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At least 253 records · Page 14

Micron2 Lab: Microfluidic Microbiology Lab Project

Microbial monitoring during spaceflight is crucial to maintain crew health and ensure water purifications systems are functioning properly. Current protocols for in-flight enumeration of bacteria in potable water systems require culture based methods. In this project, we aim to develop a flight- and microgravity-compatible flow cytometer capable of counting total microbial counts in the water supply and differentiating live from dead bacteria.

Burton, Aaron↗

Molecular Microbial Analyses of the Mars Exploration Rovers Assembly Facility

During space exploration, the control of terrestrial microbes associated with robotic space vehicles intended to land on extraterrestrial solar system bodies is necessary to prevent forward contamination and maintain scientific integrity during the search for life. Microorganisms associated with the spacecraft assembly environment can be a source of contamination for the spacecraft. In this study, we have monitored the microbial burden of air samples of the Mars Exploration Rovers' assembly facility at the Kennedy Space Center utilizing complementary diagnostic tools. To estimate the microbial burden and identify potential contaminants in the assembly facility, several microbiological techniques were used including culturing, cloning and sequencing of 16S rRNA genes, DNA microarray analysis, and ATP assays to assess viable microorganisms. Culturing severely underestimated types and amounts of contamination since many of the microbes implicated by molecular analyses were not cultivable. In addition to the cultivation of Agrobacterium, Burkholderia and Bacillus species, the cloning approach retrieved 16s rDNA sequences of oligotrophs, symbionts, and y-proteobacteria members. DNA microarray analysis based on rational probe design and dissociation curves complemented existing molecular techniques and produced a highly parallel, high resolution analysis of contaminating microbial populations. For instance, strong hybridization signals to probes targeting the Bacillus species indicated that members of this species were present in the assembly area samples; however, differences in dissociation curves between perfect-match and air sample sequences showed that these samples harbored nucleotide polymorphisms. Vegetative cells of several isolates were resistant when subjected to treatments of UVC (254 nm) and vapor H202 (4 mg/L). This study further validates the significance of non-cultivable microbes in association with spacecraft assembly facilities, as our analyses have identified several non-cultivable microbes likely to contaminate the surfaces of spacecraft hardware.

microbial diversity MER 16S rDNA DNA microarray sp↗

Genomic and Phenotypic Characterization of Chloracidobacterium Isolates Provides Evidence for Multiple Species

Chloracidobacterium is the first and until now the sole genus in the phylum Acidobacteriota (formerly Acidobacteria ) whose members perform chlorophyll-dependent phototrophy (i.e., chlorophototrophy). An axenic isolate of Chloracidobacterium thermophilum (strain B T ) was previously obtained by using the inferred genome sequence from an enrichment culture and diel metatranscriptomic profiling analyses in situ to direct adjustments to the growth medium and incubation conditions, and thereby a defined growth medium for Chloracidobacterium thermophilum was developed. These advances allowed eight additional strains of Chloracidobacterium spp. to be isolated from microbial mat samples collected from Mushroom Spring, Yellowstone National Park, United States, at temperatures of 41, 52, and 60°C; an axenic strain was also isolated from Rupite hot spring in Bulgaria. All isolates are obligately photoheterotrophic, microaerophilic, non-motile, thermophilic, rod-shaped bacteria. Chloracidobacterium spp. synthesize multiple types of (bacterio-)chlorophylls and have type-1 reaction centers like those of green sulfur bacteria. Light harvesting is accomplished by the bacteriochlorophyll a -binding, Fenna-Matthews-Olson protein and chlorosomes containing bacteriochlorophyll c . Their genomes are approximately 3.7 Mbp in size and comprise two circular chromosomes with sizes of approximately 2.7 Mbp and 1.0 Mbp. Comparative genomic studies and phenotypic properties indicate that the nine isolates represent three species within the genus Chloracidobacterium . In addition to C. thermophilum , the microbial mats at Mushroom Spring contain a second species, tentatively named Chloracidobacterium aggregatum , which grows as aggregates in liquid cultures. The Bulgarian isolate, tentatively named Chloracidobacterium validum , will be proposed as the type species of the genus, Chloracidobacterium. Additionally, Chloracidobacterium will be proposed as the type genus of a new family, Chloracidobacteriaceae , within the order Blastocatellales , the class Blastocatellia , and the phylum Acidobacteriota.

59 BASIC BIOLOGICAL SCIENCES↗

Iron acquisition and mineral transformation by cyanobacteria living in extreme environments

Iron is an essential micronutrient for most living organisms, including cyanobacteria. These microorganisms have been found in Earth's driest polar and non-polar deserts, including the Atacama Desert, Chile. Iron-containing minerals were identified in colonized rock substrates from the Atacama Desert, however, the interactions between microorganisms and iron minerals remain unclear. In the current study, we determined that colonized gypsum rocks collected from the Atacama Desert contained both magnetite and hematite phases. A cyanobacteria isolate was cultured on substrates consisting of gypsum with embedded magnetite nanoparticles. Transmission electron microscopy imaging revealed a significant reduction in the size of magnetite nanoparticles due to their dissolution, which occurred around the microbial biofilms. Concurrently, hematite was detected, likely from the oxidation of the magnetite nanoparticles. Higher cell counts and production of siderophores were observed in cultures with magnetite nanoparticles suggesting that cyanobacteria were actively acquiring iron from the magnetite nanoparticles. Magnetite dissolution and iron acquisition by the cyanobacteria was further confirmed using large bulk magnetite crystals, uncovering a survival strategy of cyanobacteria in these extreme environments.

59 BASIC BIOLOGICAL SCIENCES↗

Acronema sippewissettensis Gen. Nov. Sp. Nov., microbial mat bicosoecid (Bicosoecales = Bicosoecida)

A heterotrophic mastigote from the flat laminated Microcoleus-dominated intertidal microbial mat at the Sippewissett salt marsh, Cape Cod, Massachusetts, was isolated into monoprotist culture in the same anoxic medium that led to spirochete and other anaerobic bacterial enrichments. The protist grew vigorously and was transferred indefinitely in oxic marine medium. Videomicroscopy as well as scanning and transmission electron microscopy were used to document its features. The swimming and perching behavior, nutritional mode (bactivory) and morphology including ultra-structure identify it as an aloricate bicosoecid. The presence of heteromorphic acronematic undulipodia, bilateral bipartite tubular mastigonemes, absence of a cytostome, absence of extrusomes, and presence of "Dauerstadien" (duration stages) distinguish this from other Cafeteriaceae bicosoecids. Cell division involves a closed intranuclear spindle. The unspecialized bicosoecid morphology and behavior juxtaposed with oomycete-like vesicles and mastigonemes suggest that this protist may be an extant descendant of a common ancestor of bicosoecids and other stramenopiles (e.g. labyrinthulids, thraustochytrids and oomycetes). A new genus and species, Acronema sippewissettensis, are proposed.

Non-NASA Center↗

Assessing Microbial Communities Related to Mercury Transformations in Contaminated Streambank Soils

In nature, the bioaccumulative potent neurotoxin methylmercury (MeHg) is produced from inorganic mercury (Hg) predominantly by anaerobic microorganisms. Hg-contaminated soils are a potential source of MeHg due to microbial activity. We examine streambank soils collected from the contaminated East Fork Poplar Creek (EFPC) in Tennessee, USA, where seasonal variations in MeHg levels have been observed throughout the year, suggesting active microbial Hg methylation. In this study, we characterized the microbial community in contaminated bank soil samples collected from two locations over a period of one year and compared the results to soil samples from an uncontaminated reference site with similar geochemistry (n = 12). Microbial community composition and diversity were assessed by 16S rRNA gene amplicon sequencing. Furthermore, to isolate potential methylators from soils, enrichment cultures were prepared using selective media. A set of three clade-specific primers targeting the gene hgcA were used to detect Hg methylators among the δ-Proteobacteria in EFPC bank soils across all seasons. Two families among the δ-Proteobacteria that have been previously associated with Hg methylation, Geobacteraceae and Syntrophobacteraceae, were found to be predominant with relative abundances of 0.13% and 4.0%, respectively. However, in soil enrichment cultures, Firmicutes were predominant among families associated with Hg methylation. Specifically, Clostridiaceae and Peptococcaceae and their genera Clostridium and Desulfosporosinus were among the ten most abundant genera with relative abundances of 2.6% and 1.7%, respectively. These results offer insights into the role of microbial communities on Hg transformation processes in contaminated bank soils in EFPC. Identifying the biogeochemical drivers of MeHg production is critical for future remediation efforts.

54 ENVIRONMENTAL SCIENCES↗

Microbial degradation of parathion

An organism capable of utilizing parathion as the sole carbon and energy source was isolated by enrichment culture techniques. The bacterium was characterized and tentatively classified as Pseudomonas aeruginosa. A pH of 7.0 - 7.5 and temperature of 30 C were found to be optimum for the consumption of parathion. Virtually no oxygen utilization was observed with resting cell suspensions when nonsolubilized parathion was employed. The use of ethanol as solvent for parathion in resting cell studies or preincubation of cells in ethanol obviated this problem and rapid parathion oxidation was demonstrable. Approximately 80% of the parathion consumed by resting cells was present terminally as carbon dioxide. Permeability of the cell to parathion or its metabolites was contingent upon the use of ethanol as either solvent or denaturant. Metabolites were tentatively identified by thin layer chromatography.

Gibson, W. L.↗

Microbial maintenance energy quantified and modeled with microcalorimetry

Refining the energetic costs of cellular maintenance is essential for predicting microbial growth and survival in the environment. In this work, we evaluate a simple batch culture method to quantify energy partitioning between growth and maintenance using microcalorimetry and thermodynamic modeling. The constants derived from the batch culture system were comparable to those that have been reported from meta-analyses of data derived from chemostat studies. The model accurately predicted temperature-dependent biomass yield and the upper temperature limit of growth for Desulfovibrio alaskensis G20, suggesting the method may have broad application. An Arrhenius temperature dependence for the specific energy consumption rate, inferred from substrate consumption and heat evolution, was observed over the entire viable temperature range. By combining this relationship for specific energy consumption rates and observed specific growth rates, the model describes an increase in nongrowth associated maintenance at higher temperatures and the corresponding decrease in energy available for growth. This analytical and thermodynamic formulation suggests that simply monitoring heat evolution in batch culture could be a useful complement to the recognized limitations of estimating maintenance using extrapolation to zero growth in chemostats.

59 BASIC BIOLOGICAL SCIENCES↗

Biomarkers as tracers for life on early earth and Mars

Biomarkers in geological samples are products derived from biochemical (natural product) precursors by reductive and oxidative processes (e.g., cholestanes from cholesterol). Generally, lipids, pigments and biomembranes are preserved best over longer geological times and labile compounds such as amino acids, sugars, etc. are useful biomarkers for recent times. Thus, the detailed characterization of biomarker compositions permits the assessment of the major contributing species of extinct and/or extant life. In the case of the early Earth, work has progressed to elucidate molecular structure and carbon isotropic signals preserved in ancient sedimentary rocks. In addition, the combination of bacterial biochemistry with the organic geochemistry of contemporary and ancient hydrothermal ecosystems permits the modeling of the nature, behavior and preservation potential of primitive microbial communities. This approach uses combined molecular and isotopic analyses to characterize lipids produced by cultured bacteria (representative of ancient strains) and to test a variety of culture conditions which affect their biosynthesis. On considering Mars, the biomarkers from lipids and biopolymers would be expected to be preserved best if life flourished there during its early history (3.5-4 x 10(9) yr ago). Both oxidized and reduced products would be expected. This is based on the inferred occurrence of hydrothermal activity during that time with the concomitant preservation of biochemically-derived organic matter. Both known biomarkers (i.e., as elucidated for early terrestrial samples and for primitive terrestrial microbiota) and novel, potentially unknown compounds should be characterized.

Non-NASA Center↗

Characterization of Microbe Resistant Coatings for Use in the ISS Water System

The ISS is a highly controlled environment; however, there are still microbes on it that astronauts bring from their own microbiome. Once within the ISS water treatment system, these microbes not only pose a health risk for astronauts, but they can form biofilms, causing material degradation and system failure. Due to their protective structure, biofilms are notoriously resistant to disinfectants and antibiotics. One proposed solution is applying polymer coatings to the metal parts of the water system to prevent biofilms from forming. Two types of polyampholyte polymers with previously demonstrated nonfouling properties were sent to the ISS in 2021 to test their viability for preventing microbial adhesion in space. However, the only microbial assays performed were microscopy. To enhance this ongoing research project, I developed procedures for culturing, applying, and quantifying Ralstonia pickettii on these polymers. R. pickettii was chosen because it is one of the most prevalent microbes found in the ISS water treatment system. Optical density data was recorded to obtain a growth curve demonstrating a doubling time between 2.83 and 4.88 hours in 30°C, not shaken conditions. Optical density was also correlated to colony forming units in a plating experiment. Another result was design (and pilot testing) of a surface colonization experiment. These results provided vital knowledge about R. pickettii, which will be used for a 2023 payload of antimicrobial polymer coatings on stainless steel, and improved methods for biofilm analysis upon its return to evaluate the efficacy of the coatings.

microbiology↗

Biofilm formation and control in a simulated spacecraft water system - Two-year results

The ability of iodine to maintain microbial water quality in a simulated spacecraft water system is being studied. An iodine level of about 2.0 mg/L is maintained by passing ultrapure influent water through an iodinated ion exchange resin. Six liters are withdrawn daily and the chemical and microbial quality of the water is monitored regularly. Stainless steel coupons used to monitor biofilm formation are being analyzed by culture methods, epifluorescence microscopy, and scanning electron microscopy. Results from the first two years of operation show a single episode of high bacterial colony counts in the iodinated system. This growth was apparently controlled by replacing the iodinated ion exchange resin. Scanning electron microscopy indicates that the iodine has limited but not completely eliminated the formation of biofilm during the first two years of operation. Significant microbial contamination has been present continuously in a parallel noniodinated system since the third week of operation.

Schultz, John R.↗

Hydrogen Biogeochemistry in Anaerobic and Photosynthetic Ecosystems

The simple biochemistry of molecular hydrogen is central to a large number of microbial processes, affecting the interaction of organisms with each other and with the environment. In anoxic sediments, a great majority of microbial redox processes involve hydrogen as a reactant, product or potential by-product. Accordingly, the energetics (thermodynamics) of each of these processes is affected by variations in local H2 concentrations. It has long been established that this effect is important in governing microbe-microbe interactions and there are multiple demonstrations that "interspecies hydrogen transfer" can alter the products of, inhibit/stimulate, or even reverse microbial metabolic reactions. In anoxic sediments, H2 concentrations themselves are thought to be controlled by the thermodynamics of the predominant H2-consuming microbial process. In sediments from Cape Lookout Bight, this relationship quantitatively describes the co-variation of H2 concentrations with temperature (for methanogens and sulfate reducers) and with sulfate concentration (for sulfate reducers). The quantitative aspect is import= for two reasons: 1) it permits the modeling of H2-sensitive biogeochemistry, such as anaerobic methane oxidation or pathways of organic matter remineralization, as a function of environmental controls; 2) for such a relationship to be observed requires that intracellular biochemistry and bioenergetics are being directly expressed in a component of the extracellular medium. H2 could therefore be utilized a non-invasive probe of cellular energetic function in intact microbial ecosystems. Based on the latter principle we have measured down-core profiles of H2 and other relevant physico-chemical parameters in order to calculate the metabolic energy yields (DG) that support microbial metabolism in Cape Lookout Bight sediments. Methanogens in this system apparently function with energy yields significantly smaller than the minimum requirements suggested by pure culture studies. Our recent work has extended the study of hydrogen to cyanobacterial mat communities. The large amounts of reducing power generated during photosynthetic activity carry the potential to contribute a swamping term to the H2 economy of the anaerobic microbial populations within the mat - and thereby to alter the population structure and biogeochemical function of the mat as a whole. In hypersaline microbial mats, we observe a distinct diel cycle in H2 production and a substantial corresponding flux. On an early Earth dominated by microbial mats, this transmission of photosynthetic reducing power may have carried important implications for both biospheric and atmospheric evolution.

Hoehler, Tori M.↗

Mutualism in a Reduced Gravity Environment (MuRGE)

Mutualism in a Reduced Gravity Environment (MuRGE) is a ground research study to determine the feasibility of assessing fungi-plant (Piriformospora indica-Arabidopsis thaliana) interactions in microgravity. Seeds from the plant Arabiddospsis thaliana (At) will be grown in the presence of Piriformospora indica (Pi) an endophytic Sebacinacae family fungus. Pi is capable of colonizing the roots of a wide variety of plant species, including non-mycorrhizal hosts like At, and promoting plant growth similarly to AMF (arbusuclar mychorrizal fungi) unlike most AMF, Pi is not an obligate plant symbiont and can be grown in the absence of a host. In the presence of a suitable plant host, Pi can attach to and colonize root tips. Interaction visualization is accomplished with strong autofluorescence in the roots, followed by root colonization via fungal hyphae, and chlamydospore production. Increased root growth can be observed even before root colonization is detectable. In addition, Pi chlamydospores generated from axenic culture in microgravity will be used to inoculate roots of At grown in 1g to determine the effect of microgravity upon the inherent virulence or beneficial effects. Based on recent reports of increased virulence of S. typhimurium, P. aeruginosa, and S. Pneumoniae in reduced gravity, differences in microbial pathogenic responses and host plant systemic acquired resistance are expected. The focus of this project within MuRGE involved the development P. indica culture media evaluation and microscopy protocol development. High, clean spore harvest yields for the detection of fungi-plant interactions microscopically was the immediate goal of this experiment.

Haire, Timothy C.↗

Microbial distribution in the Environmental Control and Life Support System water recovery test conducted at NASA, MSFC

NASA/MSFC is developing a physical/chemical treatment system to reclaim wastewater for reuse on Space Station Freedom (SSF). Integrated testing of hygiene and potable water subsystems assessed the capability to reclaim water to SSF specifications. The test was conducted from May through July 1990 with a total of 47 days of system test operation. Water samples were analyzed using standard cultural methods employing membrane filtration and spread plate techniques and epifluorescence microscopy. Fatty acid methyl ester and biochemical profiles were used for microbial identification. Analysis of waste and product water produced by the subsystems demonstrated the effective reduction of viable microbial populations greater than 8.0E + 06 colony forming units (CFU) per 100 mL to an average of 5 CFU/100 mL prior to distribution into storage tanks.

Gauthier, J. J.↗

Versatile microbial communities rapidly assimilate ammonium hydroxide-treated plastic waste

Abstract Waste plastic presently accumulates in landfills or the environment. While natural microbial metabolisms can degrade plastic polymers, biodegradation of plastic is very slow. This study demonstrates that chemical deconstruction of polyethylene terephthalate (PET) with ammonium hydroxide can replace the rate limiting step (depolymerization) and by producing plastic-derived terephthalic acid and terephthalic acid monoamide. The deconstructed PET (DCPET) is neutralized with phosphoric acid prior to bioprocessing, resulting in a product containing biologically accessible nitrogen and phosphorus from the process reactants. Three microbial consortia obtained from compost and sediment degraded DCPET in ultrapure water and scavenged river water without addition of nutrients. No statistically significant difference was observed in growth rate compared to communities grown on DCPET in minimal culture medium. The consortia were dominated by Rhodococcus spp., Hydrogenophaga spp., and many lower abundance genera. All taxa were related to species known to degrade aromatic compounds. Microbial consortia are known to confer flexibility in processing diverse substrates. To highlight this, we also demonstrate that two microbial consortia can grow on similarly deconstructed polyesters, polyamides, and polyurethanes in water instead of medium. Our findings suggest that microbial communities may enable flexible bioprocessing of mixed plastic wastes when coupled with chemical deconstruction.

59 BASIC BIOLOGICAL SCIENCES↗

Multi-technique characterization of iron reduction by an Antarctic Shewanella : an analog system for putative Martian biosignature identification

ABSTRACT Microbes from terrestrial extreme environments enable testing of biosignature production in conditions relevant to astrobiological targets. Mars, which was likely more conducive to life during early warmer and wetter epochs, has inspired missions that search for signs of early life in the surficial rock record, including mineral or organic biosignatures. Microbial iron reduction is a common and ancient metabolism that may have also operated on other rocky celestial bodies. To investigate biosignature production during iron reduction, aShewanellasp. (strain BF02_Schw) isolated from a subglacial discharge known as Blood Falls, Antarctica, was incubated with the electron acceptor ferrihydrite (Fh). Biosignatures associated with Fh reduction were identified using a suite of techniques currently utilized or proposed for Mars missions, including X-ray diffraction and infrared, Mössbauer, and Raman spectroscopy. The biotic origin of features was validated by transcriptional changes observed between treatments with and without Fh and comparison to killed controls. In live treatments, Fh was reduced to magnetite and goethite, both detected in Martian lacustrine basins. Several soluble and volatile metabolites were also detected, including riboflavin and dimethyl sulfide (DMS), which could be astrobiological indicators of active microbial processes. While none of the identified biosignatures individually would serve as definitive proof of life (past or present), detecting concomitant features associated with known terrestrial biotic processes would provide compelling rationale for more targeted life detection missions. Terrestrial extremophiles can support the exploration of astrobiologically relevant microbial processes, validation of life detection instrumentation, and potentially the discovery of new biomarkers. IMPORTANCE Culture-based experiments with terrestrial extremophiles can elucidate biosignatures that may be analogous to those produced under extraterrestrial conditions, and thus inform sampling and technology strategies for future missions. Here, we demonstrate the production of several biosignatures under iron-reducing conditions byShewanellasp. BF02_Schw, originally isolated from an Antarctic analog feature. These biosignatures could be detectable using flight-ready instrumentation. Growth experiments with terrestrial extremophiles can identify biosignatures measurable by current methodologies and inform the development and optimization of techniques for detecting extant or extinct life on other worlds.

Biotechnology & Applied Microbiology↗

Mychonastes desiccatus Brown sp. nova (Chlorococcales, Chlorophyta)--an intertidal alga forming achlorophyllous desiccation-resistant cysts

An intertidal Chlorella-like alga Mychonastes desiccatus Brown sp. nova, capable of forming achlorophyllous desiccation-resistant cysts, has been grown in unialgal culture. This small alga was first isolated from a dried sample of a well-studied microbial mat. The mat, located at North Pond, Laguna Figueroa, San Quintin, Baja California, Mexico, is a vertically-stratified microbial community which forms laminated sediments. Morphology, pigment composition and G+C content are within the range typical for the genus Chlorella s. 1. Unlike other chlorellae, however, upon desiccation M. desiccatus forms an achlorophyllous, lipid-filled cyst (thick-walled resting stage) in which no plastid is evident. Rewetting leads to chloroplast differentiation, excystment and recovery of the fully green alga. During desiccation, sporopollenin is deposited within a thickening cell wall. Encystment cannot be induced by growth in the dark. The formation of desiccation-induced cysts allows the alga to survive frequent and intermittent periods of dryness. These chlorellae tolerate wide ranges of acidity and temperature; they both grow and form cysts in media in which sodium ions are replaced with potassium. Although the cysts tolerate crystalline salts, the cell grow optimally in concentrations corresponding from three-quarters to full-strength seawater.

NASA Discipline Exobiology↗

Biological potential of extraterrestrial materials. 2. Microbial and plant responses to nutrients in the Murchison carbonaceous meteorite

Meteoritic materials are investigated as potential early planetary nutrients. Aqueous extracts of the Murchison C2 carbonaceous meteorite are utilized as a sole carbon source by microorganisms, as demonstrated by the genetically modified Pseudomonas fluorescence equipped with the lux gene. Nutrient effects are observed also with the soil microorganisms Nocardia asteroides and Arthrobacter pascens that reach populations up to 5 x 10(7) CFU/ml in meteorite extracts, similar to populations in terrestrial soil extracts. Plant tissue cultures of Asparagus officinalis and Solanum tuberosum (potato) exhibit enhanced pigmentation and some enhanced growth when meteorite extracts are added to partial nutrient media, but inhibited growth when added to full nutrient solution. The meteorite extracts lead to large increases in S, Ca, Mg, and Fe plant tissue contents as shown by X-ray fluorescence, while P, K, and Cl contents show mixed effects. In both microbiological and plant tissue experiments, the nutrient and inhibitory effects appear to be best balanced for growth at about 1:20 (extracted solid : H2O) ratios. The results suggest that solutions in cavities in meteorites can provide efficient concentrated biogenic and early nutrient environments, including high phosphate levels, which may be the limiting nutrient. The results also suggest that carbonaceous asteroid resources can sustain soil microbial activity and provide essential macronutrients for future space-based ecosystems.

Non-NASA Center↗