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The effects of planetary quarantine constraints are assessed for advanced missions and unmanned planetary sample return missions. Considered are natural space environment factors, post launch recontamination effects, spacecraft microbial burden estimation and prediction, and spacecraft cleaning and decontamination techniques.
An overview is provided of the biogeochemical research. The funding, productivity, personnel and facilities are reviewed. Some of the technical areas covered are: carbon isotopic records; isotopic studies of banded iron formations; isotope effects in microbial systems; studies of organic compounds in ancient sediments; and development in isotopic geochemistry and analysis.
Gravity is a well-known, but little understood, physical force. It interacts with other physical environmental factors to impact the formation of today’s Earth, and to contribute to biological variations between water and land species. It is also involved with cell differentiation process, and is responsible for setting the boundaries for life and keeping cells small. With their simple structures and small sizes, microbes are expected to be less gravity sensitive than larger species. Nevertheless, various effects of spaceflight on bacterial growth have been reported, but the reports have been sometime inconsistent, and the underlying mechanism remains unclear to many. This paper summarizes the systematic efforts to evaluate the spaceflight effect on microbial growth, highlighting the extracellular mass transfer mechanism. It also provides a critical literature review on the topic to resolve the discrepancy in experimental observations and to further clarify the underlying mechanisms.
Microbiological background information is compiled in handbook for engineers and scientists working on bio-related projects. It is intended as aid in - /1/ evaluating effects of engineering procedures on microbial life, /2/ determining effects of decontamination and sterilization on performance of overall systems, and /3/ understanding language of microbiologists.
In situ chemical oxidation has the potential for degrading large quantities of organic contaminants and can be more effective and timely than traditional ex situ treatment methods. However, there is a need to better characterize the potential effects of this treatment on natural processes. This study focuses on potential inhibition to anaerobic dechlorination of trichloroethene (TCE) in soils from a large manufacturing facility as a result of in situ oxidation using potassium permanganate (KMn04)Previous microcosm studies established that natural attenuation occurs on-site and that it is enhanced by the addition of ethanol to the system. A potential remediation scheme for the site involves the use of potassium permanganate to reduce levels of TCE in heavily contaminated areas, then to inject ethanol into the system to "neutralize" excess oxidant and enhance microbial degradation. However, it is currently unknown whether the exposure of indigenous microbial populations to potassium permanganate may adversely affect biological reductive dechlorination by these microorganisms. Consequently, additional microcosm studies were conducted to evaluate this remediation scheme and assess the effect of potassium permanganate addition on biological reductive dechlorination of TCE. Samples of subsurface soil and groundwater were collected from a TCE-impacted area of the site. A portion of the soil was pretreated with nutrients and ethanol to stimulate microbial activity, while the remainder of the soil was left unamended. Soil/groundwater microcosms were prepared in sealed vials using the nutrient-amended and unamended soils, and the effects of potassium permanganate addition were evaluated using two permanganate concentrations (0.8 and 2.4 percent) and two contact times (1 and 3 weeks). TCE was then re-added to each microcosm and TCE and dichloroethene (DCE) concentrations were monitored to determine the degree to which microbial dechlorination occurred following chemical oxidation. Evidence of microbial degradation was generally detected within four weeks after TCE addition. Increases in DCE concentrations were consistent with decreases in TCE. The concentration of TCE in the nutrient-amended samples exposed to 2.4% KMnO4 for one week degraded somewhat more slowly than the samples exposed to the 0.8% KMnO4. The rates of degradation did not correlate with the length of KMn04 exposure for the nutrient-amended microcosms. Microbial degradation of TCE in the unamended microcosms was generally similar to that observed in the nutrient-amended microcosms. One treatment condition (unamended, one week exposure, 2.4% KMnO4) was exposed to elevated levels of ethanol and showed little evidence of degradation. It is suspected that the high levels of ethanol were toxic to the microorganisms. The results of the study indicate that exposure of indigenous soil and groundwater microbial populations to KMnO4 at concentrations of 0.8 to 2.4% do not impair the ability of the microbial populations to dechlorinate TCE. Consequently, the combination of chemical oxidation followed by enhanced biological reductive dechlorination appears to be a viable remedial strategy for highly-impacted subsurface areas of the site.
Reference cultures of 16 microorganisms obtained from the American Type Culture Collection and four clinical isolates were used in standardized solutions to inoculate 60 cards for each test strain. A set of three ID and three susceptibility cards was processed in the Vitek AutoMicrobic System (AMS) immediately after inoculation. The remaining cards were refrigerated at 4 C, and sets of six cards were removed and processed periodically for up to 17 days. The preinoculated AMS cards were evaluated for microorganism identification, percent probability of correct identification, length of time required for final result, individual substrate reactions, and antibiotic minimal inhibitory/concentration (MIC) values. Results indicate that 11 of the 20 microbes tested withstood refrigerated storage up to 17 days without detectable changes in delineating characteristics. MIC results appear variable, but certain antibiotics proved to be more stable than others. The results of these exploratory studies will be used to plan a microgravity experiment designed to study the effect of microgravity on microbial physiology and antibiotic sensitivity.
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Microorganisms grow differently in spaceflight than they do on Earth. While numerous factors likely contribute, one ubiquitous feature of the space environment is altered gravity - but because microbes are generally considered too small to detect gravity directly, they likely experience the effects of gravity via changes in their fluid environment, primarily a reduction in convective mixing. This theory is supported by evidence that individual microbial strains experience starvation and acid stress in microgravity. If it is true, then microgravity would also have measurable effects on microbes in mixed communities, because many interspecies interactions involve the exchange of soluble metabolites through their medium (cross-feeding). Specifically, cooperative cross-feeding communities would grow more slowly in microgravity, and this effect should be reproducible in ground-based rotational culture systems (rotating wall vessels, RWVs) designed to simulate the quiescent environment of microgravity. Here we present preliminary results of our efforts to test this hypothesis using using a model system of Eschericia coli and Salmonella enterica that grow only when they can exchange methionine and acetate through the medium. We report on the development of a novel laboratory system that allows us measure growth rates and species ratios in real-time using fluorescence spectrometry as the organisms grow in RWVs. We are able to track growth rates of individual species and the co-culture continuously with high temporal resolution for several hours, a substantial improvement over prior RWV systems that require destructive sampling. We vary the fidelity of the microgravity simulation by varying the rotation rate, and find that rotation rate affects growth rates of the community more than that of individual strains, consistent with the claim that RWVs can simulate a microgravity-like quiescent environment. While more work needs to be done to probe the mechanisms of this effect, we hope that our system will ultimately allow us to generate quantitative predictions about the effects of spaceflight on organisms that are tiny but essential to sustaining long-term human space exploration.
A series of experiments at several levels of relative humidity and radiation dose rates was carried out using spores of Bacillus subtilis var. niger to evaluate the effect of heat alone, radiation alone, and a combination of heat and radiation. Combined heat and radiation treatment of microorganisms yields a destruction rate greater than the additive rates of the independent agents. The synergistic mechanism shows a proportional dependency on radiation dose rate, an Arrhenius dependence on temperature, and a dependency on relative humidity. Maximum synergism occurs under conditions where heat and radiation individually destroy microorganisms at approximately equal rates. Larger synergistic advantage is possible at low relative humidities rather than at high relative humidities.
Systems for the growth of crops in closed environments are being developed and tested for potential use in space applications to provide a source of fresh food. Plant growth conditions, growth media composition and harvest methods can have an effect on the microbial population of the plant, and therefore should be considered along with the optimization of plant growth and harvest yields to ensure a safe and palatable food crop. This work examines the effect of plant cultivar, growth media, and harvest method on plant microbial populations. Twelve varieties of leafy greens and herbs were grown on a mixture of Fafard #2 and Arcillite in the pillow root containment system currently being considered for the VEGGIE plant growth unit developed by Orbitec. In addition, ,Sierra and Outredgeous lettuce varieties were grown in three different mixtures (Fafard #2, Ardllite, and Perlite/Vermiculite). The plants were analyzed for microbial density. Two harvest methods, "cut and come again" (CACA) and terminal harvest were also compared. In one set ofexpe'riments red leaf lettuce and mizuna were grown in pots in a Biomass Production System for education. Plants were harvested every two weeks by either method. Another set of experiments was performed using the rooting pillows to grow 5 varieties of leafy greens and cut harvesting at different intervals. Radishes were harvested and replanted at two-week intervals. Results indicate up to a 3 IOglO difference in microbial counts between some varieties of plants. Rooting medium resulted in an approximately 2 IOglO lower count in the lettuce grown in arscillite then those grown in the other mixtures. Harvest method and frequency had less impact on microbial counts only showing a significant increase in one variety of plant. Post harvest methods to decrease the bacterial counts on edible crops were investigated in these and other experiments. The effectiveness of PRO-SAN and UV-C radiation is compared.
To determine how dilution of microbial communities affects the diversity of the diluted assemblage a series of numerical simulations were conducted that determined the theoretical change in diversity, richness, and evenness of the community with serial dilution. The results of the simulation suggested that the effects are non linear with a high degree of dependence on the initial evenness of the community being diluted. A series of incubation experiments using a range of dilutions of raw sewage as an inoculum into sterile sewage was used for comparison to the simulations. The diluted communities were maintained in batch fed reactors (three day retention time) for nine days. The communities were harvested and examined by conventional plating and by molecular analysis of the whole-community DNA using AFLP and T-RFLP. Additional, CLPP analysis was also applied. The effects on richness predicted by the numerical simulations were confirmed by the analyses used. The diluted communities fell into three groups, a low dilution, intermediate dilution, and high dilution group, which corresponded well with the groupings obtained for community richness in simulation. The grouping demonstrated the non-linear nature of dilution of whole communities. Furthermore, the results implied that the undiluted community consisted of a few dominant types accompanied by a number of rare (low abundance) types as is typical in unevenly distributed communities.
The focus of resource recovery research at the KSC-CELSS Breadboard Project has been the evaluation of microbiologically mediated biodegradation of crop residues by manipulation of bioreactor process and environmental variables. We will present results from over 3 years of studies that used laboratory- and breadboard-scale (8 and 120 L working volumes, respectively) aerobic, fed-batch, continuous stirred tank reactors (CSTR) for recovery of carbon and minerals from breadboard grown wheat and white potato residues. The paper will focus on the effects of a key process variable, bioreactor retention time, on response variables indicative of bioreactor performance. The goal is to determine the shortest retention time that is feasible for processing CELSS crop residues, thereby reducing bioreactor volume and weight requirements. Pushing the lower limits of bioreactor retention times will provide useful data for engineers who need to compare biological and physicochemical components. Bioreactor retention times were manipulated to range between 0.25 and 48 days. Results indicate that increases in retention time lead to a 4-fold increase in crop residue biodegradation, as measured by both dry weight losses and CO2 production. A similar overall trend was also observed for crop residue fiber (cellulose and hemicellulose), with a noticeable jump in cellulose degradation between the 5.3 day and 10.7 day retention times. Water-soluble organic compounds (measured as soluble TOC) were appreciably reduced by more than 4-fold at all retention times tested. Results from a study of even shorter retention times (down to 0.25 days), in progress, will also be presented.
Algae, including blue-green algae (cyanobacteria), are the major source of fixed carbon in many aquatic ecosystems. Previous work has shown that photosynthetic carbon fixation is often enhanced in the presence of additional carbon dioxide (CO2). This study was undertaken to determine if this CO2 fertilization effect extended to microbial mats, and, if so, at what times during the day might the addition of CO2 affect carbon fixation. Four microbial mats from diverse environments were selected, including mats from a hypersaline pond (area 5, Exportadora de Sal, Mexico), the marine intertidal (Lyngbya, Laguna Ojo de Liebre, Mexico), an acidic hotspring (Cyanidium, Nymph Creek, Yellowstone National Park), and an acidic stream at ambient temperature (Zygogonium, Yellowstone National Park). Carbon fixation in the absence of additional CO2 essentially followed the rising and falling sunlight levels, except that during the middle of the day there was a short dip in carbon fixation rates. The addition of CO2 profoundly enhanced carbon fixation rates during the daylight hours, including during the midday dip. Therefore, it is unlikely that the midday dip was due to photoinhibition. Surprisingly, enhancement of carbon fixation was often greatest in the early morning or late afternoon, times when carbon fixation would be most likely to be light limited.
The effect of several environmental parameters on previously developed life detection systems is explored. Initial attempts were made to conduct all the experiments in a moist mode (high soil volume to water volume ratio). However, only labeled release and measurement of ATP were found to be feasible under conditions of low moisture. Therefore, these two life detection experiments were used for most of the environmental effects studies. Three soils, Mojave (California desert), Wyaconda (Maryland, sandy loam) and Victoria Valley (Antarctic desert) were generally used throughout. The environmental conditions studied included: incubation temperature 3 C to 80 C, ultraviolet irradiation of soils, variations in soil/liquid ratio, specific atmospheric gases, various antimetabolites, specific substrates, and variation in pH. An experiment designed to monitor nitrogen metabolism was also investigated.
One prokaryote, Methylococcus capsulatus, synthesizes both hopanoids and sterols and, thus, provides a unique opportunity to study the evolution of membrane function. When M. capsulatus was grown at different temperatures, lipid analysis of the whole cells showed that both sterol and unsaturated fatty acid levels decreased at higher growth temperatures; sterol concentrations were 0.116 micro mole/micro mole phospholipid at 30 C and 0.025 micro mole/mirco mole phospholipid at 45 C, while the saturated to unsaturated fatty acid ratio increased from 0.397 to 1.475. Hopane polyol levels were constant over this range; however, methylation of the A-ring decreased markedly in cells grown at 30 C. These results imply that sterol and hopane molecules are required for enhancement of some specific membrane function, potentially by modulating membrane fluidity.
Microbial mats were widespread during the first ca. 2 Ga. of our biosphere's history. To better understand microbial ecosystems and their biomarkers under the low sulfate levels present in early oceans, we attempted a long-term (ca. 1 year) manipulation of sulfate in modem mats. Mats collected from salt ponds at Guerrero Negro, Baja Calif. Sur were incubated in a Greenhouse "Collaboratory" at Ames. Mats were maintained in artificial seawater brine containing either: 1) sulfate levels normal for these mats (70 mM), or 2) brine in which sulfate was replaced by chloride. Sulfate concentrations in the "low sulfate" brine gradually approached their lowest (to date) value of 0. 1 mM as sulfate was consumed and/or diffused out of the mat over a period of ca. 4 months. During that period of time, a number of differences between the treatments emerged. Relative to the "low sulfate" mats, "normal sulfate" mats had: 1) lower consumption of oxygen in the lower levels of the mat, 2) higher efficiencies of oxygenic photosynthesis, and 3) higher rates of nitrogen fixation. Rates of methane production by the mats increased greatly as sulfate concentrations fell below ca. 0.2 mM. In contrast, "low" and "normal" sulfate mats had similar net rates of exchange of O2 and dissolved inorganic C between the mats and overlying water. Reduced sulfate levels have diverse impacts upon these ecosystems.
Introduction: Microorganisms are everywhere on Earth, even in the cleanest of places. Spacecraft assembly cleanrooms can harbor low levels of living and dead microbial cells (e.g., [1,2]), and cleanroom bioburden can also include organic molecules from industrial sources and in situ biomass. Life detection missions require careful attention to avoid contaminants that can be easily convoluted with analytical targets. We are evaluating epifluorescent microscopy and fluorescence in situ hybridization (FISH) as methods to complement organic contamination detection techniques. Epifluorescent cell counting offers an accurate and cost-effective way to quantify low levels of surface biomass. FISH could allow for the identification of residual organisms, and can be targeted to detect active populations of specific organisms such as bacteria known to resist cleaning procedures. This effort is part of a larger study that is concentrated on characterizing the surface and airborne molecular organic contamination background in Johnson Space Center (JSC) Astromaterials curation laboratories and Goddard Space Flight Center (GSFC) spacecraft assembly rooms, and understanding contaminants in the context of cleaning procedures and residual bioburden. Methods: Samples were collected by swabbing surfaces in ISO 5 and ISO 7 equivalent cleanrooms at JSC. Swabs for FISH were fixed in 4% paraformaldehyde (PFA) for 3 hours and then stored in 1:1 ethanol:PBS, while swabs for cell counting were stored in 4% PFA until analysis to avoid any cell loss during centrifugation that could impact quantification of very low biomass samples. Cell counting was performed with SYBR Gold as in [3], but adapted for very low biomass. FISH was performed as in [4], using DAPI as a counterstain for all DNA-containing cells. Negative controls included wells with no probe applied, to test for natural fluorescence, as well as the nonsense probe NONEUB (reverse complement of EUB338) to evaluate non-specific probe binding. Results and Discussion: Cleanroom surfaces had 102-103 cells cm-2. The extremely low biomass of these samples was challenging for enumeration, and required careful and routine use of “field” and laboratory blanks. FISH was performed with the general archaeal and bacterial probes ARCH915 and EUB338 (EUBMIX, [4]), probe GAMBET ([4]), and PSE227, which targets the genus Pseudomonas [5]). The latter two probes were selected because Pseudomonas spp. and other Gammaproteobacteria have not been isolated from cleanroom surfaces but do appear frequently in rRNA gene libraries from these surfaces. While some active bacteria were identified (Fig. 1c), most cells detectable by DAPI did not have a strong or any fluorescent signal (e.g., Fig. 1d), indicating that the vast majority of cells are dead or inactive. This suggests that cleaning protocols are effective at inactivating microbial contaminants, but that dead or inactive cells can remain on surfaces. Cells were often clumped in a weakly autofluorescent matrix, possibly biofilm material (Fig. 1c,d). We also observed other particulate material that was collected by the swabs, including apparent textile fibers (Fig. 1b). Our results are consistent with other studies that show that the bioburden present in clean rooms includes active, dormant, and dead cells. We will discuss how FISH and epifluorescent cell counting could be applied in planetary protection protocols, including the advantages and disadvantages of FISH and cell counting for routine use, as well as different possible applications for more specialized FISH procedures. References: [1] Moissl-Eichinger et al. (2015) Sci Rep, 5, 9156 [2] Hendrickson et al. (2021) Microbiome, 9, 238 [3] Jones et al. (2017) Appl Environ Microbiol, 83, e00909-17 [4] Jones et al. (2015) Appl Environ Microbiol, 81, 1242-1250. [5] Watt et al. (2006) Environ Microbiol, 8, 871-884
Ten purebred Beagle dogs (all males) were used to determine the effect of a prolonged stay in a locked environment (i.e., no exogenous microbial contamination) on the microbial flora. At monthly intervals the microbial profile (feces, nose, and throat) of each dog was assessed. After 12 months it was found there was no drastic alteration or simplification of the microbial profile of isolated or control dogs. Although isolated dogs had slightly higher levels of anaerobic bacteria and somewhat lower levels of enterococci, the major groups of anaerobic, aerobic, and facultative bacteria remained qualitatively and quantitatively similar for the 12-month study period. Although they were only minor components of the fecal flora, Candida albicans and Shigella sonnei were consistently isolated in larger numbers from the dogs in the locked environment.