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102 records · Page 6

Characterization of Biological Fallout Particles of Cleanrooms to Measure Spacecraft Cleanliness

NASA cleanrooms are humidity, temperature-regulated, and particle contaminant-controlled environments used for spacecraft assembly. Microorganisms, which are crucial contaminants shed from humans and transported through the environment, can adapt to the oligotrophic cleanroom conditions. These biogenic particles need to be identified to extend our knowledge of biological contamination for future NASA mission use. Here, we devised an aluminum coupon with polycarbonate filter setup to collect fallout particles from an ISO 5 and an ISO 7 cleanroom. We demonstrated the presence of microbial particles, as standalone entities and associated with inert particles in cleanroom environments. The contamination was confirmed as biogenic in virtue of estimating the elemental composition of individual and aggregated particles using an epifluorescence and Field Emission Scanning Electron Microscopy (FESEM) coupled with an energy dispersive X-ray analysis (EDX). Epifluorescence microscopy showed that the particles were stained less (25%) with SYTO 9 and more (75%) with propidium iodide, suggesting that the suspected biogenic particles are membrane-compromised. Remarkably, biological particles such as bacteria and fungal spores/hyphae were observed at sizes ranging from 2 to 70 µm, with abundant C and O elemental signatures and essential tracers (Na, Mg, K, Ca and Cl). Inert particles (2 to 500 µm) that were characterized included compositions such as silica (SiO2), aluminosilicates (Al, Si), and carbonaceous fibers. Furthermore, we designed and illustrated a unique correlative epifluorescence and FE-SEM method to strengthen this approach by acquiring the same biogenic particles in the same filter coupon for a one-to-one comparison. Interestingly, the SYTO 9 stained particles showed abundant C and O elemental signature implying that they are viable microbial particles. Constant evaluation of cleanroom biological and inert particles will help to characterize biological contamination for future NASA missions (e.g. Mars 2020).

Malli Mohan, Ganesh Babu↗

Airborne Radiometry for Calibration, Validation, and Research in Oceanic, Coastal, and Inland Waters

Present-day ocean color satellite sensors, which principally provide reliable data on chlorophyll, sediments, and colored dissolved organic material in the open ocean, are not well suited for coastal and inland water studies for a variety of reasons, including coarse spatial and spectral resolution plus challenges with atmospheric correction. National Aeronautics and Space Administration (NASA) airborne mission concepts tested in 2011, 2013, 2017, and 2018 over Monterey Bay, CA, and nearby inland waters have demonstrated the feasibility of improving airborne monitoring and research activities in case-1 and case-2 aquatic ecosystems through the combined use of state-of-the-art above- and in-water measurement capabilities. These competencies have evolved through time to produce a sensor-web approach: imaging spectrometer, microradiometers, and a sun photometer (airborne) with their analogous algorithms, and with corresponding in-water radiometers and ground-based sun photometry. The NASA airborne instrument suite and mission concept demonstrations, leveraging high quality above- and in-water data, significantly improves the fidelity as well as the spatial and spectral resolution of observations for studying and monitoring water quality in oceanic, coastal, and inland water ecosystems. The goal of this series of projects was to develop and fly a portable airborne sensor suite for NASA science missions focusing on a gradient of water types from oligotrophic to turbid waters addressing the challenges of an optically complex coastal ocean zone and inland waters. The airborne radiometry in this range of aquatic conditions and sites has supported improved results of studies of water quality and biogeochemistry and provides capabilities for research areas such as ocean productivity and biogeochemistry; aquatic impacts of coastal landscape alteration; coastal, estuarine, and inland waters ecosystem productivity; atmospheric correction; and regional climate variability.

atmospheric correction↗

Tropical rains controlling deposition of Saharan dust across the North Atlantic Ocean

Mineral dust plays an important role in the atmospheric radiation budget as well as in the ocean carbon cycle through fertilization and by ballasting of settling organic matter. However, observational records of open-ocean dust deposition are sparse. Here, we present the spatial and temporal evolution of Saharan dust deposition over 2 years from marine sediment traps across the North Atlantic, directly below the core of the Saharan dust plume, with highest dust fluxes observed in summer. We combined the observed deposition fluxes with model simulations and satellite observations and argue that dust deposition in the Atlantic is predominantly controlled by summer rains. The dominant depositional pathway changes from wet deposition in summer to dry deposition in winter. Wet deposition has previously been suggested to increase the release of dust-derived nutrients and their bioavailability, which may be a key contributor to surface-ocean productivity in remote and oligotrophic parts of the oceans.

Mineral dust↗

Congruence of Clusters Defined By Whole Genome Sequencing and MALDI-TOF for Bacteria Isolated From Cleanrooms

Introduction: Oligotrophic conditions can render cleanrooms inhospitable to microbes. Despite these constraints, fungi and bacteria are frequently isolated from surfaces in astromaterials cleanrooms at the Johnson Space Center. Bacillus species are of particular concern because endospores belonging to this genus are resilient and can affect astromaterials. Current monitoring programs rely on 16S rRNA sequencing and the VITEK2 Compact system. These methods have limited power to resolve Bacillus species. Matrix-assisted laser desorption - time of flight mass spectrometry (MALDI-TOF MS), provides a rapid, low cost, method of identifying bacterial isolates and has a higher resolution than 16S rRNA sequencing, particularly for Bacillus species; however, few studies have compared this method to the industry gold standard, whole genome sequencing (WGS). Methods: Based on 16S rRNA classification, we selected 14 isolates for analysis with MALDI-TOF and WGS. Mass spectra were generated with MALDI-TOF MS and processed with custom scripts to identify clusters of closely related isolates and calculate a matrix of pairwise cosine similarity scores. Hybrid Illumina and Nanopore sequencing were used to generate draft genomes. Pairwise similarity scores were calculated from these genomes based on the average amino acid identity (AAI) predicted from single copy core genes. Congruence of clustering between these methods, was assessed by calculating adjusted Rand and Wallace coefficients. Results: Clusters of species generated from MALDI-TOF MS showed good agreement of phylotypes generated with WGS. Pairs of strains that were > 94% similar to each other in terms of predicted amino acid sequences consistently showed cosine similarities of mass spectra > 0.65 and, of the 9 clusters identified with WGS, 8 were identical with MALDI-TOF. This corresponds to an adjusted Rand index of 0.95 and a 95% confidence interval of 0.80 – 1.00 for adjusted Wallace coefficients. The only discordance was for a pair of isolates that were classified as Paenibacillus species. This pair showed relatively high similarity (0.84) in terms of MALDI-TOF MS but only 85% similarity in terms of AAI. Conclusion: This study shows that MALDI-TOF and WGS exhibit a similar ability to delineate Bacillus species isolated from cleanrooms and taxonomic units described by these two methods are consistent with one another. Since MALDI-TOF MS is low in cost and high in throughput, this approach appears to be an ideal option for routine microbial monitoring and identifying Bacillus species.

Farnaz Mazhari↗

Comprehensive Measurement of Microbial Burden in Nutrient-Deprived Cleanrooms

Spacecraft surfaces that are destined to land on potential life-harboring celestial bodies are required to be rigorously cleaned and continuously monitored for spore bioburden as a proxy for spacecraft cleanliness. The NASA standard spore assay (NSA), used for spacecraft bioburden estimates, specifically measures spores that are cultivable, aerobic, resistant to heat shock, and grow at 30˚C in a nutrient-rich medium. Since the vast majority of microorganisms cannot be cultivated using the NSA assay, it is necessary to utilize state-of-the art molecular techniques to better understand the presence of all viable microorganisms, not just those measured with the NSA. In this study, the nutrient-deprived low biomass cleanrooms, where spacecraft are assembled, were used as a surrogate to spacecraft surfaces to measure the ratio of NSA spores in relation to the total viable microorganism population to compare with a 2006 space studies report that estimates that for every 1 spore there is approximately 50,000 viable organisms. Ninety-eight surface wipe samples were collected from the spacecraft assembly facility (SAF) cleanroom at the Jet Propulsion Laboratory (JPL) over a 6-month period. The samples were processed and analyzed using classical microbiology along with molecular assays. Traditional microbiology plating methods were used to determine the cultivable bacterial, fungal, and spore populations. Molecular assays were used to determine the total organisms (TO, dead and live) and the viable organisms (VO, live). The TO was measured using adenine triphosphate (ATP) and quantitative polymerase chain reaction (qPCR) assays. The VO was measured using internal ATP, propidium monoazide (PMA)-qPCR, and flow cytometry (after staining for viable microorganisms) assays. Based on the results, it was possible to establish a ratio between spore counts and VO for each viability assay. The ATP based spore to VO ratio ranged from 149 – 746 and the bacterial PMA-qPCR assay based ratio ranged from 314 – 1491 VO. The most conservative estimate came from FACS, which estimated the ratio to be 12,091 VO per 1 NSA spore. Since archaeal (<1%) and fungal (~2%) populations were negligible, the spore to VO ratios were based on bacterial population estimates. The most conservative ratio from this study can be used as a replacement for the SSB estimate on nutrient-deprived (oligotrophic) desiccated spacecraft surfaces, to estimate the VO from NSA measurements without utilizing state-of-the art molecular methods that are costly and require more biomass than is typically found of spacecraft surfaces.

Venkateswaran, Kasthuri↗

Resolution of Maldi-Tof Compared to Whole Genome Sequencing for Identification of Bacillus Species Isolated From Cleanrooms at Nasa Johnson Space Center

The Astromaterials Acquisition and Curation Office at NASA Johnson Space Center maintains cleanrooms to archive extraterrestrial materials returned from space exploration missions. Compared to typical built environments, oligotrophic conditions make these facilities inhospitable to microbes. Despite these controls, bacteria and fungi are regularly cultured from these cleanrooms. In particular, Bacillus sp. are frequently isolated during routine microbial monitoring. Endospores associated with this genus can survive extreme environments, such as cleanrooms. This microbial contamination may affect the integrity of astromaterials.

Microbiology↗

Phytoplankton Diversity and Chemotaxonomy in Contrasting North Pacific Ecosystems

Background. Phytoplankton is the base of majority of ocean ecosystems. It is responsible for half of the global primary production, and different phytoplankton taxa have a unique role in global biogeochemical cycles. In addition, phytoplankton abundance and diversity are highly susceptible to climate induced changes, hence monitoring of phytoplankton and its diversity is important and necessary. Methods. Water samples for phytoplankton and photosynthetic pigment analyses were collected in boreal winter 2017, along transect in the North Pacific Subtropical Gyre (NPSG) and the California Current System (CCS). Phytoplankton community was analyzed using light and scanning electron microscopy and photosynthetic pigments by high-performance liquid chromatography. To describe distinct ecosystems, monthly average satellite data of MODIS Aqua Sea Surface temperature and Chlorophyll a concentration, as well as Apparent Visible Wavelength were used. Results. A total of 207 taxa have been determined, mostly comprised of coccolithophores (35.5%), diatoms (25.2%) and dinoflagellates (19.5%) while cryptophytes, phytoflagellates and silicoflagellates were included in the group ``others" (19.8%). Phytoplankton spatial distribution was distinct, indicating variable planktonic dispersal rates and specific adaptation to ecosystems. Dinoflagellates, and nano-scale coccolithophores dominated NPSG, while micro-scale diatoms, and cryptophytes prevailed in CCS. A clear split between CCS and NPSG is evident in dendogram visualising LINKTREE constrained binary divisive clustering analysis done on phytoplankton counts and pigment oncentrations. Of all pigments determined, alloxanthin, zeaxanthin, divinyl chlorophyll b and lutein have highest correlation to phytoplankton counts. Conclusion. Combining chemotaxonomy and microscopy is an optimal method to determine phytoplankton diversity on a large-scale transect. Distinct communities between the two contrasting ecosystems of North Pacific reveal phytoplankton groups specific adaptations to trophic state, and support the hypothesis of shift from microto nano-scale taxa due to sea surface temperatures rising, favoring stratification and oligotrophic conditions.

oceanography↗

INSIGHTS INTO MARINE UNICELLULAR CYANOBACTERIAL AND NON-CYANOBACTERIAL DIAZOTROPHS THROUGH SINGLE-CELL ANALYSES

Nutrient availability affects primary productivity, and nitrogen (N) is the limiting nutrient over large regions of the ocean. Biological N fixation (BNF) is the conversion of atmospheric dinitrogen (N2) to bioavailable forms of N (ammonium and amino acids). A select group of Bacteria and Archaea, known as diazotrophs, are the only organisms capable of this conversion. BNF is a critical source of N in nutrient-depleted (oligotrophic) marine waters. As such, BNF studies are a fundamental component for better understanding primary productivity and carbon cycling, as well as parameterizing predictive future global ocean models. Unicellular diazotrophs are widespread in marine systems and, in some cases, important contributors of BNF. This dissertation investigates unicellular diazotrophs at the single-cell level, including: UCYN-A, a cyanobacterial diazotroph that lives in symbiosis with an algal host, non-cyanobacterial diazotrophs (NCDs), which are putative heterotrophs, as well as a specific NCD known as Gamma A, a commonly occurring organism throughout the tropics and subtropics that belongs to the class Gammaproteobacteria. The single-cell investigations used in these studies can provide high-resolution insights that are not possible from community-level analyses of BNF, such as the N2 fixation rate of individual cells and cell morphology. Singlecell nanoscale secondary ion mass spectrometry (nanoSIMS) analyses were used in chapter 2 and 3 to investigate the cell-specific N2 fixation rates of (2) UCYN-A in the viii cold waters of the Bering and Arctic Seas where N2 fixation was not previously believed to be possible, and (3) NCDs in the North Pacific Subtropical Gyre where we demonstrated the first direct evidence of NCD N2 fixation on open ocean particles. Chapter 4 focused on the uncultivated NCD, Gamma A, using geneFISH to visualize single cells in natural seawater samples. We successfully identified Gamma A by developing a fluorescent in situ hybridization (FISH) probe targeting the diazotrophic functional marker gene nifH, revealing Gamma A cells are most often attached to particles. Together these studies provide new insights into the distributions, activities, and physiologies of geographically widespread, unicellular diazotrophs, thus improving our understanding of their roles as sources of fixed N in the surface ocean.

Harding, K.↗

Positive correlation between wood δ 15 N and stream nitrate concentrations in two temperate deciduous forests

Abstract A limitation to understanding drivers of long-term trends in terrestrial nitrogen (N) availability in forests and its subsequent influence on stream nitrate export is a general lack of integrated analyses using long-term data on terrestrial and aquatic N cycling at comparable spatial scales. Here we analyze relationships between stream nitrate concentrations and wood δ 15 N records (n = 96 trees) across five neighboring headwater catchments in the Blue Ridge physiographic province and within a single catchment in the Appalachian Plateau physiographic province in the eastern United States. Climatic, acidic deposition, and forest disturbance datasets were developed to elucidate the influence of these factors on terrestrial N availability through time. We hypothesized that spatial and temporal variation of terrestrial N availability, for which tree-ring δ 15 N records serve as a proxy, affects the variation of stream nitrate concentration across space and time. Across space at the Blue Ridge study sites, stream nitrate concentration increased linearly with increasing catchment mean wood δ 15 N. Over time, stream nitrate concentrations decreased with decreasing wood δ 15 N in five of the six catchments. Wood δ 15 N showed a significant negative relationship with disturbance and acidic deposition. Disturbance likely exacerbated N limitation by inducing nitrate leaching and ultimately enhancing vegetative uptake. As observed elsewhere, lower rates of acidic deposition and subsequent deacidification of soils may increase terrestrial N availability. Despite the ephemeral modifications of terrestrial N availability by these two drivers and climate, long-term declines in terrestrial N availability were robust and have likely driven much of the declines in stream nitrate concentration throughout the central Appalachians.

54 ENVIRONMENTAL SCIENCES↗

Differences in substrate use linked to divergent carbon flow during litter decomposition

ABSTRACT Discovering widespread microbial processes that create variation in soil carbon (C) cycling within ecosystems may improve soil C modeling. Toward this end, we screened 206 soil communities decomposing plant litter in a common garden microcosm environment and examined features linked to divergent patterns of C flow. C flow was measured as carbon dioxide (CO2) and dissolved organic carbon (DOC) from 44-days of litter decomposition. Two large groups of microbial communities representing ‘high’ and ‘low’ DOC phenotypes from original soil and 44-day microcosm samples were down-selected for fungal and bacterial profiling. Metatranscriptomes were also sequenced from a smaller subset of communities in each group. The two groups exhibited differences in average rate of CO2 production, demonstrating that the divergent patterns of C flow arose from innate functional constraints on C metabolism, not a time-dependent artefact. To infer functional constraints, we identified features – traits at the organism, pathway or gene level – linked to the high and low DOC phenotypes using RNA-Seq approaches and machine learning approaches. Substrate use differed across the high and low DOC phenotypes. Additional features suggested that divergent patterns of C flow may be driven in part by differences in organism interactions that affect DOC abundance directly or indirectly by controlling community structure.

59 BASIC BIOLOGICAL SCIENCES↗

Identifying Meteorologic and Oceanic Conditions Contributing to a Unique Phytoplankton Bloom Occurrence in Micronesia during October 2013

On the first several days of October 2013, daily chlorophyll a (chl a) data acquired by the Moderate Resolution Imaging Spectroradiometer (MODIS) onboard the National Aeronautics and Space Administration (NASA) Aqua satellite detected a marked increase in chlorophyll a concentrations (chl a) in the vicinity of Chuuk Lagoon and the Federated States of Micronesia. Such an increase, likely indicative of a phytoplankton bloom, has not been observed in this location at any other time during the MODIS-Aqua mission, which commenced in 2002 and continues to present. Examination of sea surface wind data from the Modern Era Retrospective analysis for Research and Applications Version 2 (MERRA-2) reanalysis dataset indicated that the region experienced a sequence of alternating elevated and very low wind speed events prior to the observation of the bloom. The influence of the winds can be seen in MERRA-2 sea surface skin temperature data. Elevated windspeeds for several days likely induced a mixing of deeper waters with higher nutrient levels to the surface, which was followed by stratification and phytoplankton growth during low wind intervals and finally transport induced by a brief high windspeed event. Analysis of hourly MERRA-2 maximum windspeed data over a 40-year period indicated that this sequence was climatologically rare.

Remote Sensing↗