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Techniques for maintaining spacecraft sterility after addition or replacement of components - microbiological contamination of sterile environments
Sensor systems are under development for use in real-time detection and quantitation of microbes in water without need for sampling. These systems include arrays of optical sensors; miniature, portable electronic data-acquisition circuits; and optoelectronic interfaces between the sensor arrays and data-acquisition circuits. These systems are intended for original use in long-term, inline monitoring of waterborne micro-organisms in water-reclamation systems aboard future spacecraft. They could also be adapted to similar terrestrial uses with respect to municipal water supplies, stored drinking water, and swimming water; for detecting low-level biological contamination in biotechnological, semiconductor, and pharmaceutical process streams; and in verifying the safety of foods and beverages. In addition, they could be adapted to monitoring of airborne microbes and of surfaces (e.g., to detect and/or quantitate biofilms). The designs of the sensors in these systems are based partly on those of sensors developed previously for monitoring airborne biological materials. The designs exploit molecular- recognition and fluorescence-spectroscopy techniques, such that in the presence of micro-organisms of interest, fluorescence signals change and the changes can be measured. These systems are characterized as semiselective because they respond to classes of micro-organisms and can be used to discriminate among the classes. This semiselectivity is a major aspect of the design: It is important to distinguish between (1) the principle of detection and quantitation of classes of micro-organisms by use of these sensors and (2) the principle of detection and quantitation of individual microbiological species by means of prior immuno-diagnostic and/or molecular-biology techniques. Detection of classes (in contradistinction to species) is particularly valuable when the exact nature of a contaminant is unknown.
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.
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.
On Earth, light microscopy is commonly used in microbiology to identify organisms and observe their interactions with the environment; this makes it an attractive technique for in situ life detection methods on ocean worlds. As a standalone technique, brightfield microscopy, while able to provide important contextual information, has limited usefulness as a life detection technique because it is often challenging to differentiate between abiotic and biotic particles based solely on their size and shape, which may introduce risks of false positive or false negative interpretations. However, these risks can be reduced by combining brightfield microscopy with fluorescence microscopy to provide a method that correlate sample chemistry with sample morphology. In this work, we have used the Luminescence Imager for Exploration (LIfE), a brightfield and epifluorescence microscope with an integrated sample processing system (matured under the Concepts for Ocean worlds Life Detection Technology and Instrument Concepts of Europa Exploration programs) to develop methods that increase the fidelity of in situ microscopy life detection measurements through two main approaches. First, native fluorescence is excited in molecules that contain aromatic moieties such as proteins (using deep UV excitation), and energy carrying molecules and endogenous chromophores (using visible-light excitation), to correlate the location of these species with cell-like structural features (brightfield imaging). Second, fluorescent stains are used to selectively image cells and cell fragments by targeting proteins, lipids, and nucleic acids. We discuss the results of tests, obtained using ocean world analog samples, that have examined trades associated with implementing these methods autonomously in planetary missions, including the intrinsic properties of candidate fluorescence dyes and long-term storage and radiation stability.
On Earth, light microscopy is commonly used in microbiology to identify organisms and observe their interactions with the environment; this makes it an attractive technique for in situ life detection methods on ocean worlds. As a standalone technique, brightfield microscopy, while able to provide important contextual information, has limited usefulness as a life detection technique because it is often challenging to differentiate between abiotic and biotic particles based solely on their size and shape, which may introduce risks of false positive or false negative interpretations. However, these risks can be reduced by combining brightfield microscopy with fluorescence microscopy to provide a method that correlate sample chemistry with sample morphology. In this work, we have used the Luminescence Imager for Exploration (LIfE), a brightfield and epifluorescence microscope with an integrated sample processing system (matured under the Concepts for Ocean worlds Life Detection Technology and Instrument Concepts of Europa Exploration programs) to develop methods that increase the fidelity of in situ microscopy life detection measurements through two main approaches. First, native fluorescence is excited in molecules that contain aromatic moieties such as proteins (using deep UV excitation), and energy carrying molecules and endogenous chromophores (using visible-light excitation), to correlate the location of these species with cell-like structural features (brightfield imaging). Second, fluorescent stains are used to selectively image cells and cell fragments by targeting proteins, lipids, and nucleic acids. We discuss the results of tests, obtained using ocean world analog samples, that have examined trades associated with implementing these methods autonomously in planetary missions, including the intrinsic properties of candidate fluorescence dyes and long-term storage and radiation stability.
We have designed, built, and tested a sampling kit (Fig. 1) to aseptically collect microbiological samples from exterior surfaces on the ISS (International Space Station). The kit was flown to ISS as part of the NG-19 commercial cargo mission in August of 2023. Astronauts will use the kit to collect samples from six exterior surfaces on ISS. These samples will be frozen at -80°C after collection and returned to the ground for next generation DNA sequencing. The results of this experiment will help inform planetary protection requirements for crewed missions. We hypothesize that there are detectable microbial communities outside ISS and that these communities originate from inside ISS. Current life support systems do not include any components for reducing microbial leakage. Gases are vented from inside ISS without filtration and there are currently no protocols in place to minimize bioburden on space suit exteriors prior to use. It is important to quantify the bioburden on current vehicles so that achievable limits can be set for crewed missions to astrobiologically relevant locations like Mars.
Permafrost microbial research has flourished in the past decades, due in part to improvements in sampling and molecular techniques, but also the increased focus on the permafrost greenhouse gas feedback to climate change and other ecological processes in high latitude and alpine permafrost soils. Permafrost microorganisms are adapted to these extreme environments and remain active at low temperatures and when resources are limited. They are also an important component of global elemental cycles as they regulate organic matter turnover and greenhouse gas production, particularly as permafrost thaws. Here we review the permafrost microbiology literature coupled with an exploration of its historical aspects, with a particular focus on a new understanding advanced by molecular biology techniques. We further identify knowledge gaps and ways forward to improve our understanding of microbial contributions to ecosystem biogeochemistry of permafrost-affected systems.
Throughout the history of manned space flight the supply of potable water to the astronauts has presented unique problems. Of particular concern has been the microbiological quality of the potable water. This has required the development of both preflight water system servicing procedures to disinfect the systems and inflight disinfectant addition and monitoring devices to ensure continuing microbiological control. The disinfectants successfully used to date have been aqueous chlorine or iodine. Because of special system limitations the use of iodine has been the most successful for inflight use and promises to be the agent most likely to be used in the future. Future spacecraft potable, hygiene, and experiment water systems will utilize recycled water. This will present special problems for water quality control. NASA is currently conducting research and development to solve these problems.
Some of the techniques and methodology that were used on Viking to implement planetary quarantine requirements are reported. Special attention was given to techniques and approaches used to implement sterilization of the Viking probe. Quarantine procedures for unmanned planetary missions and procedures for microbiological contamination of space hardware are included. A probability of contamination of the biological instruments onboard by terrestrial organisms was examined.
Nucleic acid sequencing as a technique to determine the chemical and biological evolution of certain prokaryotic metabolic pathways is discussed. Protein in data and a microbiological organization of the prokaryotes is included.
Searching for life in astromaterials to be delivered from the future missions to extraterrestrial bodies is undoubtedly related to studies of the properties and signatures of living microbial cells and microfossils on Earth. As model terrestrial analogs of Martian polar subsurface layers are often regarded the Antarctic glacier and Earth permafrost habitats where alive microbial cells preserved viability for millennia years due to entering the anabiotic state. For the future findings of viable microorganisms in samples from extraterrestrial objects, it is important to use a combined methodology that includes classical microbiological methods, plating onto nutrient media, direct epifluorescence and electron microscopy examinations, detection of the elemental composition of cells, radiolabeling techniques, PCR and FISH methods. Of great importance is to ensure authenticity of microorganisms (if any in studied samples) and to standardize the protocols used to minimize a risk of external contamination. Although the convincing evidence of extraterrestrial microbial life will may come from the discovery of living cells in astromaterials, biomorphs and microfossils must also be regarded as a target in search of life evidence bearing in mind a scenario that alive microorganisms had not be preserved and underwent mineralization. Under the laboratory conditions, processes that accompanied fossilization of cyanobacteria were reconstructed, and artificially produced cyanobacterial stromatolites resembles by their morphological properties those found in natural Earth habitats. Regarding the vital importance of distinguishing between biogenic and abiogenic signatures and between living and fossil microorganisms in analyzed samples, it is worthwhile to use some previously developed approaches based on electron microscopy examinations and analysis of elemental composition of biomorphs in situ and comparison with the analogous data obtained for laboratory microbial cultures and fossilized microorganisms. This communication will be focused on the analysis of our experience in working with ancient microorganisms and fossils and discussion of some issues that are crucial for development of the program for future finding of extraterrestrial life and its evidence.
A description of the design and function of the Apollo potable water system is presented. The command module potable water is supplied as a byproduct of the fuel cells. The cells, located in the service module, function primarily to supply electrical energy to the spacecraft. The source of the lunar module potable water is three tanks, which are filled before lift-off. The technique of supplying the water in each of these cases and the problems associated with materials compatibility are described. The chemical and microbiological quality of the water is reviewed, as are efforts to maintain the water in a microbially safe condition for drinking and food mixing.
OSIRIS-REx is an asteroid sample return mission that delivered asteroid regolith from Bennu to Earth on 9/24/2023 [1]. Cleanroom monitoring of OSIRIS-REx Curation facilities has been ongoing since 2021 when construction of the OSIRIS-REx lab at Johnson Space Center (JSC) was completed [2]. The monitoring continues inside of the OSIRIS-REx lab in Houston. Monitoring was also conducted on a temporary cleanroom constructed inside a hangar at the Utah Test and Training Range (UTTR) to initially receive the Sample Return Capsule (SRC) upon its landing there. The following reports on cleanroom monitoring that has taken place in the months leading up to as well as after OSIRIS-REx return. Monitoring techniques used include particle counts, deployment of Si wafer witness plates (Balazs, Inc.) and Al foil witness plates, gas sampling (Balazs, Inc.) and microbiological monitoring. We collected particle counts at both the lab at JSC as well as the temporary cleanroom at UTTR. Particle counts were taken monthly from six locations in the lab at JSC. Particle counts were taken from seven locations at UTTR in July, August, and September. The UTTR cleanroom particle counts were taken daily in the five days leading to OSIRIS-REx return as well as consistently the day of return (every 2 minutes and 15 seconds 7:21 am- 6:27 pm) from one location inside the cleanroom. The particle counts at UTTR consistently stayed well within the ranges of its required ISO 7 equivalent classification (352,000 maximum particles/ m3 ≥ 0.5 μm). Most analysis showed that the particle counts were far below ISO 7 requirements (highest measured count was 61,484 particles/ m3 ≥ 0.5 um). but the elevated instances correlated to exterior hangar doors being open. Particle counts were reduced when keeping exterior hangar doors closed. The particle counts at JSC have stayed consistently within their ISO 5 equivalent classification (3,520 maximum particles/ m3 ≥ 0.5 um). In most instances, the counts are much better except in one instance where the count measured 13,381 particles/ m3 ≥ 0.5 um. The high spike in particle counts in the JSC lab correlated with the air handlers being shut down temporarily the previous day. The particle counts taken in the same location quickly reduced and measured 0 when taken a month later with the air handlers on. We deployed 8-inch semiconductor polished Si wafer witness plates for 24-hour periods inside of the OSIRIS-REx Curation lab at JSC (in July, August, September, and November 2023) and the cleanroom at UTTR (July and September 2023) to test for possible organic and inorganic contaminants. We also deployed these wafers inside of the OSIRIS-REx Touch-and-Go sample Acquisition Mechanism (TAGSAM) glovebox (where Bennu sample processing occurs) and desiccator (Bennu sample storage) at JSC in August 2023. These samples were all sent to Balazs for analysis via Thermal Desorption Gas Chromatography Mass Spectroscopy (TD-GC-MS) to quantify organic compounds and vapor phase decomposition inductively coupled plasma mass spectrometry (VPD-ICP-MS) to quantify inorganic contaminants. We collected air samples using an adsorbent tube connected to a pump for six hours (100 mL/minute) inside of both the JSC lab and the UTTR cleanroom. These samples were taken at JSC in July, August, September, November, and December and at UTTR in July and September. Additionally, a sample was taken just outside of the TAGSAM glovebox airlock door in July. These samples were sent to Balazs for analysis of volatile organics in air. Overall, the OSIRIS-REx lab at JSC and cleanroom at UTTR yielded very low organic and inorganic contaminants (similar results to Genesis lab at JSC which is ISO 4 equivalent) except for boron, which is attributed to the borosilicate glass in the fan filter units. We collected monthly surface and air samples inside the JSC lab to monitor potential microbial contamination. We report the recovery rate for each sampling event, which is defined as the number of samples exhibiting bacterial or fungal growth divided by the total number of samples collected. Since the lab was commissioned in 2021 the median recovery rate is 29%. This recovery rate is consistent with other ISO 5 equivalent labs used to curate astromaterials collections. Increases in recovery rate correspond to construction and/or extra activity in the lab. However, increases in recovery rate do not reliably correlate to increases in particle counts, which highlights the need for a dedicated microbial monitoring program for biologically sensitive collections. We sampled the temporary clean room at UTTR in July, August, and September of 2023 to monitor potential microbial contamination. The recovery rate decreased from 80% in August to 29% in September. However, in all instances, the diversity of bacteria and fungi was higher in the UTTR cleanroom than in the JSC lab. We routinely collected more than twenty different organisms from the UTTR cleanroom. In the JSC lab the median diversity is 2 organisms. Based on these results we hypothesize that microbes from UTTR could be transported back to JSC with the Bennu samples. To mitigate this risk, in September of 2023, we implemented additional cleaning procedures in the JSC lab to reduce the bioburden on surfaces that could come into direct contact with hardware used to process Bennu samples. Since adopting these additional cleaning measures, the median recovery rate has decreased to 14%. We did not observe an increase in fungal or bacterial diversity in the lab in the October sampling. We will continue to monitor this trend closely for the next several months. Supported by NASA under Award NNH09ZDA007O and Contract NNM10AA11C.
Efforts to identify the ideal candidate to serve as the biological monitor on the space station Freedom are discussed. The literature review, the evaluation scheme, descriptions of candidate monitors, experimental studies, test beds, and culture techniques are discussed. Particular attention is given to descriptions of five candidate monitors or monitoring techniques: laser light scattering, primary fluorescence, secondary fluorescence, the volatile product detector, and the surface acoustic wave detector.
The viability and dry heat resistance of indigenous microflora associated with small soil particles were investigated. An aluminum boat TDT CUP-TSA solid media system was developed for the analyses; a complete description of the technique is included. Data cited here were obtained using analyses of individual soil particles. Detailed particle viability profiles for dry heat effects were determined for Kennedy Space Center soil. At 110 C at least some particles retained viability through a heating period of between 8 and 16 hours. Single particles heated at 125 C for 80 minutes or longer did not show evidence of viability under test conditions. Preliminary aerobic, mesophilic plate counts of the 74-88 micron m soil fraction yielded mean values of 16.2 organisms per dark particle and 2.6 organisms per light particle. Heat treatment of particles in a dry atmosphere did not appear to increase the rate of inactivation for in situ soil particle microflora.
Upon the recommendation of the scientific community, the National Aeronautics and Space Administration has established as a major goal the search for extraterrestrial life. So that the data obtained in this search will not be compromised, NASA has embarked on a program designed to prevent external contamination of the planets. This National Conference on Spacecraft Sterilization Technology was convened to bring together for exchange of knowledge those scientists, engineers, and administrators who play a role in spacecraft sterilization technology. Recognizing the rapid accumulation of knowledge in this area of scientific endeavor, it was deemed essential to broadcast this information as rapidly as possible, to establish a firm base upon which newer techniques could be built, and to provide a forum in which differences could be aired and guidelines revised or established.
The design and operation of a salt-gradient solar pond power plant at the Salton Sea presents problems not encountered at small research ponds that were built in the United States. The specific characteristics of the Salton Sea site and the desire to construct the pond using the local clay as a sealant represent major deviations from previous solar pond experience. The site-specific research in support of the plant design is described. The research activity included validation of the spectrophotometric light transmission measurement technique, a search for options for clarifying the turbid and colored water of the Salton Sea, development of water clarification specifications in terms common to industry practice, quantification of gas production from microbiological reactions in the ground, a determination of the combined effects of temperature and salinity on the permeation of the local clays, and a preliminary evaluation of material corrosion.