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Technical Paper

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.
Technical Paper

Development of Vapor Phase Hydrogen Peroxide Sterilization Process for Spacecraft Applications

In order to meet microbial reduction requirements for all Mars in-situ life detection and sample return missions, entire planetary spacecraft (including planetary entry probes and planetary landing capsules) may have to be exposed to a qualified sterilization process. At JPL, we are developing a low temperature (~45°C) vapor phase hydrogen peroxide sterilization process. This process is currently being used by the medical industry and its effectiveness is well established. In order to effectively and safely apply this technology to sterilize a spacecraft, which is made out of various man-made materials and electronic circuit boards, the following technical issues need to be resolved: 1. Efficacy of sterilization process. 2. Diffusion of H2O2 under sterilization process conditions into hard to reach places. 3. Materials and components compatibility with the sterilization process. 4. Development of methodology to protect (isolate) sensitive components (i.e. electronic ) from H2O2 vapor.
Technical Paper

Viral Populations within the International Space Station's Internal Active Thermal Control System Ground Support and Potential Flight Hardware

The Internal Active Thermal Control System (IATCS) aboard the International Space Station (ISS) contains an aqueous, alkaline fluid (pH 9.5±0.5) that aids in maintaining a habitable environment for the crew. Because microbes have significant potential to cause disease, adverse effects on astronaut health, and microbe-induced corrosion, the presence of both bacteria and viruses within IATCS fluids is of concern. This study sought to detect and identify viral populations in IATCS samples obtained from the Kennedy Space Center as a first step towards characterizing and understanding potential risks associated with them. Samples were concentrated and viral nucleic acids (NA) extracted providing solutions containing 8.87-22.67 μg NA per mL of heat transfer fluid. After further amplification viral DNA and cDNA were then pooled, fluorescently labeled, and hybridized onto a Combimatrix panvira 12K microarray containing probes for ∼1,000 known human viruses.
Technical Paper

Education Payload Operations Kit C: A Miniature, Low ESM Hobby Garden for Space-Based Educational Activities

The wonder of space exploration is a sure way to catch the attention of students of all ages, and space biology is one of many sciences critical to understanding the spaceflight environment. Many systems used in the past for space-to-classroom biology activities have required extensive crew time and material resources, making space-linked education logistically and financially difficult. The new Education Payload Operations Kit C (EPO Kit C) aims to overcome obstacles to space-linked education and outreach by dramatically reducing the resources required for educational activities in plant space biology that have a true spaceflight component. EPO Kit C is expected to be flown from STS-118 to the International Space Station in June 2007. NASA and several other organizations are currently planning an outreach program to complement the flight of EPO Kit C.
Technical Paper

Development of a Gravity Independent Nitrification Biological Water Processor

Biological water processors are currently being developed for application in microgravity environments. Work has been performed to develop a single-phase, gravity independent anoxic denitrification reactor for organic carbon removal [1]. As a follow on to this work it was necessary to develop a gravity independent nitrification reactor in order to provide sufficient nitrite and nitrate to the organic carbon oxidation reactor for the complete removal of organic carbon. One approach for providing the significant amounts of dissolved oxygen required for nitrification is to require the biological reactor design to process two-phase gas and liquid in micro-gravity. This paper addresses the design and test results overview for development of a tubular, two-phase, gravity independent nitrification biological water processor.
Technical Paper

Assessment of Microbial Community Variability in Replicate Tubular Nitrifying Bioreactors using PCR and TRFLP Analysis

Bioregenerative life support systems (BLSS) may be necessary for long-term space missions due to the high costs of lifting supplies and equipment into orbit. Much of the recycling to be done in a BLSS involves microbial activity. Although most studies to date have used a culture-based approach to characterize bacteria in BLSS under development, recently work has begun utilizing non-culture-based, DNA approaches to elucidate which microbes are present. In this study, we investigated whether replicate reactors develop replicate microbial communities using a 16S rRNA gene approach and terminal restriction length polymorphism analysis for tubular, nitrifier reactors in use at JSC. Our result suggests that both individual reactor and temporal signals can be detected in the microbial populations. This information may lead to optimization of inoculation procedures and reactor operations conditions to increase predictability and reliability of biological systems.
Technical Paper

Evaluation of Sample Preservation Methods for Space Missions

This study of samples collected from Mars 01 Orbiter was conducted to gain a better understanding and practical experience in methods to process and preserve samples intended for planetary protection analysis. Samples were evaluated for the viable growth of microbes, the molecular biomarker adenosine triphosphate (ATP), and the presence of lipopolysaccharide, a bacterial cell wall component. Losses were observed in the number of viable microbes after freezing as well as in the detectable lipopolysaccharide. Two independent studies of pooled cleanroom samples demonstrate good ATP recovery and consistent values after freezing at −20 °C.
Technical Paper

Lunar-Mars Life Support Test Project Phase III Water Recovery System Operation and Results

An integrated water recovery system was operated for 91 days in support of the Lunar Mars Life Support Test Project (LMLSTP) Phase III test. The system combined both biological and physical-chemical processes to treat a combined wastewater stream consisting of waste hygiene water, urine, and humidity condensate. Biological processes were used for primary degradation of organic material as well as for nitrification of ammonium in the wastewater. Physical-chemical systems removed inorganic salts from the water and provided post-treatment. The integrated system provided potable water to the crew throughout the test. This paper describes the water recovery system and reviews the performance of the system during the test.
Technical Paper

Regenerative Water Recovery System Testing and Model Correlation

Biological wastewater processing has been under investigation by AlliedSignal Aerospace and NASA Johnson Space Center (JSC) for future use in space. Testing at JSC in the Hybrid Regenerative Water Recovery System (HRWRS) in preparation for future closed human testing has been performed. Computer models have been developed to aid in the design of a new four-person immobilized cell bioreactor. The design of the reactor and validation of the computer model is presented. In addition, the total organic carbon (TOC) computer model has been expanded to begin investigation of nitrification. This model is being developed to identify the key parameters of the nitrification process, and to improve the design and operating conditions of nitrifying bioreactors. In addition, the model can be used as a design tool to rapidly predict the effects of changes in operational conditions and reactor design, significantly reducing the number and duration of experiments required.
Technical Paper

Removal of Low Levels of Ammonium Ion From pacecraft Recycled Water

Poly (vinyl chloride) (PVC) matrix membranes which incorporate the ionophore nonactin have been evaluated as cation exchange membranes for ammonium ion transport in an electrolytic cell configuration. Interest exists for the development of cation selective membranes for removal of low levels (<200ppm) of ammonium ions commonly found in recycled effluent streams in such diverse applications as expected in a Space Station and commercial fisheries. Ammonium ions are generated as a decomposition product of urea and over time build up in concentration, thus rendering the water unsuitable for human consumption. Nonactin is commonly used in a PVC matrix for ion-selective electrodes.
Technical Paper

Extended Temperature Range Studies for Dry Heat Microbial Reduction

Dry heat microbial reduction is an approved method to reduce the microbial bioburden on space-flight hardware prior to launch to meet flight project planetary protection requirements. Microbial bioburden reduction also occurs if a spacecraft enters a planetary atmosphere (e.g., Mars) and is heated by frictional forces. However, without further studies, administrative credit for this reduction cannot be applied. The killing of Bacillus subtilis var. niger spores has been examined and lethality data has been collected by placing spores in a vacuum oven or thermal spore exposure vessels (TSEV) in a constant temperature bath. Using this lethality data, a preliminary mathematical model is being developed that can be used to predict spore killing at different temperatures. This paper will present the lethality data that has been collected at this time and the planned future studies.
Technical Paper

Microbial Burden of Commercial Aircraft Cabin Air

The microbial burdens of 69 cabin air samples collected in-flight aboard commercial airliners were assessed via culture-dependent and molecular-based microbial enumeration assays. Cabin air samples from each of four separate flights aboard two different carriers were collected via air-impingement. Microbial enumeration techniques targeting DNA, ATP, and endotoxin were employed to estimate total microbial burden. The total viable microbial population ranged from 0 to 3.6 × 104 cells per 100 liters of air, as assessed by the ATP-assay. When these same samples were plated on minimal medium, anywhere from 2 to 80% of the viable population was cultivable. Five of the 29 samples examined exhibited higher cultivable plate counts than ATP-derived viable counts, perhaps a consequence of the dormant nature (lower concentration of intracellular ATP) of cells inhabiting these air cabin samples.
Technical Paper

Q-PCR Based Bioburden Assessment of Drinking Water Throughout Treatment and Delivery to the International Space Station

Previous studies indicated evidence of opportunistic pathogens in samples obtained during missions to the International Space Station (ISS). This study utilized TaqMan quantitative PCR to determine specific gene abundance in potable and non-potable ISS waters. Probe and primer sets specific to the small subunit rRNA genes were designed and used to elucidate overall bacterial rRNA gene numbers. In addition, primer-probe sets specific for Burkholderia cepacia and Stenotrophomonas maltophilia were optimized and genes of these two opportunistic pathogens quantified in the pre- and post-flight drinking water as well as coolant waters. This Q-PCR approach supports findings of previous culture-based studies however; the culture based studies may have underestimated the microbial burden of ISS drinking water.
Technical Paper

Implications of the VBNC State of B. cepacia and S. maltophilia on Bioreduction and Microbial Monitoring of ISS Potable Waters

Certain Eubacteria enter a viable but nonculturable (VBNC) state upon encountering unfavorable environmental conditions. VBNC cells do not divide on conventional media yet remain viable and in some cases retain virulence. Here, we describe the VBNC state of two opportunistic pathogens previously isolated from ISS potable waters, Burkholderia cepacia and Stenotrophomonas maltophilia. Artificially inoculated microcosms were exposed to the biocidal agents copper (CuSO4) and iodine (I2) in an attempt to induce nonculturablility. Viability was assessed via fluorescent microscopy (direct viable count assay coupled with BacLight™ staining) and metabolic activity was monitored by quantifying both intracellular ATP and transcribed rRNA (reverse transcriptase quantitative PCR). Culturablility was lost in both B. cepacia and S. maltophilia within two days of exposure to copper or high concentrations of iodine (6 or 8 ppm).
Journal Article

ATCC 29669 Spores Show Substantial Dry Heat Survivability

Bacillus sp. ATCC 29669 was isolated from microbial fallout in clean rooms during the assembly of the Viking Spacecraft missions to Mars, making it a potential contamination concern for outbound space missions. Spores from this bacterial strain were found to be thirty times more resistant to dry heat than B. atrophaeus. Spore inactivation rates under vacuum controlled humidity were faster than rates obtained under ambient humidity. Inactivation rates for these heat resistant spores are important considerations for planetary protection implementation where temperature, time and humidity conditions are used to estimate the effectiveness of dry heat microbial reduction (DHMR) procedures.
Technical Paper

Evaluation of Methods for Remediating Biofilms in Spacecraft Potable Water Systems

Controlling microbial growth and biofilm formation in spacecraft water-distribution systems is necessary to protect the health of the crew. Methods to decontaminate the water system in flight may be needed to support long-term missions. We evaluated the ability of iodine and ozone to kill attached bacteria and remove biofilms formed on stainless steel coupons. The biofilms were developed by placing the coupons in a manifold attached to the effluent line of a simulated spacecraft water-distribution system. After biofilms were established, the coupons were removed and placed in a treatment manifold in a separate water treatment system where they were exposed to the chemical treatments for various periods. Disinfection efficiency over time was measured by counting the bacteria that could be recovered from the coupons using a sonication and plate count technique. Scanning electron microscopy was also used to determine whether the treatments actually removed the biofilm.
Technical Paper

Biofilm Formation and Control in a Simulated Spacecraft Water System: Three Year Results

Two simulated spacecraft water systems are being used to evaluate the effectiveness of iodine for controlling microbial contamination within such systems. An iodine concentration of about 2.0 mg/L is maintained in one system by passing ultrapure water through an iodinated ion exchange resin. Stainless steel coupons with electropolished and mechanically-polished sides are being used to monitor biofilm formation. Results after three years of operation show a single episode of significant bacterial growth in the iodinated system when the iodine level dropped to 1.9 mg/L. This growth was apparently controlled by replacing the iodinated ion exchange resin, thereby increasing the iodine level. The second batch of resin has remained effective in controlling microbial growth down to an iodine level of 1.0 mg/L. Scanning electron microscopy indicates that the iodine has impeded but may have not completely eliminated the formation of biofilm.
Technical Paper

Progress on Development of the Advanced Life Support Human-Rated Test Facility

NASA's Advanced Life Support Program has included as part of its long-range planning the development of a large-scale advanced life support facility capable of supporting long-duration testing of integrated, regenerative biological and physicochemical life support systems. As the designated NASA Field Center responsible for integration and testing of advanced life support systems, Johnson Space Center has undertaken the development of such a facility--the Advanced Life Support Human-Rated Test Facility (HRTF). As conceived, the HRTF is an interconnected five-chamber facility with a sealed internal environment capable of supporting a test crew of four for periods exceeding one year. The life support system which sustains the crew consists of both biological and physicochemical components and will perform air revitalization, water recovery, food production, solid waste processing, thermal management, and integrated control and monitoring functions.
Technical Paper

NASA's Approach to Integrated System Testing of Regenerative Life Support Systems

Integrating physicochemical and biological technologies into a regenerative life support system is a complex technical challenge. NASA recognizes that the depth and breadth of the challenge warrants a comprehensive investigation. NASA is implementing several ground-based projects to look at different systems integration issues. The combined efforts of these activities will enable NASA to develop regenerative life support systems for human exploration of the solar system in the 21st century. This paper provides an overview of NASA's overall approach to ground testing of integrated regenerative life support systems.
Technical Paper

Immobilized Microbe Microgravity Water Processing System (IMMWPS) Flight Experiment Integrated Ground Test Program

This paper provides an overview of the IMMWPS Integrated Ground Test Program, completed at the NASA Johnson Space Center (JSC) during October and November 2001. The JSC Crew and Thermal Systems Division (CTSD) has developed the IMMWPS orbital flight experiment to test the feasibility of a microbe-based water purifier for use in zero-gravity conditions. The IMMWPS design utilizes a Microbial Processor Assembly (MPA) inoculated with facultative anaerobes to convert organic contaminants in wastewater to carbon dioxide and biomass. The primary purpose of the ground test program was to verify functional operations and procedures. A secondary objective was to provide initial ground data for later comparison to on-orbit performance. This paper provides a description of the overall test program, including the test article hardware and the test sequence performed to simulate the anticipated space flight test program. In addition, a summary of significant results from the testing is provided.