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All sourcesNASA NTRSDOE OSTI
DOE OSTI2015

False Negative Rates of a Macrofoam-Swab Sampling Method with Low Surface Concentrations of Two Bacillus anthracis Surrogates via Real-Time PCR

Surface sampling for Bacillus anthracis spores has traditionally relied on detection via bacterial cultivation methods. Although effective, this approach does not provide the level of organism specificity that can be gained through molecular techniques. False negative rates (FNR) and limits of detection (LOD) were determined for two B. anthracis surrogates with modified rapid viability-polymerase chain reaction (mRV-PCR) following macrofoam-swab sampling. This study was conducted in parallel with a previously reported study that analyzed spores using a plate-culture method. B. anthracis Sterne (BAS) or B. atrophaeus Nakamura (BG) spores were deposited onto four surface materials (glass, stainless steel, vinyl tile, and plastic) at nine target concentrations (2 to 500 spores/coupon; 0.078 to 19.375 colony-forming units [CFU] per cm 2 ). Mean FNR values for mRV-PCR analysis ranged from 0 to 0.917 for BAS and 0 to 0.875 for BG and increased as spore concentration decreased (over the concentrations investigated) for each surface material. FNRs based on mRV-PCR data were not statistically different for BAS and BG, but were significantly lower for glass than for vinyl tile. FNRs also tended to be lower for the mRV-PCR method compared to the culture method. The mRV-PCR LOD 95 was lowest for glass (0.429 CFU/cm 2 with BAS and 0.341 CFU/cm 2 with BG) and highest for vinyl tile (0.919 CFU/cm 2 with BAS and 0.917 CFU/cm 2 with BG). These mRV-PCR LOD 95 values were lower than the culture values (BAS: 0.678 to 1.023 CFU/cm 2 and BG: 0.820 to 1.489 CFU/cm 2 ). The FNR and LOD₉₅ values reported in this work provide guidance for environmental sampling of Bacillus spores at low concentrations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Characterization Techniques for TRU Waste from the Analytical Laboratory Hot Cells at Idaho National Laboratory - 19610

Since the first reactor went hot in 1942, there has been a never ending desire to improve the nuclear reactor and the fuel that it burns. The Idaho National Laboratory has been a key player in reactor and fuel development. As more research projects begin, the spectrum of waste parameters grows. Over the years, the Analytical laboratory (AL) at the INL Materials and Fuels Complex (MFC) has supported a large range of various research projects. Each project brings its own challenge and analytical technique that, in turn, yields its own unique waste parameters. The AL Hot Cells at INL consist of 6 small cells that can support around 12 different fuel research projects at a time along with infrastructure and environmental samples. Each different project yields different levels of transuranic (TRU) material, RCRA constituents, dose, and contamination levels. The waste parameters from each project dictate a path forward for the waste as Low-Level Waste (LLW), Contact Handled TRU (CH-TRU), Mixed Contact Handled TRU (M-CH-TRU), Remote Handled TRU (RH-TRU), or Mixed Remote Handled TRU (M-RHTRU). With visual examination techniques and process knowledge as an aid, there have been waste handling methods developed that allow debris and sample material to be segregated. Unique characterization mechanisms have been applied for each debris and sample material that are generally more accurate than smear and surface contamination data. For debris found in the Hot Cells, analytical data is gathered and conservatively summarized for each fuel type and research method. This consists of data from Quadrupole Inductively Coupled Plasma Mass Spectrometry (Q-ICP-MS), Gamma Spectroscopy, and some data verification through Thermal Ionization Mass Spectrometry (TIMS) and Multi-Collector Inductively Coupled Plasma Mass Spectrometry (MC-ICP-MS). This data gives a range of TRU isotope concentrations. Fuel age is taken into account to determine the dominant TRU activity isotopes. Cesium concentrations are also gathered. Each parameter/fuel type then ties a measured Cesium number to TRU content based on historical analytical data. Debris in the hot cells is segregated by project and packaged in individual 2 gallon metal paint cans. Each can is counted by gamma spectroscopy in a calculated and measured geometry. Using process knowledge to determine the fuel or project type, newly counted cesium numbers can be applied to the historic analytical data and TRU content can be estimated. The TRU content allows waste debris to be segregated between CH-TRU and RH-TRU. Packaging techniques can allow for the minimization of RHTRU waste. Sample material received into the AL must be dissolved into a liquid matrix in order to be analyzed in most cases. When analysis is complete, these liquids go through a consolidation, complexation, neutralization, and solidification process where real time analytical data can be used to calculate TRU content and RCRA constitutes. These calculations allow for no further analytical data to be gathered. The waste matrix is considered stable and acceptable by all Acceptable Knowledge (AK) documents. The unique techniques used in characterizing and handling the Analytical Laboratories Hot Cell waste can be applied to various situations and various laboratories. They allow for more accurate data for waste constituents and also for the minimization of RH-TRU waste. (authors)

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗
DOE OSTI2019

Automated thermal extraction-desorption gas chromatography mass spectrometry: A multifunctional tool for comprehensive characterization of polymers and their degradation products

The TED-GC–MS analysis is a two-step method. A sample is first decomposed in a thermogravimetric analyzer (TGA) and the gaseous decomposition products are then trapped on a solid-phase adsorber. Subsequently, the solid-phase adsorber is analyzed with thermal desorption gas chromatography mass spectrometry (TDU-GC–MS). This method is ideally suited for the analysis of polymers and their degradation processes. A new entirely automated system is introduced which enables high sample throughput and reproducible automated fractioned collection of decomposition products. The fractionated collection together with low temperatures reduces the risk of contamination, improves instrumental stability and minimizes maintenance efforts. Through variation of the two main parameters (purge gas flow and heating rate) it is shown how the extraction process can be optimized. By measuring the decomposition products of polyethylene it is demonstrated that compounds with masses of up to 434 Da can be detected. This is achieved despite the low temperature (˜40 °C) of the solid-phase adsorber and the low thermal desorption temperature of 200 °C in the TDU unit. It is now shown that automated TED-GC–MS represents a new flexible multi-functional method for comprehensive polymer analyses. Comparable polymer characterization was previously only achievable through a combination of multiple independent analytical methods. This is demonstrated by three examples focused on practical challenges in materials analysis and identification: The first one is the analysis of wood plastic composites for which the decomposition processes of the polymer and the bio polymer (wood) could be clearly distinguished by fractionated collection using sequential adsorbers. Secondly, a fast quantitative application is shown by determining the weight concentrations of an unknown polyolefin blend through comparison with a reference material. Additionally, the determination of microplastic concentrations in environmental samples is becoming an increasingly important analytical necessity. It is demonstrated that with TED-GC–MS calibration curves showing good linearity for the most important precursors for microplastic, even complex matrix materials (suspended particulate matter) can be successfully analyzed.

36 MATERIALS SCIENCE↗
DOE OSTI2013

Defining cell culture conditions to improve human norovirus infectivity assays

Significant difficulties remain for determining whether human noroviruses (hNoV) recovered from water, food, and environmental samples are infectious. Three-dimensional tissue culture of human intestinal cells has shown promise in developing an infectivity assay, but reproducibility, even within a single laboratory, remains problematic. From the literature and our observations, we hypothesized that the common factors that leads to more reproducible hNoV infectivity in vitro requires that the cell line be 1) of human gastrointestinal origin, 2) expresses apical microvilli, and 3) be a positive secretor cell line. The C 2 BBe1 cell line, which is a brush-border producing clone of Caco-2, meets these three criteria. When challenged with Genogroup II viruses, we observed a 2 Log10 increase in viral RNA titer. A passage experiment with GII viruses showed evidence of the ability to propagate hNoV by both reverse transcription quantitative PCR (qRT-PCR) and microscopy. Using 3-D C 2 BBe1 cells improves reproducibility of the infectivity assay for hNoV, but the assay can still be variable. Two sources of variability include the cells themselves (mixed phenotypes of small and large intestine) and initial titer measurements using quantitative reverse transcription PCR (qRT-PCR) that measures all RNA vs. plaque assays that measure infectious virus.

C2BBe1 cells↗
DOE OSTI2011

Underwater Application of Quantitative PCR on an Ocean Mooring

The Environmental Sample Processor (ESP) is a device that allows for the underwater, autonomous application of DNA and protein probe array technologies as a means to remotely identify and quantify, in situ, marine microorganisms and substances they produce. Here, we added functionality to the ESP through the development and incorporation of a module capable of solid-phase nucleic acid extraction and quantitative PCR (qPCR). Samples collected by the instrument were homogenized in a chaotropic buffer compatible with direct detection of ribosomal RNA (rRNA) and nucleic acid purification. From a single sample, both an rRNA community profile and select gene abundances were ascertained. To illustrate this functionality, we focused on bacterioplankton commonly found along the central coast of California and that are known to vary in accordance with different oceanic conditions. DNA probe arrays targeting rRNA revealed the presence of 16S rRNA indicative of marine crenarchaea, SAR11 and marine cyanobacteria; in parallel, qPCR was used to detect 16S rRNA genes from the former two groups and the large subunit RuBisCo gene (rbcL) from Synecchococcus. The PCR-enabled ESP was deployed on a coastal mooring in Monterey Bay for 28 days during the spring-summer upwelling season. The distributions of the targeted bacterioplankon groups were as expected, with the exception of an increase in abundance of marine crenarchaea in anomalous nitrate-rich, low-salinity waters. The unexpected co-occurrence demonstrated the utility of the ESP in detecting novel events relative to previously described distributions of particular bacterioplankton groups. The ESP can easily be configured to detect and enumerate genes and gene products from a wide range of organisms. This study demonstrated for the first time that gene abundances could be assessed autonomously, underwater in near real-time and referenced against prevailing chemical, physical and bulk biological conditions.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2011

High-Throughput In Vitro Glycoside Hydrolase (HIGH) Screening for Enzyme Discovery

A high-throughput protein-expression and screening method (HIGH method, see picture) provides a rapid approach to the discovery of active glycoside hydrolases in environmental samples. Finally, HIGH screening combines cloning, protein expression, and enzyme hydrolysis in one pot; thus, the entire process from gene expression to activity detection requires only three hours.

Kim, Tae-Wan↗
DOE OSTI2022

Position-specific carbon isotope analysis of serine by gas chromatography/Orbitrap mass spectrometry, and an application to plant metabolism

Position-specific 13 C/ 12 C ratios within amino acids remain largely unexplored in environmental samples due to methodological limitations. We hypothesized that natural-abundance isotope patterns in serine may serve as a proxy for plant metabolic fluxes including photorespiration. Here we describe an Orbitrap method optimized for the position-specific carbon isotope analysis of serine to test our hypothesis and discuss the generalizability of this method to other amino acids. Position-specific carbon isotope ratios of serine were measured using a Thermo Scientific™ Q Exactive™ GC Orbitrap™. Amino acids were hydrolyzed from Arabidopsis biomass, purified from potential matrix interferences, and derivatized alongside standards. Derivatized serine (N,O-bis(trifluoroacetyl)methyl ester) was isolated using gas chromatography, trapped in a reservoir, and purged into the electron ionization source over tens of minutes, producing fragment ions containing different combinations of atoms from the serine-derivative molecule. The 13 C/ 12 C ratios of fragments with monoisotopic masses of 110.0217, 138.0166, and 165.0037 Da were monitored in the mass analyzer and used to calculate position-specific δ 13 C values relative to a working standard. This methodology constrains position-specific δ 13 C values for nanomole amounts of serine isolated from chemically complex mixtures. The δ 13 C values of fragment ions of serine were characterized with ≤1‰ precisions, leading to propagated standard errors of 0.7–5‰ for each carbon position. Position-specific δ 13 C values differed by up to ca 28 ± 5‰ between serine molecules hydrolyzed from plants grown under contrasting pCO 2 , selected to promote different fluxes through photosynthesis and photorespiration. The method was validated using pure serine standards characterized offline. Here this study presents the first Orbitrap-based measurements of natural-abundance, position-specific carbon isotope variation in an amino acid isolated from a biological matrix. We present a method for the precise characterization of isotope ratios in serine and propose applications probing metabolism in plants. We discuss the potential for extending these approaches to other amino acids, paving the way for novel applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Substrate, temperature, and geographical patterns among nearly 2000 natural yeast isolates

Yeasts have broad importance as industrially and clinically relevant microbes and as powerful models for fundamental research, but we are only beginning to understand the roles yeasts play in natural ecosystems. Yeast ecology is often more difficult to study compared to other, more abundant microbes, but growing collections of natural yeast isolates are beginning to shed light on fundamental ecological questions. In this work, we used environmental sampling and isolation to assemble a dataset of 1962 isolates collected from throughout the contiguous United States of America (USA) and Alaska, which were then used to uncover geographic patterns, along with substrate and temperature associations among yeast taxa. We found some taxa, including the common yeasts Torulaspora delbrueckii and Saccharomyces paradoxus, to be repeatedly isolated from multiple sampled regions of the USA, and we classify these as broadly distributed cosmopolitan yeasts. A number of yeast taxon-substrate associations were identified, some of which were novel and some of which support previously reported associations. Further, we found a strong effect of isolation temperature on the phyla of yeasts recovered, as well as for many species. We speculate that substrate and isolation temperature associations reflect the ecological diversity of and niche partitioning by yeast taxa.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Concepts for Alpha Coincidence Detection

The effectiveness of conventional measurement techniques for environmental monitoring is limited by background and other interferences. We are exploring a new measurement approach involving the detection of α particles in coincidence with conversion electrons as a means to simultaneously assay environmental samples for actinides without chemical separation. The initial target isotopes studied in this work are 238Pu, 239Pu, 240Pu and 241Am. We explore various aspects of the design, such as impact of the mounting of the source material, resolution requirements and impact of a background on isotopic uncertainties. We conclude that a dual gas proportional counter and a dual-sided, large-area silicon detector could provide similar performance for the measurement scenario examined.

Warren, Glen A.↗
DOE OSTI2017

Effects of urban debris material on the extraction chromatographic separation of strontium—part I: steel

An incident involving a radiation dispersal device (RDD) would most likely occur in an urban or metropolitan area. However, the majority of radiochemical separation schemes available have been developed for environmental samples that are not necessarily representative of those found in an urban environment. 90 Sr is one of the possible radioisotopes that could be used in an RDD. It is therefore important to find efficient and accurate ways to separate it from debris in the area of deployment. Finally, this research demonstrates that the presence of steel in dissolved samples shows no appreciable interference with extraction chromatographic separations. Instead, it indicates that the separation could actually be improved by the presence of the steel constituents.

36 MATERIALS SCIENCE↗
DOE OSTI2017

Effects of urban debris material on the extraction chromatographic separation of strontium: Part II: cement and concrete

The majority of radiochemical separation schemes available have been developed for environmental samples that are not necessarily representative of those found in an urban environment. However, it is much more likely that an incident involving a radiation dispersal device (RDD) would occur in an urban or metropolitan area. It is unclear if the currently available separation schemes would be effective in such an event. It is therefore important to determine if the current schemes would be adequate, or to find efficient and accurate ways to separate radiological material from urban debris. One important radiological material that could be used in an RDD is 90 Sr. Part I of this work investigated the effects steel had on strontium separations, while this work investigates cement and concrete. This research demonstrates that the individual elements present in a cement and concrete sample matrix can give rise to significant interferences with extraction chromatographic separations. In conclusion, solutions of the constituents mixed in representative ratios; however, show fewer problems

36 MATERIALS SCIENCE↗
DOE OSTI2026

High-gradient magnetic filtration of UO 2 colloids from aqueous suspensions

Environmental samples often contain innocuous components that make trace actinide analysis difficult and expensive. Therefore, it is expedient to separate these materials from actinide particles. One characteristic of many actinides is that they are paramagnetic. High-gradient magnetic filtration (HGMF) is a non-destructive technique that selectively captures magnetic constituents from a matrix containing non-magnetic species. Here, in this work, a novel HGMF device made of a permanent magnet array was used to effectively separate UO 2 particles from undesirable soil species. Up to 88% of the UO 2 particles present in suspensions were effectively captured by HGMF, resulting in their enrichment and a substantial reduction in the concentrations of other constituents.

Actinide separations↗
DOE OSTI2025

Uncertainty quantification for nuclear forensics with population analyses

Although neural networks offer cutting-edge predictive power, their deployment in high-consequence nuclear forensic applications is limited, partly because of their black-box nature. Incorporating robust uncertainty quantification methods into the predictive frameworks of neural networks is progress towards their future deployment in such scenarios. This work integrates uncertainty quantification into neural networks for nuclear reactor core-average burnup estimation from simulated environmental samples. We test two regimes (homogeneous and heterogeneous events) on DeepSets and Set Transformer architectures, we find both quantify predictive uncertainty effectively, but Set Transformer excels in partitioning latent events, offering superior predictive power and more informative uncertainty estimates.

Hatton, Conner [ORNL] (ORCID:0009000804970959)↗
DOE OSTI2018

The ionic liquid 1-octyl-3-methylimidazolium (M8OI) is an activator of the human estrogen receptor alpha

Highlights: • M8OI activates the human ERα and induces TFF1 in MCF7 cells. • M8OI is a super-activator of the mouse ERβ variant 2 receptor. • M8OI does not activate the human ERβ. Recent environmental sampling around a landfill site in the UK demonstrated that unidentified xenoestrogens were present at higher levels than control sites; that these xenoestrogens were capable of super-activating (resisting ligand-dependent antagonism) the murine variant 2 ERβ and that the ionic liquid 1-octyl-3-methylimidazolium chloride (M8OI) was present in some samples. To determine whether M8OI was a contributor to the xenoestrogen pool in the soils, activation of human estrogen receptors by M8OI was examined. M8OI activated the human ERα in MCF7 cells in a dose-response manner. These effects were inhibited by the ER antagonist ICI182780; occurred in the absence of any metabolism of M8OI and were confirmed on examination of ER-dependent induction of trefoil factor 1 mRNA in MCF7 cells. M8OI also super-activated the murine variant 2 ERβ in a murine hepatopancreatobiliary cell line. The human ERβ was not activated by M8OI when expressed in HEK293 cells. These data demonstrate that M8OI is a xenoestrogen capable of activating the human ERα and super-activating the murine variant 2 ERβ.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2011

Influence of Combustion Conditions on Yields of Solvent-Extractable Anhydrosugars and Lignin Phenols in Chars: Implications for Characterizations of Biomass Combustion Residues

Anhydrosugars, such as levoglucosan and its isomers (mannosan, galactosan), as well as the solvent-extractable lignin phenols (methoxylated phenols) are thermal degradation products of cellulose/hemicellulos and lignin, respectively. These two groups of biomarkers are often used as unique tracers of combusted biomass inputs in diverse environmental media. However, detail characterization of the relative proportion and signatures of these compounds in highly heterogeneous plant-derived chars/charcoals are still scarce. Here we conducted a systematic study to investigate the yields of solvent-extractable anhydrosugars and lignin phenols in twenty-five lab-made chars produced from different plant materials under different combustion conditions. Solvent extractable anhydrosugars and lignin phenols were only observed in chars formed below 350 C and yields were variable across different combustion temperatures. The yields mannosan (M) and galactosan (G) decreased more rapidly than those of levoglucosan (L) under increasing combustion severity (temp. and duration), resulting in variable L/M and L/(M+G) ratios, two diagnostic ratios often used for identification of combustion sources (e.g. hardwoods vs. softwoods vs. grasses). On the other hand, the results of this study suggest that the ratios of the major solvent-extractable lignin phenols (vanillyls (V), syringyls (S), cinnamyls (C)) provide additional source reconstruction potential despite observed variations with combustion temperature. We thus propose using a property property plot (L/M vs. S/V) as an improved means for source characterization of biomass combustion residues. The L/M-S/V plot has shown to be effective in environmental samples (soil organic matter, atmospheric aerosols) receiving substantial inputs of biomass combustion residues.

09 BIOMASS FUELS↗
DOE OSTI2026

Evaluation of chromatographic resins for high efficacy separation of radium from chemical analogues

The separation of Radium (Ra) from interfering chemical analogues is of major importance for both the accurate measurement of Ra-226 in environmental samples and the preparation of radium targets destined for medical isotope production. The adsorption behavior of Ra, Barium (Ba), and Strontium (Sr) was investigated on multiple crown-ether-based proprietary extraction chromatographic resins developed by TrisKem Int. Among these, the TK101 resin, characterized by a novel room-temperature ionic liquid (RTIL) dual extractant–solvent system, demonstrated enhanced affinity and selectivity, particularly at low acid concentrations, achieving a D w of 3477 ± 73 for Ra in 0.04 M HNO₃, whereas the maximum Ra D w obtained with the traditional Sr resin in 8 M HNO 3 was only 12.33 ± 0.4. In 0.04 M HNO 3 , a Ra distribution ratio (D w ) of 776 was achieved within 1 min, indicating rapid uptake kinetics. The adsorption capacities were estimated at 19.6 ± 0.1 mg/g for Ba and 12.0 ± 0.2 mg/g for Sr. However, competing alkali metals significantly reduced alkaline earth metal uptake, indicating that the TK101 resin is best suited for radium purification following a preconcentration step to minimize matrix effects in high salinity samples. Dynamic column studies confirmed the strong chromatographic performance of the TK101 resin, with no significant Ba or Sr breakthrough in the Ra fraction and a Ra recovery of 87 ± 2.8%. These results demonstrate the potential of the TK101 resin for selective radium separation in analytical and radiochemical applications.

and nuclear chemistry↗
DOE OSTI2024

Visualizing metagenomic and metatranscriptomic data: A comprehensive review

The fields of Metagenomics and Metatranscriptomics involve the examination of complete nucleotide sequences, gene identification, and analysis of potential biological functions within diverse organisms or environmental samples. Despite the vast opportunities for discovery in metagenomics, the sheer volume and complexity of sequence data often present challenges in processing analysis and visualization. This article highlights the critical role of advanced visualization tools in enabling effective exploration, querying, and analysis of these complex datasets. Emphasizing the importance of accessibility, the article categorizes various visualizers based on their intended applications and highlights their utility in empowering bioinformaticians and non-bioinformaticians to interpret and derive insights from meta-omics data effectively.

59 BASIC BIOLOGICAL SCIENCES↗