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NASA NTRS2002

Comparative ecology of H2 cycling in sedimentary and phototrophic ecosystems

The simple biochemistry of H2 is critical to a large number of microbial processes, affecting the interaction of organisms with each other and with the environment. The sensitivity of each of these processes to H2 can be described collectively, through the quantitative language of thermodynamics. A necessary prerequisite is to understand the factors that, in turn, control H2 partial pressures. These factors are assessed for two distinctly different ecosystems. In anoxic sediments from Cape Lookout Bight (North Carolina, USA), H2 partial pressures are strictly maintained at low, steady-state levels by H2-consuming organisms, in a fashion that can be quantitatively predicted by simple thermodynamic calculations. In phototrophic microbial mats from Baja California (Mexico), H2 partial pressures are controlled by the activity of light-sensitive H2-producing organisms, and consequently fluctuate over orders of magnitude on a daily basis. The differences in H2 cycling can subsequently impact any of the H2-sensitive microbial processes in these systems. In one example, methanogenesis in Cape Lookout Bight sediments is completely suppressed through the efficient consumption of H2 by sulfate-reducing bacteria; in contrast, elevated levels of H2 prevail in the producer-controlled phototrophic system, and methanogenesis occurs readily in the presence of 40 mM sulfate.

Review, Tutorial↗

NASA NTRS2000

Depositional facies and aqueous-solid geochemistry of travertine-depositing hot springs (Angel Terrace, Mammoth Hot Springs, Yellowstone National Park, U.S.A.)

Petrographic and geochemical analyses of travertine-depositing hot springs at Angel Terrace, Mammoth Hot Springs, Yellowstone National Park, have been used to define five depositional facies along the spring drainage system. Spring waters are expelled in the vent facies at 71 to 73 degrees C and precipitate mounded travertine composed of aragonite needle botryoids. The apron and channel facies (43-72 degrees C) is floored by hollow tubes composed of aragonite needle botryoids that encrust sulfide-oxidizing Aquificales bacteria. The travertine of the pond facies (30-62 degrees C) varies in composition from aragonite needle shrubs formed at higher temperatures to ridged networks of calcite and aragonite at lower temperatures. Calcite "ice sheets", calcified bubbles, and aggregates of aragonite needles ("fuzzy dumbbells") precipitate at the air-water interface and settle to pond floors. The proximal-slope facies (28-54 degrees C), which forms the margins of terracette pools, is composed of arcuate aragonite needle shrubs that create small microterracettes on the steep slope face. Finally, the distal-slope facies (28-30 degrees C) is composed of calcite spherules and calcite "feather" crystals. Despite the presence of abundant microbial mat communities and their observed role in providing substrates for mineralization, the compositions of spring-water and travertine predominantly reflect abiotic physical and chemical processes. Vigorous CO2 degassing causes a +2 unit increase in spring water pH, as well as Rayleigh-type covariations between the concentration of dissolved inorganic carbon and corresponding delta 13C. Travertine delta 13C and delta 18O are nearly equivalent to aragonite and calcite equilibrium values calculated from spring water in the higher-temperature (approximately 50-73 degrees C) depositional facies. Conversely, travertine precipitating in the lower-temperature (< approximately 50 degrees C) depositional facies exhibits delta 13C and delta 18O values that are as much as 4% less than predicted equilibrium values. This isotopic shift may record microbial respiration as well as downstream transport of travertine crystals. Despite the production of H2S and the abundance of sulfide oxidizing microbes, preliminary delta 34S data do not uniquely define the microbial metabolic pathways present in the spring system. This suggests that the high extent of CO2 degassing and large open-system solute reservoir in these thermal systems overwhelm biological controls on travertine crystal chemistry.

Non-NASA Center↗

NASA NTRS1993

Extreme environments and exobiology

Ecological research on extreme environments can be applied to exobiological problems such as the question of life on Mars. If life forms (fossil or extant) are found on Mars, their study will help to solve fundamental questions about the nature of life on Earth. Extreme environments that are beyond the range of adaptability of their inhabitants are defined as "absolute extreme". Such environments can serve as terrestrial models for the last stages of life in the history of Mars, when the surface cooled down and atmosphere and water disappeared. The cryptoendolithic microbial community in porous rocks of the Ross Desert in Antarctica and the microbial mats at the bottom of frozen Antarctic lakes are such examples. The microbial communities of Siberian permafrost show that, in frozen but stable communities, long-term survival is possible. In the context of terraforming Mars, selected microorganisms isolated from absolute extreme environments are considered for use in creation of a biological carbon cycle.

NASA Discipline Exobiology↗

NASA NTRS1992

Novel long-chain anteiso-alkanes and anteiso-alkanoic acids in Antarctic rocks colonized by living and fossil cryptoendolithic microorganisms

Saponified extracts of rock samples colonized by cryptoendolithic microbial communities from the McMurdo Dry Valleys of Southern Victoria Land, Antarctica, were separated into hydrocarbon and fatty acid fractions by silica gel column chromatography. Hydrocarbons and methyl esters of fatty acids were analyzed by capillary gas chromatography-mass spectrometry. Unusually, a suite of long-chain anteiso-alkanes (a-C20 to a-C30) and anteiso-alkanoic acids (a-C20 to a-C30) were detected in many samples, together with straight-chain, branched and/or cyclic and acyclic isoprenoid compounds. These novel compounds are probably derived from unidentified heterotrophic bacteria or symbiotic processes in a unique microbial community in the Antarctic cold desert and suggest the occurrence of a special biosynthetic pathway. Long-chain anteiso-alkanes are probably formed through microbial decarboxylation of corresponding anteiso-alkanoic acids. They may serve as new biomarkers in environmental and geochemical studies.

NASA Program Exobiology↗

NASA NTRS2004

Astrobiology of Comets

We review the current state of knowledge concerning microbial extremophiles and comets and the potential significance of comets to Astrobiology. We model the thermal history of a cometary body, regarded as an assemblage of boulders, dust, ices and organics, as it approaches a perihelion distance of - IAU. The transfer of incident energy from sunlight into the interior leads to the melting of near surface ices, some under stable porous crust, providing possible habitats for a wide range of microorganisms. We provide data concerning new evidence for indigenous microfossils in CI meteorites, which may be the remains of extinct cometary cores. We discuss the dominant microbial communities of polar sea-ice, Antarctic ice sheet, and cryoconite environments as possible analogs for microbial ecosystems that may grow in sub-crustal pools or in ice/water films in comets.

Hoover, Richard B.↗

NASA NTRS2005

Microbiological Characterization and Concerns of the International Space Station Internal Active Thermal Control System

Since January 1999, the chemical the International Space Station Thermal Control System (IATCS) and microbial state of (ISS) Internal Active fluid has been monitored by analysis of samples returned to Earth. Key chemical parameters have changed over time, including a drop in pH from the specified 9.5 +/- 0.5 ta = 58.4, an increase in the level of total inorganic carbon (TIC), total organic carbon (TOC) and dissolved nickel (Ni) in the fluid, and a decrease in the phosphate (PO,) level. In addition, silver (AS) ion levels in the fluid decreased rapidly as Ag deposited on internal metallic surfaces of the system. The lack of available Ag ions coupled with changes in the fluid chemistry has resulted in a favorable environment for microbial growth. Counts of heterotrophic bacteria have increased from less than 10 colony-forming units (CFUs)/l00 mL to l0(exp 6) to l0(exp 7) CFUs/100 mL. The increase of the microbial population is of concern because uncontrolled microbiological growth in the IATCS can contribute to deterioration in the performance of critical components within the system and potentially impact human health if opportunistic pathogens become established and escape into the cabin atmosphere. Micro-organisms can potentially degrade the coolant chemistry; attach to surfaces and form biofilms; lead to biofouling of filters, tubing, and pumps; decrease flow rates; reduce heat transfer; initiate and accelerate corrosion; and enhance mineral scale formation. The micro- biological data from the ISS IATCS fluid, and approaches to addressing the concerns, are summarized in this paper.

Roman, Monsi C.↗

NASA NTRS2006

Irradiating of Bulk Soybeans: Influence on Their Functional and Sensory Properties for Soyfood Processing

Soybeans were chosen for lunar and planetary missions, where soybeans will be supplied in bulk or grown locally, 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 consumption. Radiation that soybeans would be exposed to during bulk storage prior to and during a Mars mission may influence their germination and functional properties. The influence of radiation includes the affect of surface pasteurization to ensure the astronauts safety from food-borne illnesses (HACCP, CCP), and the affect of the amount of radiation the soybeans receive during a Mars mission. Decreases in the amount of natural antioxidants free radical formation, and oxidation-induced changes in the soybean will influence the nutritional value, texture, color, and aroma of soyfoods. The objective of this study was to determine the influence of pasteurization and sterilization surface radiation on whole soybeans using gamma and electron beam radiation. The influence of 0, 1, 5, 10, and 30kGy on microbial load, germination rate, ease of processing, and quality of soymilk and tofu were determined. Surface radiation of whole dry soybeans using electron beam or gamma rays from 1-30kGy did provide microbial safety for the astronauts. However, the lower dose levels had surviving yeasts and molds. These doses caused oxidative changes that resulted in soymilk and tofu with rancid aromas. GC-MS of the aroma compounds using SPME Headspace confirmed the presence of lipid oxidation compounds. Soybean germination ability was reduced as radiation dosage increased. While lower doses may reduce these problems, the ability to insure microbial safety of bulk soybeans will be lost. Counter measures could include vacuum packaging, nitrogen flushing, added antioxidants, and radiating under freezing conditions. Doses below 1kGy need to be investigated further to determine the influence of the radiation encountered during Mars missions.

Chia, Chiew-Ling↗

NASA NTRS2006

Planned Environmental Microbiology Aspects of Future Lunar and Mars Missions

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

Ott, C. Mark↗

NASA NTRS2006

The Influence of Microbiology on Spacecraft Design and Controls: A Historical Perspective of the Shuttle and International Space Station Programs

For over 40 years, NASA has been putting humans safely into space in part by minimizing microbial risks to crew members. Success of the program to minimize such risks has resulted from a combination of engineering and design controls as well as active monitoring of the crew, food, water, hardware, and spacecraft interior. The evolution of engineering and design controls is exemplified by the implementation of HEPA filters for air treatment, antimicrobial surface materials, and the disinfection regimen currently used on board the International Space Station. Data from spaceflight missions confirm the effectiveness of current measures; however, fluctuations in microbial concentrations and trends in contamination events suggest the need for continued diligence in monitoring and evaluation as well as further improvements in engineering systems. The knowledge of microbial controls and monitoring from assessments of past missions will be critical in driving the design of future spacecraft.

Castro, Victoria A.↗

NASA NTRS2007

Super-long Anabiosis of Ancient Microorganisms in Ice and Terrestrial Models for Development of Methods to Search for Life on Mars, Europa and other Planetary Bodies

Successful missions to Mars, Europe and other bodies of the Solar system have created a prerequisite to search for extraterrestrial life. The first attempts of microbial life detection on the Martian surface by the Viking landed missions gave no biological results. Microbiological investigations of the Martian subsurface ground ice layers seem to be more promising. It is well substantiated to consider the Antarctic ice sheet and the Antarctic and Arctic permafrost as terrestrial analogues of Martian habitats. The results of our long-standing microbiological studies of the Antarctic ice would provide the basis for detection of viable microbial cells on Mars. Our microbiological investigations of the deepest and thus most ancient strata of the Antarctic ice sheet for the first time gave evidence for the natural phenomenon of long-term anabiosis (preservation of viability and vitality for millennia years). A combination of classical microbiological methods, epifluorescence microscopy, SEM, TEM, molecular diagnostics, radioisotope labeling and other techniques made it possible for us to obtain convincing proof of the presence of pro- and eukaryotes in the Antarctic ice sheet. In this communication, we will review and discuss some critical issues related to the detection of viable microorganisms in cold terrestrial environments with regard to future searches for microbial life and/or its biological signatures on extraterrestrial objects.

Abyzov, S. S.↗

NASA NTRS2008

Purifying Nucleic Acids from Samples of Extremely Low Biomass

A new method is able to circumvent the bias to which one commercial DNA extraction method falls prey with regard to the lysing of certain types of microbial cells, resulting in a truncated spectrum of microbial diversity. By prefacing the protocol with glass-bead-beating agitation (mechanically lysing a much more encompassing array of cell types and spores), the resulting microbial diversity detection is greatly enhanced. In preliminary studies, a commercially available automated DNA extraction method is effective at delivering total DNA yield, but only the non-hardy members of the bacterial bisque were represented in clone libraries, suggesting that this method was ineffective at lysing the hardier cell types. To circumvent such a bias in cells, yet another extraction method was devised. In this technique, samples are first subjected to a stringent bead-beating step, and then are processed via standard protocols. Prior to being loaded into extraction vials, samples are placed in micro-centrifuge bead tubes containing 50 micro-L of commercially produced lysis solution. After inverting several times, tubes are agitated at maximum speed for two minutes. Following agitation, tubes are centrifuged at 10,000 x g for one minute. At this time, the aqueous volumes are removed from the bead tubes and are loaded into extraction vials to be further processed via extraction regime. The new method couples two independent methodologies in such as way as to yield the highest concentration of PCR-amplifiable DNA with consistent and reproducible results and with the most accurate and encompassing report of species richness.

La Duc, Myron↗

NASA NTRS2009

A Comprehensive Characterization of Microorganisms and Allergens in Spacecraft Environment

The determination of risk from infectious disease during long-duration missions is composed of several factors including the concentration and the characteristics of the infectious agent. Thus, a thorough knowledge of the microorganisms aboard spacecraft is essential in mitigating infectious disease risk to the crew. While stringent steps are taken to minimize the transfer of potential pathogens to spacecraft, several medically significant organisms have been isolated from both the Mir and International Space Station (ISS). Historically, the method for isolation and identification of microorganisms from spacecraft environmental samples depended upon their growth on culture media. Unfortunately, only a fraction of the organisms may grow on a culture medium, potentially omitting those microorganisms whose nutritional and physical requirements for growth are not met. Thus, several pathogens may not have been detected, such as Legionella pneumophila, the etiological agent of Legionnaire s disease. We hypothesize that environmental analysis using non-culture-based technologies will reveal microorganisms, allergens, and microbial toxins not previously reported in spacecraft, allowing for a more complete health assessment. The development of techniques for this flight experiment, operationally named SWAB, has already provided advances in NASA laboratory processes and beneficial information toward human health risk assessment. The translation of 16S ribosomal DNA sequencing for the identification of bacteria from the SWAB experiment to nominal operations has increased bacterial speciation of environmental isolates from previous flights three fold compared to previous conventional methodology. The incorporation of molecular-based DNA fingerprinting using repetitive sequence-based polymerase chain reaction (rep-PCR) into the capabilities of the laboratory has provided a methodology to track microorganisms between crewmembers and their environment. Both 16S ribosomal DNA identification and bacterial fingerprinting have improved NASA s capability to better understand spacecraft environments and determine the source of contamination events. Preflight sampling has been completed for air, surface, and water samples. In-flight sample collection has been completed for a total of 8 air and surface sample collection sessions. In-flight hardware has performed well and the surface sampling device received positive feedback from the crew for its ease of use. While processing and analysis continue for these samples, early results have begun to provide information on the spacecraft environment. Using a method called Denaturing Gradient Gel Electrophoresis (DGGE), several air and samples were evaluated to determine the types of organisms that were present. Using only molecular techniques, DGGE does not depend on any microbial growth on culture media, allowing a more comprehensive assessment of the spacecraft interior. Preliminary results have identified several microorganisms that would not have been isolated using current technology, though none of these organisms would be considered medically significant. Interestingly, the isolation of Gram negative organisms is greater using DGGE than conventional media based isolation. The cause of this finding is unclear, though it may be the result of the technique s ability to isolate both viable and non-viable bacteria. The next phase of the SWAB sample analysis is the use of quantitative polymerase chain reaction (QPCR) to look for specific medically significant organisms. While not as broad as DGGE, QPCR is much more sensitive and may reveal findings that were not seen during the initial evaluation. Together, this information will lead toward an accurate microbial risk assessment to help set flight requirements to protect the safety, health, and performance of the crew.

Castro, V.A.↗

NASA NTRS2009

Panel Discussion: Life in the Cosmos

Water appears to be essential to all life on Earth. For this reason, "Follow the Water" has been adopted as a mantra for the search for Life in the Cosmos. Expeditions have helped to establish the limits and biodiversity of life in the most extreme environments on Earth. Microbial extremophiles inhabit acidic streams; hypersaline and hyperalkaline lakes and pools; the cold deep sea floor, permafrost, rocks, glaciers, and perennially ice-covered lakes of the polar environments; geysers, volcanic fumaroles, hydrothermal vents and hot rocks deep within the Earth's crust. The ESA Venus Express Spacecraft entered Venusian Orbit in 2006 and continues to produce exciting results. The Visible and Infrared Thermal Imaging Spectrometer (VIRTIS) instrument made the first detection of hydroxyl in the atmosphere of Venus, indicating it is much more similar to Earth and Mars than previously thought. Huge hurricane-like vortices have been found above the poles of the planet and as yet unidentified UV absorbers that form mysterious dark bands in the upper atmosphere. At 70 km and below, water vapor and sulfur dioxide combine to form sulfuric acid droplets that create a haze above the cloud tops. Thermophilic acidophiles, such as have recently been discovered on Earth, could possibly survive in the hot sulfuric acid droplets that exist in the upper atmosphere of Venus. In order to understand how to search for life elsewhere in the Solar System, over 40 VIRTIS images of Earth from Venus have been obtained to search for evidence of life on Earth. The signatures of water and molecular Oxygen were detected in the Earth s atmosphere, but the atmosphere of Venus also exhibits these signatures. The water and water ice are far more abundant on comet, the polar caps and permafrost of Mars and the icy moons of Jupiter and Saturn. These "frozen worlds" of our Solar System, are much more promising regimes where extant or extinct microbial life may exist. The ESA Mars Advanced Radar for Subsurface and Ionospheric Sounding (MARSIS) probe has found that both the North and South Polar Caps of Mars are approximately 3.5 km thick and are composed almost entirely of water ice. In winter, a thin dry ice layer covers the caps, but it sublimates directly to CO2 in the spring. The ESA Mars Express Orbiter images reveal Rupes Tenuis to be a vast snow-laden region on the southern edge of the Martian North Polar Cap. The Mars Exploration Rover Spirit found alkaline volcanic rocks in the Gusev Crater and the Phoenix Mars Lander has shown that the soil of Mars is much more alkaline than previously expected. The Phoenix Mars Lander has also made direct observations of frozen and liquid water on Mars. It is known that microorganisms from Alaska, Siberia and Antarctica can remain alive frozen in permafrost or ice for long periods of time. These discoveries increase the possibility that the Labeled Release Experiment may have discovered life on Mars during the Viking Mission and provide strong impetus for the return of life detection experiments to Mars. Changes in the spin rate of Saturn's moon Titan indicate that it may also harbor a 300 km thick liquid water ocean beneath its icy crust. The NASA/ESA/Italian Space Agency Cassini Spacecraft has imaged geysers containing water vapor, methane, carbon dioxide and organics erupting from the "tiger stripe" regions near the South Pole of Saturn's moon Enceladus. The high temperatures observed, the water vapor and large number of ice particles expelled suggest that a liquid water lake may exist beneath the "tiger stripe" ice cracks of Enceladus. The NASA Deep Impact probe found the surface temperature of comet 9P/Temple 1 at 1.5 AY was slightly above the ice/water phase change temperature (273 K). This suggests melting of water ice near the comet surface. A spectrometer the spacecraft detected a mixture of clay and carbonate minerals (that form in the presence of liquid water) streaming off the comet after the collision with the pactor. The study of chemical and mineral biomarkers, chiral amino acids and possible indigenous microfossils in SNC and carbonaceous meteorites continues. These results suggest that comets should be considered prime targets in the search for Life in the Cosmos. The ESA Rosetta mission is on track to rendezvous with comet 67P/Churyumov-Gerasimenko. The recent space observations combined with new information about the ability of microbial extremophiles to thrive in polar environments suggest that life may be far more widely distributed in the Cosmos than previously thought possible. The Panelists will review recent discoveries and provide their own insights about Life in the Cosmos -- followed by a question and answer session with the audience.

Hoover, Richard B.↗

NASA NTRS2010

Root Zone Respiration on Hydroponically Grown Wheat Plant Systems

Root respiration is a biological phenomenon that controls plant growth and physiological development during a plant's lifespan. This process is dependent on the availability of oxygen in the system where the plant is located. In hydroponic systems, where plants are submerged in a solution containing vital nutrients but no type of soil, the availability of oxygen arises from the dissolved oxygen concentration in the solution. This oxygen concentration is dependent on the , gas-liquid interface formed on the upper surface of the liquid, as given by Henry's Law, depending on pressure and temperature conditions. Respiration rates of the plants rise as biomass and root zone increase with age. The respiration rate of Apogee wheat plants (Triticum aestivum) was measured as a function of light intensity (catalytic for photosynthesis) and CO2 concentration to determine their effect on respiration rates. To determine their effects on respiration rate and plant growth microbial communities were introduced into the system, by Innoculum. Surfactants were introduced, simulating gray-water usage in space, as another factor to determine their effect on chemical oxygen demand of microbials and on respiration rates of the plants. It is expected to see small effects from changes in CO2 concentration or light levels, and to see root respiration decrease in an exponential manner with plant age and microbial activity.

Soler-Crespo, R. A.↗

NASA NTRS2011

Microbiological Lessons Learned from the Space Shuttle

After 30 years of being the centerpiece of NASA s human spacecraft, the Space Shuttle will retire. This highly successful program provided many valuable lessons for the International Space Station (ISS) and future spacecraft. Major microbiological risks to crewmembers include food, water, air, surfaces, payloads, animals, other crewmembers, and ground support personnel. Adverse effects of microorganisms are varied and can jeopardize crew health and safety, spacecraft systems, and mission objectives. Engineering practices and operational procedures can minimize the negative effects of microorganisms. To minimize problems associated with microorganisms, appropriate steps must begin in the design phase of new spacecraft or space habitats. Spacecraft design must include requirements to control accumulation of water including humidity, leaks, and condensate on surfaces. Materials used in habitable volumes must not contribute to microbial growth. Use of appropriate materials and the implementation of robust housekeeping that utilizes periodic cleaning and disinfection will prevent high levels of microbial growth on surfaces. Air filtration can ensure low levels of bioaerosols and particulates in the breathing air. The use of physical and chemical steps to disinfect drinking water coupled with filtration can provide safe drinking water. Thorough preflight examination of flight crews, consumables, and the environment can greatly reduce pathogens in spacecraft. The advances in knowledge of living and working onboard the Space Shuttle formed the foundation for environmental microbiology requirements and operations for the International Space Station (ISS) and future spacecraft. Research conducted during the Space Shuttle Program resulted in an improved understanding of the effects of spaceflight on human physiology, microbial properties, and specifically the host-microbe interactions. Host-microbe interactions are substantially affected by spaceflight. Astronaut immune functions were found to be altered. Selected microorganisms were found to become more virulent during spaceflight. The increased knowledge gained on the Space Shuttle resulted in further studies of the host-microbe interactions on the ISS to determine if countermeasures were necessary. Lessons learned from the Space Shuttle Program were integrated into the ISS resulting in the safest space habitat to date.

Pierson, Duane L.↗

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 NTRS2012

Next Generation Microbiology Requirements

As humans continue to explore deep into space, microorganisms will travel with them. The primary means to mitigate the risk of infectious disease are a combination of prudent spacecraft design and rigorous operational controls. The effectiveness of these methods are evaluated by microbiological monitoring of spacecraft, food, water, and the crew that is performed preflight, in-flight, and post-flight. Current NASA requirements associated with microbiological monitoring are based on culture-based methodology where microorganisms are grown on a semi-solid growth medium and enumerated. Subsequent identification of the organisms requires specialized labor and large equipment, which historically has been performed on Earth. Requirements that rely strictly on culture-based units limit the use of non-culture based monitoring technology. Specifically, the culture-based "measurement criteria" are Colony Forming Units (CFU, representing the growth of one microorganism at a single location on the agar medium) per a given volume, area, or sample size. As the CFU unit by definition is culture-based, these requirements limit alternative technologies for spaceflight applications. As spaceflight missions such as those to Mars extend further into space, culture-based technology will become difficult to implement due to the (a) limited shelf life of the culture media, (b) mass/volume necessary to carry these consumables, and (c) problems associated with the production of biohazardous material in the habitable volume of the spacecraft. In addition, an extensive amount of new knowledge has been obtained during the Space Shuttle, NASA-Mir, and International Space Station Programs, which gave direction for new or modified microbial control requirements for vehicle design and mission operations. The goal of this task is to develop and recommend a new set of requirements for vehicle design and mission operations, including microbiological monitoring, based upon "lessons learned" and new technology. During 2011, this study focused on evaluating potable water requirements by assembling a forum of internal and external experts from NASA, other federal agencies, and academia. Key findings from this forum included: (1) Preventive design and operational strategies should be stringent and the primary focus of NASA's mitigation efforts, as they are cost effective and can be attained with conventional technology. (2) Microbial monitoring hardware should be simple and must be able to measure the viability of microorganisms in a sample. Multiple monitoring technologies can be utilized as long as at the microorganisms being identified can also be confirmed as viable. (3) Evidence showing alterations in the crew immune function and microbial virulence complicates risk assessments and creates the need for very conservative requirements. (4) One key source of infectious agents will always be the crew, and appropriate preventative measures should be taken preflight. (5) Water systems should be thoroughly disinfected (sterilized if possible) preflight and retain a residual biocide throughout the mission. Future forums will cover requirements for other types of samples, specifically spaceflight food and environmental samples, such as vehicle air and vehicle and cargo surfaces. An interim report on the potable water forum has been delivered to the Human Research Program with a final report on the recommendations for all sample types being delivered in September 2013.

Ott, C. M.↗

NASA NTRS2012

Nonhazardous Urine Pretreatment Method

A method combines solid phase acidification with two non-toxic biocides to prevent ammonia volatilization and microbial proliferation. The safe, non-oxidizing biocide combination consists of a quaternary amine and a food preservative. This combination has exhibited excellent stabilization of both acidified and unacidified urine. During pretreatment tests, composite urine collected from donors was challenged with a microorganism known to proliferate in urine, and then was processed using the nonhazardous urine pre-treatment method. The challenge microorganisms included Escherichia coli, a common gram-negative bacteria; Enterococcus faecalis, a ureolytic gram-positive bacteria; Candida albicans, a yeast commonly found in urine; and Aspergillus niger, a problematic mold that resists urine pre-treatment. Urine processed in this manner remained microbially stable for over 57 days. Such effective urine stabilization was achieved using non-toxic, non-oxidizing biocides at higher pH (3.6 to 5.8) than previous methods in use or projected for use aboard the International Space Station (ISS). ISS urine pretreatment methods employ strong oxidants including ozone and hexavalent chromium (Cr(VI)), a carcinogenic material, under very acidic conditions (pH = 1.8 to 2.4). The method described here offers a much more benign chemical environment than previous pretreatment methods, and will lower equivalent system mass (ESM) by reducing containment volume and mass, system complexity, and crew time needed to handle pre-treatment chemicals. The biocides, being non-oxidizing, minimize the potential for chemical reactions with urine constituents to produce volatile, airborne contaminants such as cyanogen chloride. Additionally, the biocides are active under significantly less acidic conditions than those used in the current system, thereby reducing the degree of required acidification. A simple flow-through solid phase acidification (SPA) bed is employed to overcome the natural buffering capacity of urine, and to lower the pH to levels that fix ammoniacal nitrogen in the non-volatile and highly water soluble NH4 + form. Citric acid, a highly soluble, solid tricarboxylic acid essential to cellular metabolism, and typically used as a food preservative, has also been shown to efficiently acidify urine in conjunction with non-oxidizing biocides to provide effective stabilization with respect to both microbial growth and ammonia volatilization.

Akse, James R.↗