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At least 289 records · Page 16

Microbial Characterization of Heat Melt Compaction for Treatment of Space Generated Solid Wastes

One treatment process in development for solid waste management in space has been the Trash Compaction Processing System (TCPS). Human space mission wastes typically contain large percentages of contaminated wet solid waste. The Heat Melt Compactor (HMC) is being developed to be a multi-function means of water recovery, volume reduction, and the making safe of contaminant-rich trash with the potential for waste stabilization and/or sterilization. To determine the efficacy of the HMC treatment to kill microorganisms in solid waste and remain biologically stable, testing was conducted on three tiles produced by HMC-Gen 2 at Ames Research Center. Samples were shipped to Kennedy Space Center to test for microbial viability after compaction, determine the bio-stability of the HMC disks during storage (43 days), and assess potential airborne contaminate microbial growth on surfaces at low and high humidity conditions. In addition to the products of solid waste processing technologies, there is a concern that the crew might come into contact with hardware surfaces that have been contaminated by microorganisms during waste processing. The extent of microbial surface contamination of waste processing hardware was determined by surface sample swabbing and analysis for total bacterial and yeast counts and cultivable counts of aerobic and anaerobic bacteria, spore-forming bacteria, and fungi. Results indicate that trash processing increased bacterial counts on the surfaces of the compacter. All but one biological indicator spore strip imbedded in the HMC produced tiles were negative for growth after incubation for five days indicating effective sterilization through the heat melt compaction process. Analysis of core samples as well as surface growth of tiles inoculated with Aspergillus niger fungal spores incubated at three levels of humidity indicate that HMC created tiles did not support the proliferation of bacterial and fungal growth.

Mary E Hummerick↗

Unlocking the Spacecraft and Human Habitat Microbiome to Enable the Next Generation of Space Exploration

Planetary protection is the discipline that prevents harmful contamination of the solar system during exploration activities. The current international guidelines and NASA policy addressing biological contamination on spacecraft surfaces contains prescriptive guidelines of spore requirements (e.g., 300 spores/m2, 5×105 spores per spacecraft) applicable to spacecraft bound for Mars. To verify these requirements spacecraft engineers sample spacecraft surfaces throughout the assembly, test and launch operations phase of the mission using damp water cotton swabs and polyester wipes. After sampling, the potential biological contamination is enumerated using a series of traditional microbiology techniques to include sonication, heat shocking at 80°C for 15min to select for spores, and growth on tryptic soy agar at 32°C for 72 hours. To enable crewed missions to Mars and robotic exploration of the Ocean Worlds a risk informed decision making / performance-based approach to assess biological contamination offers a promising solution in the trade space. Recognizing the need for a performance-based approach, NASA’s new Planetary Protection policies now incorporate the agility for missions to be able to leverage a performance or prescriptive approach. One of top contenders in the option space is a coupled quantitative, descriptive and functional based approach to be able to assess the quantity, types and capabilities of the biological contamination present on spacecraft surfaces. A tailored, mission by mission assurance case could then be formulated by building an argument around the target body, projected capabilities surrounding the types of organisms their potential for survival and proliferation, and ability to be transported on the target body to contaminate an area of biological interest. A performance-based requirement would then be used to demonstrate the mission’s compliance in protecting the planetary environment safety objectives. This symposium talk will showcase the background and need case for NASA to develop such a capability as well as provide an update on the efforts underway in developing a transparent and responsible performance-based approach to biological contamination assessments on spacecraft surfaces.

Habitat Microbiome↗

High temperature flow-through device for rapid solubilization and analysis

Devices and methods for thermally lysing of biological material, for example vegetative bacterial cells and bacterial spores, are provided. Hot solution methods for solubilizing bacterial spores are described. Systems for direct analysis are disclosed including thermal lysers coupled to sample preparation stations. Integrated systems capable of performing sample lysis, labeling and protein fingerprint analysis of biological material, for example, vegetative bacterial cells, bacterial spores and viruses are provided.

59 BASIC BIOLOGICAL SCIENCES↗

Complete genome sequence of Mahella australiensis type strain (50-1 BON T )

Mahella australiensis Bonilla Salinas et al. 2004 is the type species of the genus Mahella, which belongs to the family Thermoanaerobacteraceae. The species is of interest because it differs from other known anaerobic spore-forming bacteria in its G+C content, and in certain phenotypic traits, such as carbon source utilization and relationship to temperature. Moreover, it has been discussed that this species might be an indigenous member of petroleum and oil reservoirs. This is the first completed genome sequence of a member of the genus Mahella and the ninth completed type strain genome sequence from the family Thermoanaerobacteraceae. The 3,135,972 bp long genome with its 2,974 protein-coding and 59 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

59 BASIC BIOLOGICAL SCIENCES↗

Filtration of bioaerosols using a granular metallic filter with micrometer-sized collectors

Several experimental studies with granular bed filters composed of micrometer-sized spherical or sintered metallic granules have demonstrated their use in aerosol filtration. However, the effectiveness of these metallic membrane filters against bioaerosols has not been established. In this work, the filtration efficiency and filter quality of these filters against airborne B. subtilis endospore and MS2 virus were determined as a function of face velocity and loading time. In experiments, a physical removal efficiency greater than 99.9% and a viable removal efficiency of greater than 5-log were observed for both bacterial spore and viral aerosols. A lower face velocity produced both higher collection efficiency and filter quality for virus but was not statistically significant for spore filtration. Although the filter had high filtration efficiency of the test bioaerosols, the filter's high pressure drop resulted in a low filter quality (0.25-0.75 kPa- 1). Overall, filters with micrometer-sized collectors capture bioaerosols effectively but their applications in aerosol filtration may be limited by their high pressure drop.

aerosol↗

The transcriptional landscape of basidiosporogenesis in mature Pisolithus microcarpus basidiocarp

Background: Pisolithus microcarpus (Cooke & Massee) G. Cunn is a gasteromycete that produces closed basidiocarps in symbiosis with eucalypts and acacias. The fungus produces a complex basidiocarp composed of peridioles at different developmental stages and an upper layer of basidiospores free of the hyphae and ready for wind dispersal upon the rupture of the basidiocarp pellis. During basidiosporogenesis, a process that takes place inside the basidiocarp peridioles, a conspicuous reserve of fatty acids is present throughout development. While several previous studies have described basidiosporogenesis inside peridioles, very little is known about gene expression changes that may occur during this part of the fungal life cycle. The objective of this work was to analyze gene transcription during peridiole and basidiospore development, while focusing specifically on cell cycle progression and lipid metabolism. Results: Throughout different developmental stages of the peridioles we analyzed, 737 genes were regulated between adjacent compartments (>5 fold, FDR-corrected p-value < 0.05) corresponding to 3.49% of the genes present in the P. microcarpus genome. We identified three clusters among the regulated genes which showed differential expression between the peridiole developmental stages and the basidiospores. During peridiole development, transcripts for proteins involved in cellular processes, signaling, and information storage were detected, notably those for coding transcription factors, DNA polymerase subunits, DNA repair proteins, and genes involved in chromatin structure. For both internal embedded basidiospores (hereto referred to as “Internal spores”, IS) and external free basidiospores (hereto referred to as “Free spores”, FS), upregulated transcripts were found to involve primary metabolism, particularly fatty acid metabolism (FA). High expression of transcripts related to β-oxidation and the glyoxylate shunt indicated that fatty acids served as a major carbon source for basidiosporogenesis. Conclusion: Our results show that basidiocarp formation in P. microcarpus involves a complex array of genes that are regulated throughout peridiole development. We identified waves of transcripts with coordinated regulation and identified transcription factors which may play a role in this regulation. This is the first work to describe gene expression patterns during basidiocarp formation in an ectomycorrhizal gasteromycete fungus and sheds light on genes that may play important roles in the developmental process.

59 BASIC BIOLOGICAL SCIENCES↗

FRET-Based Quantum Dot Immunoassay for Rapid and Sensitive Detection of Aspergillus amstelodami

In this study, a fluorescence resonance energy transfer (FRET)-based quantum dot (QD) immunoassay for detection and identification of Aspergillus amstelodami was developed. Biosensors were formed by conjugating QDs to IgG antibodies and incubating with quencher-labeled analytes; QD energy was transferred to the quencher species through FRET, resulting in diminished fluorescence from the QD donor. During a detection event, quencher-labeled analytes are displaced by higher affinity target analytes, creating a detectable fluorescence signal increase from the QD donor. Conjugation and the resulting antibody:QD ratios were characterized with UV-Vis spectroscopy and QuantiT protein assay. The sensitivity of initial fluorescence experiments was compromised by inherent autofluorescence of mold spores, which produced low signal-to-noise and inconsistent readings. Therefore, excitation wavelength, QD, and quencher were adjusted to provide optimal signal-to-noise over spore background. Affinities of anti-Aspergillus antibody for different mold species were estimated with sandwich immunoassays, which identified A. fumigatus and A. amstelodami for use as quencher-labeled- and target-analytes, respectively. The optimized displacement immunoassay detected A. amstelodami concentrations as low as 10 3 spores/mL in five minutes or less. Additionally, baseline fluorescence was produced in the presence of 10 5 CFU/mL heat-killed E. coli O157:H7, demonstrating high specificity. This sensing modality may be useful for identification and detection of other biological threat agents, pending identification of suitable antibodies. Overall, these FRET-based QD-antibody biosensors represent a significant advancement in detection capabilities, offering sensitive and reliable detection of targets with applications in areas from biological terrorism defense to clinical analysis.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Full-Scale Drinking Water System Decontamination at the Water Security Test Bed

The US Environmental Protection Agency's (USEPA's) Water Security Test Bed (WSTB) facility is a full-scale representation of a drinking water distribution system. In collaboration with the Idaho National Laboratory, USEPA designed the WSTB facility to support full-scale evaluations of water infrastructure, premise plumbing, and appliance decontamination. Thus far, USEPA's research has focused on decontamination of Bacillus globigii (BG) spores, a nonpathogenic surrogate for Bacillus anthracis, and Bakken crude oil. Flushing and disinfection effectively removed 7–8 log 10 BG spores from the bulk water, but only 2–3 log 10 from the pipe wall coupons. Bakken crude oil components were then removed from distribution pipe and premise plumbing water by flushing, although 55% of the injected oil components persisted in the dishwasher and refrigerator water dispenser. Using the WSTB facility allows USEPA to test contaminants without any human health or ecological risk and to inform water systems on effective methodologies for responding to contamination incidents.

42 ENGINEERING↗

Molecular microbial diversity of a spacecraft assembly facility

In ongoing investigations to map and archive the microbial footprints in various components of the spacecraft and its accessories, we have examined the microbial populations of the Jet Propulsion Laboratory's Spacecraft Assembly Facility (JPL-SAF). Witness plates made up of spacecraft materials, some painted with spacecraft qualified paints, were exposed for approximately 7 to 9 months at JPL-SAF and examined the particulate materials collected for the incidence of total cultivable aerobic heterotrophs and heat-tolerant (80 degrees C for 15-min.) spore-formers. The results showed that the witness plates coated with spacecraft qualified paints attracted more dust particles than the non-coated stainless steel witness plates. Among the four paints tested, witness plates coated with NS43G accumulated the highest number of particles, and hence attracted more cultivable microbes. The conventional microbiological examination revealed that the JPL-SAF harbors mainly Gram-positive microbes and mostly spore-forming Bacillus species. Most of the isolated microbes were heat resistant to 80 degrees C and proliferate at 60 degrees C. The phylogenetic relationships among 23 cultivable heat-tolerant microbes were examined using a battery of morphological, physiological, molecular and chemotaxonomic characterizations. By 16S rDNA sequence analysis, the isolates fell into seven clades: Bacillus licheniformis, B. pumilus, B. cereus, B. circulans, Staphylococcus capitis, Planococcus sp. and Micrococcus lylae. In contrast to the cultivable approach, direct DNA isolation, cloning and 16S rDNA sequencing analysis revealed equal representation of both Gram-positive and Gram-negative microorganisms.

Industry↗

Surface characteristics of spacecraft components affect the aggregation of microorganisms and may lead to different survival rates of bacteria on Mars landers

Layers of dormant endospores of Bacillus subtilis HA101 were applied to eight different spacecraft materials and exposed to martian conditions of low pressure (8.5 mbar), low temperature (-10 degrees C), and high CO(2) gas composition and irradiated with a Mars-normal ultraviolet (UV-visible- near-infrared spectrum. Bacterial layers were exposed to either 1 min or 1 h of Mars-normal UV irradiation, which simulated clear-sky conditions on equatorial Mars (0.1 tau). When exposed to 1 min of Mars UV irradiation, the numbers of viable endospores of B. subtilis were reduced three to four orders of magnitude for two brands of aluminum (Al), stainless steel, chemfilm-treated Al, clear-anodized Al, and black-anodized Al coupons. In contrast, bacterial survival was reduced only one to two orders of magnitude for endospores on the non-metal materials astroquartz and graphite composite when bacterial endospores were exposed to 1 min of Mars UV irradiation. When bacterial monolayers were exposed to 1 h of Mars UV irradiation, no viable bacteria were recovered from the six metal coupons listed above. In contrast, bacterial survival was reduced only two to three orders of magnitude for spore layers on astroquartz and graphite composite exposed to 1 h of Mars UV irradiation. Scanning electron microscopy images of the bacterial monolayers on all eight spacecraft materials revealed that endospores of B. subtilis formed large aggregates of multilayered spores on astroquartz and graphite composite, but not on the other six spacecraft materials. It is likely that the formation of multilayered aggregates of endospores on astroquartz and graphite composite is responsible for the enhanced survival of bacterial cells on these materials.

Mars↗

Microbial Cell Imaging

Atomic force microscopy (AFM) is finding increasing application in a variety of fields including microbiology. Until the emergence of AFM, techniques for ivnestigating processes in single microbes were limited. From a biologist's perspective, the fact that AFM can be used to generate high-resolution images in buffers or media is its most appealing feature as live-cell imaging can be pursued. Imaging living cells by AFM allows dynamic biological events to be studied, at the nanoscale, in real time. Few areas of biological research have as much to gain as microbiology from the application of AFM. Whereas the scale of microbes places them near the limit of resolution for light microscopy. AFM is well suited for the study of structures on the order of a micron or less. Although electron microscopy techniques have been the standard for high-resolution imaging of microbes, AFM is quickly gaining favor for several reasons. First, fixatives that impair biological activity are not required. Second, AFM is capable of detecting forces in the pN range, and precise control of the force applied to the cantilever can be maintained. This combination facilitates the evaluation of physical characteristics of microbes. Third, rather than yielding the composite, statistical average of cell populations, as is the case with many biochemical assays, the behavior of single cells can be monitored. Despite the potential of AFM in microbiology, there are several limitations that must be considered. For example, the time required to record an image allows for the study of gross events such as cell division or membrane degradation from an antibiotic but precludes the evaluation of biological reactions and events that happen in just fractions of a second. Additionally, the AFM is a topographical tool and is restricted to imaging surfaces. Therefore, it cannot be used to look inside cells as with opticla and transmission electron microscopes. other practical considerations are the limitation on the maximum scan size (roughly 100 x 100 {mu}m) and the restricted movement of the cantilever in the Z (or height) direction. In most commercial AFMs, the Z range is restricted to roughly 10 {mu}m such that the height of cells to be imaged must be seriously considered. Nevertheless, AFM can provide structural-functional information at nanometer resolution and do so in physiologically relevant environments. Further, instrumentation for scanning probe microscopy continues to advance. Systems for high-speed imaging are becoming available, and techniques for looking inside the cells are being demonstrated. The ability to combine AFM with other imaging modalities is likely to have an even greater impact on microbiological studies. AFM studies of intact microbial cells started to appear in the literature in the 1990s. For example, AFM studies of Saccharomyces cerevisiae examined buddings cars after cell division and detailed changes related to cell growth processes. Also, the first AFM studies of bacterial biofilms appeared. In the late 1990s, AFM studies of intact fungal spores described clear changes in spore surfaces upon germination, and studies of individual bacterial cells were also described. These early bacterial imaging studies examined changes in bacterial morphology due to antimicrobial peptides exposure and bacterial adhesion properties. The majority of these early studies were carried out on dried samples and took advantage of the resolving power of AFM. The lack of cell mounting procedures presented an impediment for cell imaging studies. Subsequently, several approaches to mounting microbial cells have been developed, and these techniques are described later. Also highlighted are general considerations for microbial imaging and a description of some of the various applications of AFM to microbiology.

60 APPLIED LIFE SCIENCES↗

Antimicrobial mechanisms of g‐C 3 N 4 @ ZnO against oomycetes Phytophthora capsici: from its metabolism, membrane structures and growth

Abstract BACKGROUND Phytophthora capsici , a refractory and model oomycete plant pathogen, especially threatens multiple vegetable crops. A limited number of chemical pesticides play a vital role in controlling oomycete plant diseases. However, this approach often leads to excessive use of chemical agent, exacerbates environmental issues and more and more drug‐resistant strains of oomycete. Therefore, it is imperative to devise innovative solutions that can effectively address the infection of oomycete while maintaining high levels of environmental sustainability and low toxicity. RESULTS In this study, g‐C 3 N 4 @ZnO heterostructure was synthesized and characterized. The g‐C 3 N 4 @ZnO showed higher toxicity on Phytophthora capsici than graphitic carbon nitride (g‐C 3 N 4 ) nanosheets and zinc oxide (ZnO) nanoparticles in vitro and in vivo . Except the hyphal growth of Phytophthora capsici , their germination rate of spores, sporangium formation and number of spores were all suppressed by g‐C 3 N 4 @ZnO heterostructure. Furthermore, we found that this g‐C 3 N 4 @ZnO heterostructure has higher photocatalytic activity under visible light, which potentially enhanced the reactive oxygen species (ROS) mediated stress on Phytophthora capsici . Ultrastructural morphology, global changes of gene expression and weighted gene co‐expression network analysis all supported that the anti‐oomycete activity of g‐C 3 N 4 @ZnO was manifested in the destruction of membrane system and inhibition of multiple metabolisms of Phytophthora capsici under visible irradiation, which also could be attributed to the ROS and zinc ion (Zn 2+ ) mediated stress. CONCLUSION This works offers a novel oomycete disease management strategy by using g‐C 3 N 4 @ZnO, which were attributed to the ROS stress, destruction of membrane system and inhibition of multiple metabolisms. © 2023 Society of Chemical Industry.

Cai, Lin↗

The histone demethylase KdmB is part of a trimeric protein complex and mediates virulence and mycotoxin production in Penicillium expansum

Epigenetic modification of chromosome structure has increasingly been associated with alterations in secondary metabolism and sporulation defects in filamentous fungal pathogens. Recently, the epigenetic reader protein SntB was shown to govern virulence, spore production and mycotoxin synthesis in the fruit pathogen Penicillium expansum. Through immunoprecipitation-coupled mass spectrometry, we found that SntB is a member of a protein complex with KdmB, a histone demethylase and the essential protein RpdA, a histone deacetylase. Deletion of kdmB phenocopied some but not all characteristics of the ΔsntB mutant. KdmB deletion strains exhibited reduced lesion development on Golden Delicious apples and this was accompanied by decreased production of patulin and citrinin in host tissue. In addition, ΔkdmB mutants were sensitive to several cell wall stressors which possibly contributed to the decreased virulence observed on apples. In conclusion, slight differences in spore production and germination rates of ΔkdmB mutants in vitro did not impact overall diameter growth in culture.

59 BASIC BIOLOGICAL SCIENCES↗

Fusion of Laboratory and Textual Data for Investigative Bioforensics

Chemical and biological forensic programs focus on the identification of a threat and acquisition of laboratory measurements to determine how a threat agent may have been produced. However, to generate investigative leads, it might also be useful to identify institutions where the same agent has been produced by the same or a very similar process, since the producer of the agent may have learned methods at a university or similar institution. We have developed a Bayesian network framework that fuses hard and soft data sources to assign probability to production practices. It combines the results of laboratory measurements with an automatic text reader to scan scientific literature and rank institutions that had published papers on the agent of interest in order of the probability that the institution has the capability to generate the sample of interest based on laboratory data. We demonstrate the Bayesian network on an example case from microbial forensics, predicting the methods used to produce Bacillus anthracis spores based on mass spectrometric measurements and identifying institutions that have a history of growing Bacillus spores using the same or highly similar methods. We illustrate that the network model can assign a higher posterior probability than expected by random chance to appropriate institutions when trained using only a small set of manually analyzed documents. This is the first example of an automated methodology to integrate experimental and textual data for the purpose of investigative forensics.

Webb-Robertson, Bobbie-Jo M.↗

Fungal diversity and function in metagenomes sequenced from extreme environments

Fungi are increasingly recognized as key players in various extreme environments. Here we present an analysis of publicly-sourced metagenomes from global extreme environments, focusing on fungal taxonomy and function. The majority of 855 selected metagenomes contained scaffolds assigned to fungi. Relative abundance of fungi was as high as 10% of protein-coding genes with taxonomic annotation, with up to 289 fungal genera per sample. Despite taxonomic clustering by environment, fungal communities were more dissimilar than archaeal and bacterial communities, both for within- and between-environment comparisons. Relatively abundant fungal classes in extreme environments included Dothideomycetes, Eurotiomycetes, Leotiomycetes, Pezizomycetes, Saccharomycetes, and Sordariomycetes. Broad generalists and prolific aerial spore formers were the most relatively abundant fungal genera detected in most of the extreme environments, bringing up the question of whether they are actively growing in those environments or just surviving as spores. More specialized fungi were common in some environments, such as zoosporic taxa in cryosphere water and hot springs. Relative abundances of genes involved in adaptation to general, thermal, oxidative, and osmotic stress were greatest in soda lake, acid mine drainage, and cryosphere water samples.

60 APPLIED LIFE SCIENCES↗

Microanalysis of Primary Biological Particles from Model Grass over Its Life Cycle

Airborne biological aerosols are an integral part of the atmosphere–biosphere interface and significantly impact the environment and Earth’s climate. Primary biological particles such as fungal spores, bacteria, and fragments of plant debris are emitted directly from the biosphere into the atmosphere. The phyllosphere (aboveground plant surfaces) is one of the major habitats for different microbes. Here, atmospheric emissions of primary biological particles from model grass Brachypodium distachyon were monitored by online particle number concentration counting at eight principal growth stages starting from leaf development to senescence. Particles collected at each of the growth stages were then characterized using microspectroscopy techniques. Our findings indicate that the morphological and compositional characteristics of emitted biological particles are highly variable at the different growth stages. We identified fungal spores as the most abundant biological particles in the heading stage, while bacteria were most abundant in the flowering and fruit development stages. Understanding differences in the composition and morphology of biological particles during the plant life cycle provides more accurate and reliable insights on structural, functional, and biochemical properties of plant systems, as well as their interactions with microbial communities. This study also provides insights into Earth geoscience system models that represent primary biological particle emissions from the biosphere.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Physical and Chemical Morphology of Passively Sampled Environmental Films

Environmental surface films are important mediators in the fate and transport of persistent organic pollutants (POP) via largely uncharacterized mechanisms. This work presents the first analysis of the physical and chemical morphology of passively sampled environmental films. The films are developed in urban, sub-urban, and metropolitan locations for timescales ranging from 1 to 52 weeks by exposing polished silicon wafers to collect passively deposited tropospheric particulate matter. The time-dependent spatial morphology of the films is analyzed by bright-field, scanning electron, and atomic force microscopies. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging data are analyzed to report the film’s chemical morphology and speciation. We find that the film matrix presents an extremely complex physical and chemical system. Deposited PM sizes span six orders of magnitude (millimeter to nanometer regimes), and surface coverages range from ca. 5 % for one week films and ca. 98 % for 52 week films. We observe significant amounts of mineral dust, biogenic organics, and occasional indications of flora growth (i.e., spores). The films exhibit nanoscale surface features indicative of small particulate depositions as well as films of (semi)volatile molecules. SIMS imaging reveals diverse and sometimes segregated chemical domains of hydrocarbons, inorganics, metals, and proteinaceous macromolecules. This work presents the first spatially resolved physical and chemical surface characterization of native environmental films, beginning a study of mechanisms by which these films form, persist, and mature in the environment.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗