival of cleanroom microbes. Shockingly, several non-spore forming isolates have demonstrated the ability to survive many extreme conditions (manuscript in preparation). These short duration exposures must be augmented with larger and more gradual studies to replicate what microbes might experience in the transition from cruise, to surface habitats, to induced surface environments, and finally true Martian environments. While no Earth-based experiment can perfectly replicate the Martian environment, nor could we test every possible microbe in simulation experimental regimes, efforts should be made to examine the evolutionary potential of the “usual suspects” seen on the ISS or in other crewed environments to begin to fill this important knowledge gap.

Chelsi D. Cassilly↗

NASA NTRS2015

Life Beyond the Planet of Origin and Implications for the Search for Life on Mars

Outer space is vast, cold, devoid of matter, radiation filled with essentially no gravity. These factors present an environmental challenge for any form of life. Earth's biosphere has evolved for more than 3 billion years shielded from the hostile environment of outer space by the protective blanket of the atmosphere and magnetosphere. Space is a nutritional wasteland with no liquid water and readily available organic carbon. Moving beyond a life's planet of origin requires a means for transport, the ability to withstand transport, and the ability to colonize, thrive and ultimately evolve in the new environment. Can life survive beyond its home planet? The key to answering this question is to identify organisms that first have the ability to withstand space radiation, space vacuum desiccation and time in transit, and second the ability to grow in an alien environment. Within the last 60 years space technology allowed us to transport life beyond Earth's protective shield so we may study, in situ, their responses to selected conditions of space. To date a variety of microbes ranging from viruses, to Bacteria, to Archaea, to Eukarya have been tested in the space environment. Most died instantly, but not all. These studies revealed that ultraviolet radiation is the near-term lethal agent, while hard radiation is the long-term lethal agent when the organism is shielded from ultraviolet radiation. In fact, bacterial spores, halophilic cyanobacteria and Archaea as well as some lichens survive very well if protected from ultraviolet radiation [1]. Some microbes, then, may be able to survive the trip in outer space to Mars on a spacecraft or in a meteorite. Once on Mars can a terrestrial microbe survive? Although the conditions on Mars are not as harsh as those in space, they are not hospitable for a terrestrial microbe. Studies, however, have shown that certain microbes that can survive in space for several years may also be able to survive on Mars if protected from ultraviolet radiation [1]. Laboratory simulation experiments using a mock-up of the Phoenix lander have shown that microbes transported to the surface of Mars on a spacecraft come off the spacecraft and mix into the Martian regolith [2]. Additionally, studies simulating Martian dust storms demonstrate that microbes can survive in the Martian wind blown dust and be scattered across the Martian surface away from the spacecraft. Would these microbes that may survive on Mars metabolize and propagate? Growth requires liquid water, a carbon source and an energy source. Survival on Mars also requires protection from ultraviolet radiation. In the cold, dry environment of Mars the probability of microbial metabolism and growth at or just beneath the surface is extremely low. Although the probability is low, Mars may be contaminated with potentially live terrestrial organisms. In light of that statistic we must be extremely diligent and cautious in our search for Martian life. If we are not cautious we may find life on Mars and it may be a contaminant from Earth.

Survival in Space↗

NASA NTRS2000

Microscopic physical biomarkers in carbonate hot springs: implications in the search for life on Mars

Physical evidence of life (physical biomarkers) from the deposits of carbonate hot springs were documented at the scale of microorganisms--submillimeter to submicrometer. The four moderate-temperature (57 to 72 degrees C), neutral pH springs reported on in this study, support diverse communities of bacteria adapted to specific physical and chemical conditions. Some of the microbes coexist with travertine deposits in endolithic communities. In other cases, the microbes are rapidly coated and destroyed by precipitates but leave distinctive mineral fabrics. Some microbes adapted to carbonate hot springs produce an extracellular polymeric substance which forms a three-dimensional matrix with living cells and cell remains, known as a biofilm. Silicon and iron oxides often coat the biofilm, leading to long-term preservation. Submicrometer mineralized spheres composed of calcium fluoride or silica are common in carbonate hot spring deposits. Sphere formation is biologically mediated, but the spheres themselves are apparently not fossils or microbes. Additionally, some microbes selectively weather mineral surfaces in distinctive patterns. Hot spring deposits have been cited as prime locations for exobiological exploration of Mars. The presence of preserved microscopic physical biomarkers at all four sites supports a strategy of searching for evidence of life in hot spring deposits on Mars.

NASA Center JSC↗

NASA NTRS2011

PMA-PhyloChip DNA Microarray to Elucidate Viable Microbial Community Structure

Since the Viking missions in the mid-1970s, traditional culture-based methods have been used for microbial enumeration by various NASA programs. Viable microbes are of particular concern for spacecraft cleanliness, for forward contamination of extraterrestrial bodies (proliferation of microbes), and for crew health/safety (viable pathogenic microbes). However, a "true" estimation of viable microbial population and differentiation from their dead cells using the most sensitive molecular methods is a challenge, because of the stability of DNA from dead cells. The goal of this research is to evaluate a rapid and sensitive microbial detection concept that will selectively estimate viable microbes. Nucleic acid amplification approaches such as the polymerase chain reaction (PCR) have shown promise for reducing time to detection for a wide range of applications. The proposed method is based on the use of a fluorescent DNA intercalating agent, propidium monoazide (PMA), which can only penetrate the membrane of dead cells. The PMA-quenched reaction mixtures can be screened, where only the DNA from live cells will be available for subsequent PCR reaction and microarray detection, and be identified as part of the viable microbial community. An additional advantage of the proposed rapid method is that it will detect viable microbes and differentiate from dead cells in only a few hours, as opposed to less comprehensive culture-based assays, which take days to complete. This novel combination approach is called the PMA-Microarray method. DNA intercalating agents such as PMA have previously been used to selectively distinguish between viable and dead bacterial cells. Once in the cell, the dye intercalates with the DNA and, upon photolysis under visible light, produces stable DNA adducts. DNA cross-linked in this way is unavailable for PCR. Environmental samples suspected of containing a mixture of live and dead microbial cells/spores will be treated with PMA, and then incubated in the dark. Thereafter, the sample is exposed to visible light for five minutes, so that the DNA from dead cells will be cross-linked. Following this PMA treatment step, the sample is concentrated by centrifugation and washed (to remove excessive PMA) before DNA is extracted. The 16S rRNA gene fragments will be amplified by PCR to screen the total microbial community using PhyloChip DNA microarray analysis. This approach will detect only the viable microbial community since the PMA intercalated DNA from dead cells would be unavailable for PCR amplification. The total detection time including PCR reaction for low biomass samples will be a few hours. Numerous markets may use this technology. The food industry uses spore detection to validate new alternative food processing technologies, sterility, and quality. Pharmaceutical and medical equipment companies also detect spores as a marker for sterility. This system can be used for validating sterilization processes, water treatment systems, and in various public health and homeland security applications.

Venkateswaran, Kasthuri J.↗

NASA NTRS2012

From Kennedy, to Beyond: Growing Plants in Space

Astronauts cannot have their cake and eat it too, but what about growing a salad and eating it? As NASA continues to push the envelope on Space exploration and inhabitance the need for a fresh food source becomes more vital. The Life Support team at NASA is using a system developed by ORBITEC the VEGGIE, in which astronauts aboard the ISS, and potentially the Moon and Mars, will be capable of growing food. The introduction of plants not only gives astronauts a means of independently supplying food, but also recreation, oxygen replenishment and psychological benefits. The plants were grown in "pillows", the system used for growing plants within the VEGGIE. This test included 4 types of media mixtures that are composed of a clay based media called Arcilite and Fafard #2, which is a peat moss-based media ( <1 mm Arcilite, 1-2 mm of Arcilite, 1:1 <1 mm & 1-2 mm mixture and 1:1 Arcilite & Fafard mixture). Currently, 3 lettuce cultivars are being grown in 4 mixtures of media. Tests were being conducted to see which form of media has the ratio of best growth and least amount of microbes that are harmful. That is essential because a person's body becomes more susceptible to illness when they leave Earth. As a result, test must be conducted on the "pillow" system to assess the levels of microbial activity. The cultivars were tested at different stages during their growing process for microbes. Datum show that the mix of Fafard and Arcilite had the best growth, but also the most microbes. This was due to the fact that Fafard is an organic substance so it contains material necessary for microbes to live. Data suggest that the <1 mm Arcilite has an acceptable amount of growth and a lower level of microbes, because it is non-organic.

Flemming, Cedric, II↗

NASA NTRSDate not supplied

Planetary Protection at Marshall Space Flight Center

Introduction: NASA Marshall Space Flight Center (MSFC) is historically known for its role in propulsion. While this is still the mainstay of MSFC’s expertise, many unique capabilities exist at MSFC which pertain to Planetary Protection (PP), including 1) identifying PP threats, and 2) developing novel methods to neutralize those threats. Furthermore, because these capabilities exist among diverse groups at MSFC, this work promotes collaboration both within and outside MSFC to expand and develop PP studies related to a full spectrum of NASA research, design, manufacture, and test interests. This abstract describes the PP research ongoing at MSFC and describes how it contributes to NASA’s overall PP objectives. Microbial Identification in Cleanrooms: One of the greatest threats to successful implementation of PP requirements is recontamination post bioburden reduction. One method to prevent recontamination is to keep the spacecraft in clean environments (i.e. cleanrooms) as much as possible during assembly and integration. However, cleanrooms are not without their own sources of contamination, which is why NASA is interested in monitoring the cleanliness of cleanrooms and characterizing the microbial species present. Such information allows a greater understanding of the resistance of these microbes to cleaning methods, as well as the risk of their contaminating the targeted planetary body of a given mission. MSFC has multiple cleanrooms of various ISO cleanliness levels onsite. We sampled the air and surfaces of three of these rooms, isolated microbes, and then sequenced the 16S rRNA gene or ITS region of the 18S rRNA gene for bacterial and fungal isolates, respectively. This has resulted in a microbial library which currently includes nearly 100 isolates. Microbial Enumeration of Spacecraft Materials: Currently, there are only a couple bioburden reduction methods approved by NASA, and often the harshness of these methods presents additional concerns or risks related to material properties. The goal of this research is to assess the microbial content of solid rocket motor (SRM) materials potentially used for lander missions. This work aims to more accurately define the risk of planetary contamination by providing empirical data associated with commonly used SRM raw materials. In this study, we pulverized nonmetallic SRM materials using a cryogenic grinder, then analyzed the resulting substrate for microbial colony forming units (CFU). We found that many SRM nonmetallic materials do not harbor detectable bioburden, though a range existed depending on the material. The results from this work provide quantitative data to potentially reduce concerns of contamination, while also providing a foundation for follow up studies into additional sterilization methods and molecular identification of contaminating microbes. Space Environmental Effects on Microbial Survival: One potential area of microbial reduction is the space environment. Understanding the survivability of hardy microbes in space-like conditions is a crucial first step in answering how space may reduce bioburden and if it can be relied upon for adherence to PP requirements. This work studied the effects of ultraviolet (UV) and ionizing radiation on survival of Bacillus atrophaeus spores. Microbes were dried on relevant polymeric materials then exposed to space environmental stressors. Coupons were submerged in water, diluted, and plated to determine survival compared with controls. We found that both UV and ionizing radiation were capable of reducing viability by nearly 99%, but there were still survivors, some with changed morphology indicating resistance mechanisms within certain cells. Manufacturing credit: Finally, given the above-mentioned limitations of the NASA-approved bioburden reduction methods, there is interest in understanding if manufacturing processes may provide enough bioburden reduction without additional PP-specific bakeouts. For instance, some material additives may be antimicrobial. Given this, we investigated the effects of several commonly used rubber additives on the growth of B. atrophaeus spores. We found that some of the materials inhibited growth of the spores, possibly supporting the use of these additives on missions with PP constraints. Future work into manufacturing credit for bioburden reduction includes inoculation of green insulation with B. atrophaeus spores, followed by a typical cure. Thermal profiles will be verified for appropriate temperature and durations to meet PP requirements, and cured samples will be analyzed using a cryogenic grinder to determine survivability of spores.

Chelsi D. Cassilly↗

NASA NTRS2006

A Comprehensive Assessment of Biologicals Contained Within Commercial Airliner Cabin Air

Both culture-based and culture-independent, biomarker-targeted microbial enumeration and identification technologies were employed to estimate total microbial and viral burden and diversity within the cabin air of commercial airliners. Samples from each of twenty flights spanning three commercial carriers were collected via air-impingement. When the total viable microbial population was estimated by assaying relative concentrations of the universal energy carrier ATP, values ranged from below detection limits (BDL) to 4.1 x 106 cells/cubic m of air. The total viable microbial population was extremely low in both of Airline A (approximately 10% samples) and C (approximately 18% samples) compared to the samples collected aboard flights on Airline A and B (approximately 70% samples). When samples were collected as a function of time over the course of flights, a gradual accumulation of microbes was observed from the time of passenger boarding through mid-flight, followed by a sharp decline in microbial abundance and viability from the initiation of descent through landing. It is concluded in this study that only 10% of the viable microbes of the cabin air were cultivable and suggested a need to employ state-of-the art molecular assay that measures both cultivable and viable-but-non-cultivable microbes. Among the cultivable bacteria, colonies of Acinetobacter sp. were by far the most profuse in Phase I, and Gram-positive bacteria of the genera Staphylococcus and Bacillus were the most abundant during Phase II. The isolation of the human pathogens Acinetobacter johnsonii, A. calcoaceticus, Janibacter melonis, Microbacterium trichotecenolyticum, Massilia timonae, Staphylococcus saprophyticus, Corynebacterium lipophiloflavum is concerning, as these bacteria can cause meningitis, septicemia, and a handful of sometimes fatal diseases and infections. Molecular microbial community analyses exhibited presence of the alpha-, beta-, gamma-, and delta- proteobacteria, as well as Gram-positive bacteria, Fusobacteria, Cyanobacteria, Deinococci, Bacterioidetes, Spirochetes, and Planctomyces in varying abundance. Neisseria meningitidis rDNA sequences were retrieved in great abundance from Airline A followed by Streptococcus oralis/mitis sequences. Pseudomonas synxantha sequences dominated Airline B clone libraries, followed by those of N. meningitidis and S. oralis/mitis. In Phase II, Airline C, sequences representative of more than 113 species, enveloping 12 classes of bacteria, were retrieved. Proteobacterial sequences were retrieved in greatest frequency (58% of all clone sequences), followed in short order by those stemming from Gram-positives bacteria (31% of all clone sequences). As for overall phylogenetic breadth, Gram-positive and alpha-proteobacteria seem to have a higher affinity for international flights, whereas beta-and gamma-proteobacteria are far more common about domestic cabin air parcels in Airline C samples. Ultimately, the majority of microbial species circulating throughout the cabin airs of commercial airliners are commensal, infrequently pathogenic normal flora of the human nasopharynx and respiratory system. Many of these microbes likely originate from the oral and nasal cavities, and lungs of passengers and flight crew and are disseminated unknowingly via routine conversation, coughing, sneezing, and stochastic passing of fomites. The data documented in this study will be useful to generate a baseline microbial population database and can be utilized to develop biosensor instrumentation for monitoring microbial quality of cabin or urban air.

microbial diversity↗

NASA NTRS2017

Urban Biomining Meets Printable Electronics: End-To-End at Destination Biological Recycling and Reprinting

Space missions rely utterly on metallic components, from the spacecraft to electronics. Yet, metals add mass, and electronics have the additional problem of a limited lifespan. Thus, current mission architectures must compensate for replacement. In space, spent electronics are discarded; on earth, there is some recycling but current processes are toxic and environmentally hazardous. Imagine instead an end-to-end recycling of spent electronics at low mass, low cost, room temperature, and in a non-toxic manner. Here, we propose a solution that will not only enhance mission success by decreasing upmass and providing a fresh supply of electronics, but in addition has immediate applications to a serious environmental issue on the Earth. Spent electronics will be used as feedstock to make fresh electronic components, a process we will accomplish with so-called 'urban biomining' using synthetically enhanced microbes to bind metals with elemental specificity. To create new electronics, the microbes will be used as 'bioink' to print a new IC chip, using plasma jet electronics printing. The plasma jet electronics printing technology will have the potential to use martian atmospheric gas to print and to tailor the electronic and chemical properties of the materials. Our preliminary results have suggested that this process also serves as a purification step to enhance the proportion of metals in the 'bioink'. The presence of electric field and plasma can ensure printing in microgravity environment while also providing material morphology and electronic structure tunabiity and thus optimization. Here we propose to increase the TRL level of the concept by engineering microbes to dissolve the siliceous matrix in the IC, extract copper from a mixture of metals, and use the microbes as feedstock to print interconnects using mars gas simulant. To assess the ability of this concept to influence mission architecture, we will do an analysis of the infrastructure required to execute this concept on Mars, and additional opportunities it could offer mission design from the biological and printing technologies. In addition, we will do an analysis of the impact of this technology for terrestrial applications addressing in particular environmental concerns and availability of metals.

Space mission↗

NASA NTRS1996

Definition of exobiology experiments for future Mars missions

During the past year we have concentrated on two objectives. The first objective is ongoing and is to define the experimental parameters that are necessary to conduct autonomously a mineralogical analysis of the Martian surface in situ using differential thermal analysis coupled with gas chromatography (DTA/GC). The rationale in support of this objective is that proper interpretation of the mineralogical data from the DTA/GC can be used to better describe the present and past environments of Mars, leading to a better assessment of the probability of life evolving on Mars. To meet these objectives we have analyzed a number of samples collected from nature using the DTA/GC. One of the more significant findings was that in samples of desert varnish we detected magnetite and maghemite that may serve as potential biomarkers applicable to DTA/GC analyses of Martian surface material during landed missions. The second objective follows from the first and is to better understand microbe-environment interactions by determining the response of microbes to changes in their environment, including extreme desiccation and solar UV-radiation. The rationale behind this is to develop hypotheses regarding what may have happened to life that may have arose on Mars, and microbial life that may get to the surface of Mars via spacecraft, or meteors from Earth. To accomplish this objective we have exposed microbes, collected from NaCl and gypsum-halite crystals, to the space environment aboard the ESA-German Biopan facility for 15 days. The most significant finding was that these microbes survived the exposure better than others.

Mancinelli, Rocco L.↗

NASA NTRS2001

Molecular microbial diversity of a spacecraft assembly facility

In ongoing investigations to map and archive the microbial footprints in various components of the spacecraft and its accessories, we have examined the microbial populations of the Jet Propulsion Laboratory's Spacecraft Assembly Facility (JPL-SAF). Witness plates made up of spacecraft materials, some painted with spacecraft qualified paints, were exposed for approximately 7 to 9 months at JPL-SAF and examined the particulate materials collected for the incidence of total cultivable aerobic heterotrophs and heat-tolerant (80 degrees C for 15-min.) spore-formers. The results showed that the witness plates coated with spacecraft qualified paints attracted more dust particles than the non-coated stainless steel witness plates. Among the four paints tested, witness plates coated with NS43G accumulated the highest number of particles, and hence attracted more cultivable microbes. The conventional microbiological examination revealed that the JPL-SAF harbors mainly Gram-positive microbes and mostly spore-forming Bacillus species. Most of the isolated microbes were heat resistant to 80 degrees C and proliferate at 60 degrees C. The phylogenetic relationships among 23 cultivable heat-tolerant microbes were examined using a battery of morphological, physiological, molecular and chemotaxonomic characterizations. By 16S rDNA sequence analysis, the isolates fell into seven clades: Bacillus licheniformis, B. pumilus, B. cereus, B. circulans, Staphylococcus capitis, Planococcus sp. and Micrococcus lylae. In contrast to the cultivable approach, direct DNA isolation, cloning and 16S rDNA sequencing analysis revealed equal representation of both Gram-positive and Gram-negative microorganisms.

Industry↗

NASA NTRS2004

Molecular Microbial Analyses of the Mars Exploration Rovers Assembly Facility

During space exploration, the control of terrestrial microbes associated with robotic space vehicles intended to land on extraterrestrial solar system bodies is necessary to prevent forward contamination and maintain scientific integrity during the search for life. Microorganisms associated with the spacecraft assembly environment can be a source of contamination for the spacecraft. In this study, we have monitored the microbial burden of air samples of the Mars Exploration Rovers' assembly facility at the Kennedy Space Center utilizing complementary diagnostic tools. To estimate the microbial burden and identify potential contaminants in the assembly facility, several microbiological techniques were used including culturing, cloning and sequencing of 16S rRNA genes, DNA microarray analysis, and ATP assays to assess viable microorganisms. Culturing severely underestimated types and amounts of contamination since many of the microbes implicated by molecular analyses were not cultivable. In addition to the cultivation of Agrobacterium, Burkholderia and Bacillus species, the cloning approach retrieved 16s rDNA sequences of oligotrophs, symbionts, and y-proteobacteria members. DNA microarray analysis based on rational probe design and dissociation curves complemented existing molecular techniques and produced a highly parallel, high resolution analysis of contaminating microbial populations. For instance, strong hybridization signals to probes targeting the Bacillus species indicated that members of this species were present in the assembly area samples; however, differences in dissociation curves between perfect-match and air sample sequences showed that these samples harbored nucleotide polymorphisms. Vegetative cells of several isolates were resistant when subjected to treatments of UVC (254 nm) and vapor H202 (4 mg/L). This study further validates the significance of non-cultivable microbes in association with spacecraft assembly facilities, as our analyses have identified several non-cultivable microbes likely to contaminate the surfaces of spacecraft hardware.

microbial diversity MER 16S rDNA DNA microarray sp↗

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 NTRSDate not supplied

ISS External Microorganisms: A Payload to Close Planetary Protection Knowledge Gaps for Crewed Missions

Before NASA or COSPAR is able to set planetary protection requirements for crewed missions to locations like Mars there are a number of critical knowledge gaps that must be addressed (1). One of the most important knowledge gaps is an understanding of microbial leakage from crewed habitats and space suits. Current ECLSS (Environmental Control and Life Support System) and PLSS (Portable Life Support System) requirements do not include any provisions to control microbes that may escape along with vented or leaked gasses. The current generation of NASA space suits can leak at rates as high as 100 cm2 /min. during nominal operation (2). ISS (International Space Station) intentionally vents atmospheric gases like CO2 to maintain habitable conditions for the crew. Furthermore, every time an airlock is used for EVA (extravehicular activity)there is an accompanying release of internal atmosphere. Since it is not possible to sterilize a crewed mission, it is important that we understand what if any microbes are entrained in these vented and leaked products. It is also important to understand if these microbes can survive on exterior surfaces. Recent sampling of the Russian segments of ISS suggest that bacteria and fungi from inside ISS may be capable of surviving on external surfaces(3). NASA is developing an aseptic sampling tool for use during EVA and plans to collect samples from vents on ISS to build on these results. The results of this work will be used to develop planetary protection requirements for vented and leaked gasses from crewed volumes. NASA has developed and tested a tool kit for collecting microbiological samples during EVA(4). This tool kit contains eight commercially available, 23 mm. diameter, foam swabs that can be used to aseptically collect samples while at vacuum. The swabs are individually housed in aluminum canisters that are equipped with 0.2 μm Teflon filters. These filters allow the canisters to equilibrate to pressure changes while preventing microbiological contamination. The canisters will be cleaned and sterilized before flight. Results from ground-based testing indicate that this tool kit is capable of aseptically collecting microbes while at vacuum without becoming contaminated during pressure changes(5). Based on the results of this ground testing we have modified the tool kit to meet NASA safety requirements and improve the ergonomics. We added additional mounting points to the tool kit to give astronauts more options for securing it during use. We also changed the opening mechanism to improve the precision with which swabs can be extracted from the tool kit. We plan to use this kit on an upcoming EVA to collect samples from non-propulsive vents and areas near the U.S. airlock on ISS. These samples will be frozen at -80 ̊C and stored on station until they can be returned to Earth. We will analyze these returned samples using next generation DNA sequencing to determine the community composition and function of external ISS environments. The results of this study will close planetary protection knowledge gaps for crewed missions and will help NASA determine appropriate planetary protection requirements for life support systems. The tool kit will also be useful for collecting aseptic samples on upcoming crewed or robotic missions and could easily be modified to collect samples with organic contamination control requirements as well.

A B Regberg↗

NASA NTRSDate not supplied

ISS External Microorganisms: A Payload to Close Planetary Protection Knowledge Gaps for Crewed Missions

A B Regberg↗

NASA NTRSDate not supplied

Retrospectives: Intersection of Spaceflight Stressors and Microbial Risk to Crew and Craft

OVERVIEW The spaceflight environment has several unique stressors that affect the health of both the crew and the spacecraft. An area of continued, albeit incomplete, study is the interaction of these stressors on microbial populations inherent to both astronauts and spacecraft surfaces and systems. A primary concern is the potential for the spaceflight environment to perturb the phenotype of these populations towards negative outcomes for crew and craft. In order to effectively mitigate these potential risks, they must first be characterized. We performed a retrospective literature analysis to assess the current state of knowledge regarding the affects of ionizing radiation and elevated CO2 on relevant microbial populations. The results of these retrospectives will guide next steps in the decisions of what (if any) further studies should be pursued and to guide decisions of the need for countermeasures. STRESSORS Ionizing radiation. The health risk involved with increased exposure to cosmic radiation has been studied in crew for 35+ years, with human health and cancer risk being the main focus. However, space radiation could also affect both the resident microorganisms aboard the ISS and the normal, healthy astronaut microbiomes that are of direct concern for crew health. A retrospective review of over 250 publications was accomplished looking at the impact of cumulative ionizing radiation doses lower than 3 Gy (chronic or acute) on microbial populations. Elevated CO2. The health risk involved with elevated atmospheric CO2 in spacecraft, primarily focusing on human toxicological risks, is understudied. The current Spaceflight Maximum Allowance Concentration for 24-hour average CO2 is 0.4% (3 mm Hg), which is significantly higher than terrestrial levels (0.04%). Whether these elevated ambient CO2 levels aboard spacecraft influence the diversity and phenotypic responses of the resident microbial communities from both the spacecraft environment (air, surface, water) and crew members (gut, nasal,skin microbiomes) is not known. A retrospective review was accomplished looking at the impact of chronic CO2 exposure up to 0.7% (5 mm Hg) for up to 6 months and acute exposure up to 2.6% (20 mm Hg) for up to 24 hours. CONCLUSIONS: MICROBIOME OF THE BUILT ENVIRONMENT The microbiome of the built spacecraft environment has been sampled consistently over the course of human spaceflight and significant advancements have been made in identifying microbial populations on the ISS. The dominant source of microbes on spacecraft surfaces are human-derived. Once in the spacecraft built environment,the extreme environment selects for features that enhance survival. While efforts to understand potential antibiotic resistance and pathogenicity of ISS isolates is robust, there is little to no understanding of which spaceflight environmental stressors, to include ionizing radiation or elevated CO2, drive the evolutionary trajectory of spacecraft-associated microbial populations. CONCLUSIONS: MICROBE-HOST INTERACTIONS The host-microbiome field has emerged as an important factor in human health on Earth as well in spaceflight. The field is struggling with the complexity of the system under investigation as there is substantial taxonomic and functional heterogeneity in these communities, making it difficult to establish clear stimulus-response dynamics.Taxonomic characterization is the norm; however, the functional role of each community member is key to linking environmental perturbations to potential dysbiosis. For both ionizing radiation and elevated CO2, the likely target of the perturbation is the host tissue, not the microbes themselves.. Any resulting changes to the microbial community composition and/or function is likely a result of adapting to those changes in the host physiology. RECOMMENDATIONS Emphasize functional characterization as opposed to taxonomic characterization of microbial communities.Increase the number of investigations using chronic, spaceflight-relevant doses of ionizing radiation. MoBE studies should move away from observational studies towards predictive modeling of community dynamics. Continue to develop scale-down models, such as tissues-on-a-chip & defined microbial communities. Focus on the crew response to elevated CO2 over MoBE considerations. Assess how direct contact with the hypercapnic environment affects skin microbiome dynamics.

Countermeasures↗

NASA NTRS1989

Telemycology - A novel approach to monitoring environmental microbial load in Space Station Freedom

The currently available methods for monitoring environmental microbial load call for the cultivation of microbes on laboratory media, a time- and material-consuming task that is potentially hazardous. Telemycology proposed in this communication is designed to eliminate the need for growing microbes, especially fungi, on board the spacecraft and to shift the bulk of the work-load to the ground-based Microbiology Laboratory. The system is based on the principle of trapping microbial propagules on a membrane filter, treating it with a microbe-enhancing reagent, and examining under a microscope down-linked to the central laboratory equipped with a synchronized televideo, telerobotics, and image banking system.

Mishra, S. K.↗

NASA NTRS1998

Biomarkers in Carbonate Thermal Springs: Implications for Mars

Evidence of possible relict biogenic activity has been reported in carbonate inclusions within martian meteorite ALH 84001. The initial evidence included ovoid and elongated forms 50 - 500 nanometers in length, morphologically similar to but significantly smaller than many terrestrial microbes. More recently, thin structures resembling the remains of organic biofilms have been reported in the same meteorite. Carbonates have also been discussed in the context of Mars sample return missions. Thermal spring deposits have often been cited as prime locations for exobiological exploration. By analogy to Earth, specialized microbes may have existed in the heated, mineralized waters, and precipitates of carbonate and/or silica from these waters may have trapped and preserved evidence of life. Since the geological interactions that produce thermal springs can be recognized in orbital imagery, directed searches for microfossils in such deposits are deemed possible. We are engaged in a study of the signatures produced by contemporary biogenic activity (biomarkers) in carbonate thermal springs. We are examining the microbes that live in such environments and the preservation of microbial forms, biofilms, and petrographic fabrics indicative of life in thermal spring mineral deposits. This work is part of a much more extensive study to refine the appropriate tools, techniques, and approaches to seek evidence of life in a range of planetary samples. A deeper understanding of biological signatures will prepare us for the detailed search for life on Mars and eventually on other planets. Overall. the study of biomarkers in rocks and soils will provide insight into the evolution of life because such signatures are a record of how life interacts with its environment, how it adapts to changing conditions, and how life can influence geology and climate.

Allen, C. C.↗

NASA NTRS2001

Potential Evaporite Biomarkers from the Dead Sea

The Dead Sea is located on the northern branch of the African-Levant Rift systems. The rift system, according to one model, was formed by a series of strike slip faults, initially forming approximately two million years ago. The Dead Sea is an evaporite basin that receives freshwater from springs and from the Jordan River. The Dead Sea is different from other evaporite basins, such as the Great Salt Lake, in that it possesses high concentrations of magnesium and has an average pH of 6.1. The dominant cation in the Great Salt Lake is sodium, and the pH is 7.7. Calcium concentrations are also higher in the Dead Sea than in the Great Salt Lake. Both basins are similar in that the dominant anion is chlorine and the salinity levels are approximately 20 %. Other common cations that have been identified from the waters of the Dead Sea and the Great Salt Lake include sodium and potassium. A variety of Archea, Bacteria, and a single genus of a green algal, Dunaliella, has been described from the Dead Sea. Earlier studies concentrated on microbial identification and analysis of their unique physiology that allows them to survive in this type of extreme environment. Potential microbial fossilization processes, microbial fossils, and the metallic ions associated with fossilization have not been studied thoroughly. The present study is restricted to identifying probable microbial morphologies and associated metallic ions. XRD (X Ray Diffraction) analysis indicates the presence of halite, quartz, and orthoclase feldspar. In addition to these minerals, other workers have reported potassium chloride, magnesium bromide, magnesium chloride, calcium chloride, and calcium sulfate. Halite, calcium sulfate, and orthoclase were examined in this report for the presence of microbes, microbially induced deposits or microbial alteration. Neither the gypsum nor the orthoclase surfaces possesses any obvious indications of microbial life or fossilization. The sand-sized orthoclase particles are weathered with 122 extensive fan-shaped mineral deposits. The gypsum deposits are associated with halite minerals and also exhibit extensive weathering. Halite minerals represent the only substrates that have probable rod-shaped microbial structures with long, filamentous, apical extensions. EDS (energy dispersive x-ray) analysis of the putative microbes indicates elevated calcium levels that are enriched with magnesium. The rod-shaped structures exhibit possible fossilization stages. Rhombohedralshaped minerals of magnesium-enriched calcium carbonate are deposited on the microbial surfaces, and eventually coat the entire microbial surface. The sodium chloride continues to crystallize on nearby halite surface and even crystallizes on the fossilized microbial remains. The putative fossils are found exclusively on halite surfaces, and all contained elevated levels of calcium magnesium cations. Both of these metallic cations are associated with microbial activity and fossilization. Their morphological diversity is low in comparison with the reported living Dead Sea microbial population. If we examine the fossil record for multicellular organisms, fossilization rates are lower for soft-bodied organisms than for those possessing hard parts, i.e. shells, bones. For example, smaller, single celled organisms would have a smaller chance of fossilization; their fossilized shapes could be mistaken for abiotic products. Another consideration is that dead organisms in the water column are probably utilized as a food source by other microbes before fossilization processes are completed. This may be an important consideration as we attempt to model and interpret ancient microbial environments either on Earth or on Mars.

Morris, Penny A.↗