Environmental microbiology as related to planetary quarantine Semiannual progress report, 1 Dec. 1968 - 31 May 1969
Environmental microbiology applied to planetary quarantine
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Environmental microbiology applied to planetary quarantine
The annual report presents the fiscal year (FY) 1990 research activities and accomplishments for the Catalysis and Biocatalysis Program of the Advanced Industrial Concepts Division (AICD), Office of Industrial Technologies of the Department of Energy (DOE). The mission of the AICD is to create a balanced program of high risk, long term, directed interdisciplinary research and development that will improve energy efficiency and enhance fuel flexibility in the industrial sector. The Catalysis and Biocatalysis Program's technical activities were organized into five work elements: the Molecular Modeling and Catalysis by Design element; the Applied Microbiology and Genetics element; the Bioprocess Engineering element; the Separations and Novel Chemical Processes element; and the Process Design and Analysis element.
The microbiological aspects of clean room technology as applied to surgery were reviewed. The following pertinent subject areas were examined: (1) clean room technology per se and its utilization for surgery, (2) microbiological monitoring of the clean room surgical environment, (3) clean rooms and their impact on operating room environmental microbiology, and (4) the effect of the technology on surgical wound infection rates. Conclusions were drawn for each topic investigated.
The genus Shewanella has been studied since 1931 with regard to a variety of topics of relevance to both applied and environmental microbiology. Recent years have seen the introduction of a large number of new Shewanella-like isolates, necessitating a coordinated review of the genus. In this work, the phylogenetic relationships among known shewanellae were examined using a battery of morphological, physiological, molecular and chemotaxonomic characterizations. This polyphasic taxonomy takes into account all available phenotypic and genotypic data and integrates them into a consensus classification. Based on information generated from this study and obtained from the literature, a scheme for the identification of Shewanella species has been compiled. Key phenotypic characteristics were sulfur reduction and halophilicity. Fatty acid and quinone profiling were used to impart an additional layer of information. Molecular characterizations employing small-subunit 16S rDNA sequences were at the limits of resolution for the differentiation of species in some cases. As a result, DNA-DNA hybridization and sequence analyses of a more rapidly evolving molecule (gyrB gene) were performed. Species-specific PCR probes were designed for the gyrB gene and used for the rapid screening of closely related strains. With this polyphasic approach, in addition to the ten described Shewanella species, two new species, Shewanella oneidensis and 'Shewanella pealeana', were recognized; Shewanella oneidensis sp. nov. is described here for the first time.
The Astromaterials Acquisition and Curation Office maintains seven cleanrooms for curating extraterrestrial samples from a variety of sample return missions. These cleanrooms are monitored for inorganic and particulate contamination and are maintained at ISO 7 to ISO 4 equivalent standards as dictated by collection requirements. As part of the advanced curation effort and to prepare for future sample collections from carbonaceous asteroids and eventually Mars we have begun monitoring the microbiology of these cleanrooms. Insights gained from monitoring these labs can be applied to improving planetary protection efforts.
Circuit voltage output is reduced by voltage equivalent to ampere resistance drop of nonlogarithmic resistances of practical diodes; therefore, output is same as circuit using ideal diode. Circuit applies to meter and electronic recorder movements, and improves performance of radiation detectors and several microbiological monitoring devices
The purpose of this plan is to provide a conceptual strategy for NASA's Life Sciences Division research and development activities in environmental health. It covers the significant research areas critical to NASA's programmatic requirements for the Extended Duration Orbiter, Space Station Freedom, and exploration mission science activities. These science activities include ground-based and flight; basic, applied, and operational; animal and human subjects; and research and development. This document summarizes the history and current status of the program elements, outlines available knowledge, establishes goals and objectives, identifies scientific priorities, and defines critical questions in the three disciplines: (1) Barophysiology, (2) Toxicology, and (3) Microbiology. This document contains a general plan that will be used by both NASA Headquarters Program Officers and the field centers to review and plan basic, applied, and operational research and development activities, both intramural and extramural, in this area. The document is divided into sections addressing these three disciplines.
The effects of microbial fouling on treatment bed (TB) performance are being studied. Fouling of activated carbon (AC) and ion exchange resins (IEX) by live and devitalized bacteria can cause decreased capacity for selected sorbates with AC and IEX TB. More data are needed on organic species removal in the trace region of solute sorption isotherms. TB colonization was prevented by nonclassical chemical disinfectant compositions (quaternary ammonium resins) applied in suitable configurations. Recently, the protection of carbon beds via direct disinfectant impregnation has shown promise. Effects (of impregnation) upon bed sorption/removal characteristics are to be studied with representative contaminants. The potential need to remove solutes added or produced during water disinfection and/or TB microbiological control must be investigated.
The International Space Station (ISS) now has platforms for conducting research on horticultural plant species, and those capabilities continue to grow. The Veggie vegetable production system will be deployed to the ISS in Spring of 2014 to act as an applied research platform with goals of studying food production in space, providing the crew with a source of fresh food, allowing behavioral health and plant microbiology experimentation, and being a source of recreation and enjoyment for the crew. Veggie was conceived, designed, and constructed by Orbital Technologies Corporation (ORBITEC, Madison, WI). Veggie is the largest plant growth chamber that NASA has flown to date, and is capable of growing a wide array of horticultural crops. It was designed for low energy usage, low launch mass and stowage volume, and minimal crew time requirements. The Veggie flight hardware consists of a light cap containing red (630 nanometers), blue, (455 nanometers) and green (530 nanometers) light emitting diodes. Interfacing with the light cap is an extendable bellows baseplate secured to the light cap via magnetic closures and stabilized with extensible flexible arms. The baseplate contains vents allowing air from the ISS cabin to be pulled through the plant growth area by a fan in the light cap. The baseplate holds a Veggie root mat reservoir that will supply water to plant pillows attached via elastic cords. Plant pillows are packages of growth media and seeds that will be sent to ISS dry and installed and hydrated on orbit. Pillows can be constructed in various sizes for different plant types. Watering will be via passive wicking from the root mat to the pillows. Science procedures will include photography or videography, plant thinning, pollination, harvesting, microbial sampling, water sampling, etcetera. Veggie is one of the ISS flight options currently available for research investigations on plants. The Plant Habitat (PH) is being designed and constructed through a NASA-ORBITEC collaboration, and is scheduled to fly on ISS around 2016. This large plant chamber will control light quality, level, and timing, temperature, CO2, relative humidity, and irrigation, while scrubbing ethylene. Additional monitoring capabilities include leaf temperature sensing and root zone moisture and oxygen sensing. The PH light cap will have red (630 nanometers), blue (450 nanometers), green (525 nanometers), far red (730 nanometers) and broad spectrum white light emitting diodes. There will be several internal cameras to monitor and record plant growth and operations.
Mobile sampling cart automatically swabs surfaces for the recovery of microorganisms. Unit operates without human involvement and provides for control of swabbing speed, rotation of cotton swab, and the pressure and angle applied to swab. Capability of reverse direction is also available. Sampling cart use is limited to flat surfaces.
Returning samples from Mars has long been a major planetary science objective due to the high scientific value and transformative potential of the resulting data. An exciting dimension of this objective is the potential for the detection of ancient microbiological life, and the possibility of improving our understanding of the evolution of habitable environments on Mars and the development of life on Earth. To ensure that returned samples meet stringent planetary protection requirements and do not expose Earth to potential biohazards, the joint NASA/ESA Sample Receiving Project (SRP) assembled the Sample Safety Assessment Protocol Tiger Team (SSAP-TT). Members were recruited with the specific goal of creating a multi-disciplinary and internationally distributed team of experts in their respective fields across the federal government, academia, and private industry. This team was chartered with reassessing previous sample safety assessment strategies, defining what constitutes a biological hazard, developing a protocol to test for potential biohazards, and establishing a statistical framework to determine if samples are “safe” for release. The team developed a three-step protocol, supported by a Bayesian statistical framework, to assess whether returned samples contain potential biohazards that could present a risk to Earth’s biosphere. Initial conclusions indicated that an effective and comprehensive safety assessment protocol is feasible using modern techniques and does not require an excessive amount of sample consumption or traditional microbiological detection methodology. Herein, we will present an overview of the MSR SRP, the proposed safety assessment protocol, and how aspects of this novel approach can be applied to biological assessment in healthcare product manufacturing practices.
"Sterilization" is a term applied to any process or procedure that produces a condition of sterility. Sterility is an absolute concept and refers to the destruction or removal of all life. However, when sterility as an absolute concept is applied to the sterilization of space hardware, it ceases to be operational. Absolute proof of sterility of flight hardware is impossible. Hence, for an operational approach to certification of sterility of space hardware, it is necessary to apply the probability concept. The present report is concerned with the applicability of mathematics to predict the probability of achieving sterility in a complex space hardware system. As such, this approach is concerned with the manipulation of microbiological data rather than mathematical rigor.
Wolf Trap device determining growth of microorganisms by measuring intensity of light scattered by organisms
All organisms require energy. Characterizing and quantifying the biological demand for energy places constraints on the possible interactions of organisms with each other and with the environment. This talk will consider energetic and mass transfer constraints on the ecology of hydrothermal vent microbes. Following a general introduction to the biological energy requirements and their link to environmental conditions, energy constraints will be applied to several vent-relevant case studies.
Through KSC IRTD funding in 2022, this project brought a list of 18, 3D printed filaments into formal characterization testing to provide a reference for their behaviors under relevant applications. The project format set up a series of tests to expose 3D printed specimens. A total of 1,989 individual 3D printed test specimens were sent across KSC to be scrutinized by three laboratories to fulfill a multidisciplinary assessment of each material TRL. Testing started with 18 materials. Initially, seed germination assays in the PPA, sample materials were enclosed in petri dishes with lettuce seeds on damp germination paper. No significant impacts on lettuce seed germination were observed in this testing. Next, sample coupons were printed and sent for materials testing to the KSC Analysis/Mechanical and Environmental Testing Laboratory, where they were subjected to 14- and 30-day soak periods in solutions used to provide nutrients to plants or to sanitize hardware before and after use. Following a long soak typical of a 30-day plant growout in Hoagland’s solution, 14 materials gained more than 10% of their own mass. This indicated an increased potential for leaching or providing conditions that are not food safe. Materials that exceeded 15% absorption by mass were eliminated from further testing. Based off this result, the team continued with a core list of nine filaments to fulfill Tensile, Flexural, Biofilm formation, and plant growth testing. Those materials were PLA (Raise3D), ABS (Raise3D), PETG (PolyethyleneTerephthalate Glycol) (Raise3D), ASA (Acrylonitrile Styrene Acrylate) (Raise3D), PC (Polycarbonate) (Raise3D), TPU (Thermoplastic polyurethane)-95 (Raise3D), PLA Copper (Gizmodorks), PP (Polypropylene) (Braskem), and HIPS (High Impact Polystyrene) (Gizmodorks). Testing also quantified the spectral impact of using different color 3D printed surfaces in a growth chamber. The material used for spectral testing was PLA. Printing employed a standard surface texture representative of all materials. It was shown through Tensile Testing (ASTM D638-22) that the breaking force of a 3D printed part greatly varied depending on layer orientation. This is common through all materials, and demonstrates that the strength of a 3D printed component can be maximized by layering the material normal to the primary force on the part. Four-point flexural testing (ASTM D790) provided quantities of interest, Flexural modulus, Flexural strength, Flexural stress, and strain at break within a 5% strain limit from each of nine materials. Biofilm formation testing was conducted in the Molecular and Microbiological Laboratory. Testing completed on specimens from each material showed equal formation on the surface. Additional plant growth testing was conducted in the PPA beyond the initial germination testing. The final assessment documents that three materials (PLA, ABS, and PC) have reached TRL 6 through extensive testing, and ultimate end-to-end applied use in experimental or testing conditions (flight and ground). TRL 5 materials (ASA, TPU-95, PLA Copper, PP, PETG, and HIPS) have all been successfully applied in Research and Development for crop growth applications and are ready to be applied in formal testing. TRL 4 materials Nylon910, PLA Carbon Fiber, PPA CF, PPA Glass Fiber (GF), NinjaFlex, and P-filament 721 are materials that were able to be printed and tested, but have yet to show data meeting applied requirements. TRL 3 NylonX, Flex TPE-185, and Nylon were unable to be reliably printed to fulfill testing. These results provide researchers with reference for materials to use during plant growth experimentation, and also set a standard for future characterization work applying 3D printing and materials to testing, research, and experimentation.
The technical development of a computer program for predicting microbial burden on unmanned planetary spacecraft is outlined. The discussion includes the derivation of the basic analytical equations, the selection of a method for handling several random variables, the macrologic of the computer programs and the validation and verification of the model. The prediction model was developed to (1) supplement the biological assays of a spacecraft by simulating the microbial accretion during periods when assays are not taken; (2) minimize the necessity for a large number of microbiological assays; and (3) predict the microbial loading on a lander immediately prior to sterilization and other non-lander equipment prior to launch. It is shown that these purposes not only were achieved but also that the prediction results compare favorably to the estimates derived from the direct assays. The computer program can be applied not only as a prediction instrument but also as a management and control tool. The basic logic of the model is shown to have possible applicability to other sequential flow processes, such as food processing.
The risk of infectious disease to select individuals has historically been difficult to predict in either spaceflight or on Earth with health care efforts relying on broad-based prevention and post-infection treatment. Over the past 10 years, quantitative microbial risk assessment evaluations have evolved to formalize the assessment process and quantify the risk. This process of hazard identification, exposure assessment, dose-response assessment, and risk characterization has been applied by the water and food safety industries to address the public health impacts associated with the occurrence of and human exposure to pathogens in water and food for the development of preventive strategies for microbial disease. NASA is currently investigating the feasibility of using these techniques to better understand the risks to astronauts and refine their microbiological requirements. To assess these techniques, NASA began an evaluation of the potable water system on the International Space Station to determine how the microbial risk from water consumption during flight differed from terrestrial sources, such as municipal water systems. The ultimate goal of this work is to optimize microbial requirements which would minimize unnecessary cargo and use of crew time, while still protecting the health of the crew. Successful demonstration of this risk assessment framework with the water system holds the potential to maximize the use of available resources during spaceflight missions and facilitate investigations into the evaluation of other routes of infection, such as through the spaceflight foods system.
Introduction : The goal of planetary protection at NASA is to mitigate the risk of contaminating sensitive target bodies with biological life. While many cleaning procedures have been put in place to reduce bioburden on spacecraft, microbes are experts at evolving to survive harsh conditions. Specifically, the dry, low-nutrient environment of a cleanroom (commonly used for assembly of spacecraft) can represent an environment where extremophiles can survive. Methods : Scientists at NASA MSFC wished to gather a snapshot of the microbial population within a variety of cleanrooms on site. A study was undertaken to collect air, surface, and floor samples from clean-rooms and isolate unique morphologies. From this study, 95 isolates were collected and saved in a microbial library. About 86% of these were identified at least to a genus level. Following identification, 24 microbes were selected, based on a literature review, as potential extremophiles. These were grown in liquid cultures, diluted to a set optical density, washed with water, and then applied to a sterilized Kapton coupon. Droplets were allowed to dry overnight in a biosafety cabinet. Coupons were then installed in a pelletron and pumped down to high vacuum (~1E-6 Torr). Samples were then subjected 100 keV protons at a fluence of 2x10 15 p+/cm 2 up to 4x10 15 p+/cm 2 . Following exposure, samples were returned to the microbiology lab where they were pro-cessed by submerging in water, vortexing, and then plating either droplets or spread plates. Recovery data collected was qualitative with a ranking or +, minor, or – for growth. Some selected radiotolerant strains were sequenced using the Illumina sequencing platform. The resulting genomes were annotated with the Rapid Annotations using Subsystems Technology (RAST) server and analyzed for conserved and unique stress response relevant genomic signatures to identify clues related to specific tolerances. Results and Discussion : After five rounds of proton radiation, we narrowed our isolates to five, non-spore forming bacteria that demonstrated survival: Arthrobacter koreensis, Paenarthrobacter nitroguajacolicus, Mycetocola manganoxydans , and an Erwinia sp. Furthermore, we exposed these four microbes to 254 nm wavelength light at an intensity of 80 W/m 2 at a distance of ~18 cm for 10 minutes. Only A. koreensis demonstrated survival following UV exposure. Finally, we performed whole genome sequencing on the four strains to look for genetic markers of stress resistance. When we compared the genomes of the four strains, we found that genes coding for GGDEF and EAL domains with PAS/PAC sensors were only found in A. koreensis . These domains, modulated by PAS/PAC sensors, are hypothesized to facilitate survival under drying, desiccation, and proton irradiation. Drying and Desiccation : PAS domains sense hydration changes and modulate GGDEF and EAL domain activity to adjust c-di-GMP levels, enhancing resistance to desiccation. For instance, in Pseudomonas aeruginosa , the PAS domain of RbdA modulates activity under varying hydration conditions, affecting stress responses [1]. Proton Irradiation : Proton irradiation causes oxidative stress, leading to ROS generation. PAS domains detect this stress and modulate GGDEF and EAL domains to manage oxidative stress responses. In Shewanella , EAL domain proteins modulated by PAS sensors help bacteria adapt to extreme conditions [2]. These genes upregulate other stress response genes, protecting membrane function, protein stability, DNA repair, and antioxidant defenses. The modulation of c-di-GMP by PAS domains is crucial for bacterial adaptation to stress conditions, enabling dynamic physio-logical adjustments [3]. Understanding these mechanisms provides insights into bacterial stress responses and strategies for controlling bacterial growth [4]. Conclusions : These findings indicate that clean-rooms harbor extremophile microbes that may be able to survive conditions in deep space. Furthermore, while we identified certain stress-response genes that may be at least partly responsible for the phenotypes observed in this study, there are likely unidentified genes or characteristics about A. koreensis , and other bacteria, that may allow them to survive in harsh environments. Future studies will focus on identifying these unknown genes and characteristics, further elucidating the mechanisms of extremophile survival and potentially informing the development of new biotechnologies for space exploration and other extreme environments.