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Long-term Investigations of {sup 134}Cs and {sup 137}Cs Activity Concentrations in Honey from Croatia

This paper presents the results of long-term post-Chernobyl investigations of {sup 134}Cs and {sup 137}Cs activity concentrations in multifloral and chestnut honey sampled in northwest Croatia. For both radionuclides, the activity concentrations peaked in May 1986, decreasing exponentially until the mid-1990 s, when they fell under the detection limit for both radionuclides. After the Fukushima-Daiichi accident in 2011, the presence of both radionuclides in honey was detected once again. The ecological half-life was estimated to be 1.67 and 1.45 years for {sup 137}Cs and {sup 134}Cs, respectively. The correlation between {sup 134}Cs and {sup 137}Cs activity concentrations in fallout and honey was very good, indicating fallout to be the main source of honey contamination. The observed {sup 134}Cs/{sup 137}Cs activity ratio in honey was similar to the ratio found in other environmental samples. The estimated collective effective doses for the Croatian population incurred by honey consumption indicate that honey was not a critical pathway for the transfer of {sup 134}Cs and {sup 137}Cs from fallout to humans.

54 ENVIRONMENTAL SCIENCES↗

Background contamination by coplanar polychlorinated biphenyls (PCBs) in trace level high resolution gas chromatography/high resolution mass spectrometry (HRGC/HRMS) analytical procedures

The addition of the "dioxin-like" polychlorinated biphenyl (PCB) congeners to the assessment of risk associated with the 2,3,7,8-chlorine substituted dioxins and furans has dramatically increased the number of laboratories worldwide that are developing analytical procedures for their detection and quantitation. Most of these procedures are based on established sample preparation and analytical techniques employing high resolution gas chromatography/high resolution mass spectrometry (HRGC/HRMS), which are used for the analyses of dioxin/furans at low parts-per-trillion (ppt) levels. A significant and widespread problem that arises when using these sample preparation procedures for the analysis of coplanar PCBs is the presence of background levels of these congeners. Industrial processes, urban incineration, leaking electrical transformers, hazardous waste accidents, and improper waste disposal practices have released appreciable quantities of PCBs into the environment. This contamination has resulted in the global distribution of these compounds via the atmosphere and their ubiquitous presence in ambient air. The background presence of these compounds in method blanks must be addressed when determining the exact concentrations of these and other congeners in environmental samples. In this study reliable procedures were developed to accurately define these background levels and assess their variability over the course of the study. The background subtraction procedures developed and employed increase the probability that the values reported accurately represent the concentrations found in the samples and were not biased due to this background contamination.

Polychlorinated Biphenyls/analysis/metabolism↗

Targeted amplification for enhanced detection of biothreat agents by next-generation sequencing

Background: Historically, identification of causal agents of disease has relied heavily on the ability to culture the organism in the laboratory and/or the use of pathogen-specific antibodies or sequence-based probes. However, these methods can be limiting: Even highly sensitive PCR-based assays must be continually updated due to signature degradation as new target strains and near neighbors are sequenced. Thus, there has been a need for assays that do not suffer as greatly from these limitations and/or biases. Recent advances in library preparation technologies for Next-Generation Sequencing (NGS) are focusing on the use of targeted amplification and targeted enrichment/ capture to ensure that the most highly discriminating regions of the genomes of known targets (organism-unique regions and/or regions containing functionally important genes or phylogenetically-discriminating SNPs) will be sequenced, regardless of the complex sample background. Results: In the present study, we have assessed the feasibility of targeted sequence enhancement via amplification to facilitate detection of a bacterial pathogen present in low copy numbers in a background of human genomic material. Our results indicate that the targeted amplification of signature regions can effectively identify pathogen genomic material present in as little as 10 copies per ml in a complex sample. Importantly, the correct species and strain calls could be made in amplified samples, while this was not possible in unamplified samples. Conclusions: The results presented here demonstrate the efficacy of a targeted amplification approach to biothreat detection, using multiple highly-discriminative amplicons per biothreat organism that provide redundancy in case of variation in some primer regions. Importantly, strain level discrimination was possible at levels of 10 genome equivalents. Similar results could be obtained through use of panels focused on the identification of amplicons targeted for specific genes or SNPs instead of, or in addition to, those targeted for specific organisms (ongoing gene-targeting work to be reported later). Note that without some form of targeted enhancement, the enormous background present in complex clinical and environmental samples makes it highly unlikely that sufficient coverage of key pathogen(s) present in the sample will be achieved with current NGS technology to guarantee that the most highly discriminating regions will be sequenced.

59 BASIC BIOLOGICAL SCIENCES↗

Determination of methane in ambient air by multiplex gas chromatography

A multiplex gas chromatographic technique for the determination of methane in ambient air over extended periods is reported. A modest gas chromatograph which uses air as the carrier gas was modified by adding a silver oxide sample modulator for multiplex operation. The modulator selectively catalyzes the decomposition of methane in air. The resulting analytical system requires no consumables beyond power. A profile of the methane concentration in this laboratory was obtained for an 8-day period. During this period, methane concentration varied with an approximately daily period from a low of 1.53 + or - 0.60 ppm to a high of 4.63 + or - 0.59 ppm over the entire 8 days. Some of the measured concentrations are higher than those reported elsewhere indicating the presence of some local source or sources for methane. This work has demonstrated the utility of a relatively simple multiplex gas chromatograph for the analysis of environmental samples. The technique should be applicable to other trace components in air through use of other selective modulators.

Valentin, J. R.↗

Streamlined Approach for Environmental Restoration (SAFER) Plan for Corrective Action Unit 411: Double Tracks Plutonium Dispersion (Nellis), Nevada Test and Training Range, Nevada (Rev. 0)

This Streamlined Approach for Environmental Restoration (SAFER) Plan addresses the actions needed to achieve closure for Corrective Action Unit (CAU) 411, Double Tracks Plutonium Dispersion (Nellis). CAU 411 is located on the Nevada Test and Training Range and consists of a single corrective action site (CAS), NAFR-23-01, Pu Contaminated Soil. There is sufficient information and historical documentation from previous investigations and the 1996 interim corrective action to recommend closure of CAU 411 using the SAFER process. Based on existing data, the presumed corrective action for CAU 411 is clean closure. However, additional data will be obtained during a field investigation to document and verify the adequacy of existing information, and to determine whether the CAU 411 closure objectives have been achieved. This SAFER Plan provides the methodology to gather the necessary information for closing the CAU. The results of the field investigation will be presented in a closure report that will be prepared and submitted to the Nevada Division of Environmental Protection (NDEP) for review and approval. The site will be investigated based on the data quality objectives (DQOs) developed on November 20, 2014, by representatives of NDEP, the U.S. Air Force (USAF), and the U.S. Department of Energy (DOE), National Nuclear Security Administration Nevada Field Office. The DQO process was used to identify and define the type, amount, and quality of data needed to determine whether CAU 411 closure objectives have been achieved. The following text summarizes the SAFER activities that will support the closure of CAU 411; Collect environmental samples from designated target populations to confirm or disprove the presence of contaminants of concern (COCs) as necessary to supplement existing information; If COCs are no longer present, establish clean closure as the corrective action; If COCs are present, the extent of contamination will be defined and further corrective actions will be evaluated with the stakeholders (NDEP, USAF); and Confirm the preferred closure option is sufficient to protect human health and the environment.

54 ENVIRONMENTAL SCIENCES↗

Determination of the gram-positive bacterial content of soils and sediments by analysis of teichoic acid components

Many gram-positive bacteria form substituted polymers of glycerol and ribitol phosphate esters known as teichoic acids. Utilizing the relative specificity of cold concentrated hydrofluoric acid in the hydrolysis of polyphosphate esters it proved possible to quantitatively assay the teichoic acid-derived glycerol and ribitol from gram-positive bacteria added to various soils and sediments. The lipids are first removed from the soils or sediments with a one phase chloroform-methanol extraction and the lipid extracted residue is hydrolyzed with cold concentrated hydrofluoric acid. To achieve maximum recovery of the teichoic acid ribitol, a second acid hydrolysis of the aqueous extract is required. The glycerol and ribitol are then acetylated after neutralization and analyzed by capillary gas-liquid chromatography. This technique together with measures of the total phospholipid, the phospholipid fatty acid, the muramic acid and the hydroxy fatty acids of the lipopolysaccharide lipid A of the gram-negative bacteria makes it possible to describe the community structure environmental samples. The proportion of gram-positive bacteria measured as the teichoic acid glycerol and ribitol is higher in soils than in sediments and increases with depth in both.

Ribitol/analogs & derivatives↗

Virus-Like Particle: Evolving Meanings in Different Disciplines

Virus-like particle (VLP) is a term that has been in use for about 80 years. Usually, VLP has meant a particle that is like a virus, generally by appearance, but without either proven or actual virus functionality. Initially VLP referred to particles seen in electron microscope images of tissues. More recently, VLP has come to mean other things to other researchers. A key divergence has been use of VLP in association with vaccine and biotechnology applications versus use of VLP in enumeration of viruses in environmental samples. To these viral ecologists, a VLP is a particle that is virus sized, has nucleic acid, and could be a functional virus. But to vaccine developers and biotechnology researchers a VLP instead is a viral structure that intentionally lacks a viral genome. Here, we look at the history of use of VLP, following changes in meaning as the technology to study VLPs changed.

59 BASIC BIOLOGICAL SCIENCES↗

Streamlined approach for environmental restoration (SAFER) plan for corrective action unit 412: clean slate I plutonium dispersion (TTR) tonopah test range, Nevada, revision 0

This Streamlined Approach for Environmental Restoration (SAFER) Plan addresses the actions needed to achieve closure for Corrective Action Unit (CAU) 412. CAU 412 is located on the Tonopah Test Range and consists of a single corrective action site (CAS), TA-23-01CS, Pu Contaminated Soil. There is sufficient information and historical documentation from previous investigations and the 1997 interim corrective action to recommend closure of CAU 412 using the SAFER process. Based on existing data, the presumed corrective action for CAU 412 is clean closure. However, additional data will be obtained during a field investigation to document and verify the adequacy of existing information and determine whether the CAU 412 closure objectives have been achieved. This SAFER Plan provides the methodology to gather the necessary information for closing the CAU.The following summarizes the SAFER activities that will support the closure of CAU 412:• Collect environmental samples from designated target populations to confirm or disprove the presence of contaminants of concern (COCs) as necessary to supplement existing information.• If no COCs are present, establish clean closure as the corrective action. • If COCs are present, the extent of contamination will be defined and further corrective actions will be evaluated with the stakeholders (NDEP, USAF).• Confirm the preferred closure option is sufficient to protect human health and the environment.

01 COAL, LIGNITE, AND PEAT↗

Analysis of sensitivity and rapid hybridization of a multiplexed Microbial Detection Microarray

Microarrays have proven to be useful in rapid detection of many viruses and bacteria. Pathogen detection microarrays have been used to diagnose viral and bacterial infections in clinical samples and to evaluate the safety of biological drug materials. A multiplexed version of the Lawrence Livermore Microbial Detection Array (LLMDA) was developed and evaluated with minimum detectable concentrations for pure unamplified DNA viruses, along with mixtures of viral and bacterial DNA subjected to different whole genome amplification protocols. In addition the performance of the array was tested when hybridization time was reduced from 17 h to 1 h. The LLMDA was able to detect unamplified vaccinia virus DNA at a concentration of 14 fM, or 100,000 genome copies in 12 μL of sample. With amplification, positive identification was made with only 100 genome copies of input material. When tested against human stool samples from patients with acute gastroenteritis, the microarray detected common gastroenteritis viral and bacterial infections such as rotavirus and E. coli. Accurate detection was found but with a 4-fold drop in sensitivity for a 1 h compared to a 17 h hybridization. The array detected 2 ng (equivalent concentration of 15.6 fM) of labeled DNA from a virus with 1 h hybridization without any amplification, and was able to identify the components of a mixture of viruses and bacteria at species and in some cases strain level resolution. Sensitivity improved by three orders of magnitude with random whole genome amplification prior to hybridization; for instance, the array detected a DNA virus with only 20 fg or 100 genome copies as input. This multiplexed microarray is an efficient tool to analyze clinical and environmental samples for the presence of multiple viral and bacterial pathogens rapidly.

59 BASIC BIOLOGICAL SCIENCES↗

Psychrotolerant Anaerobes from Lake Podprudnoe, Antarctica and Penguin Spheniscus demersus Colony, South Africa

The study of a sample collected from a wind-made ice sculpture near Lake Podprudnoe, Antarctica led to the isolation of the psychrotolerant strain ISLP-3. Cells of the new isolate are vibrio-shaped that measure 0.5 x 1.0-3.0 micron in size. Growth occurs within the temperature range 5-35 C with the optimum at 22 C. Salinity range for growth is 0-2 % NaCl with the optimum at 0.25 %. The new isolate grows within a pH range from 6.0 to 9.5 with the optimum at 7.5. Strain ISLP-3 is saccharolytic, growing on the following substrates: D-glucose, D-ribose, D-fructose, D-arabinose, maltose, sucrose, D-trehalose, D-mannose, D-cellobiose, lactose, starch, chitin, triethylamine, N-acetylglucosamine, and urea. The best growth occurred on D-cellobiose. An environmental sample of pond water near a colony of the endemic species of African penguins, Spheniscus demersus, was collected in February 2008 and delivered directly to the Astrobiology laboratory at NSSTC. The microbiological study of this sample led to the isolation of two psychrotolerant strains ARHSd-7G and ARHSd-9G. Both strains are strictly anaerobic bacteria and are able to grow at high pH and low temperatures. The cells of strain ARHSd-7G are motile, vibrio-shaped, spore-forming cells. Optimal growth of this strain occurs at 30 C, 3 % NaCl, and pH 8.9. The isolate ARHSd-7G combines sugarlytic and proteolytic metabolisms, growing on some proteolysis products including peptone and yeast extract and a number of sugars. The second isolate, ARHSd-9G, exhibits thin, elongated rods that measure 0.4 x 3-5 micron. The cells are motile and spore-forming. Optimal growth of strain ARHSd-9G occurs at 30 C, 1.75 % NaCl, and pH 8.5. The strain ARHSd-9G is sugarlytic, growing well on substrates such as D-glucose, sucrose, D-cellobiose, maltose, fructose, D-mannose, and trehalose (the only exception is positive growth on yeast extract). In this report, the physiological and morphological characteristics of the novel psychrotolerant, alkaliphilic, and neutrophilic isolates from the Antarctica 2008 expedition will be discussed.

Guisler, Melissa↗

Sensing Techniques for Organochlorides through Intermolecular Interaction with Bicyclic Amidines

Toxic organochloride molecules are widely used in industry for various purposes. With their high volatility, the direct detection of organochlorides in environmental samples is challenging. Here, a new organochloride detection mechanism using 1,5-diazabicyclo[4.3.0]non-5-ene (DBN) is introduced to simplify a sensing method with higher detection sensitivity. Three types of organochloride compounds-trichloroethylene (TCE), dichloromethane (DCM), and dichlorodiphenyltrichloroethane (DDT)—were targeted to understand DCM conjugation chemistry by using nuclear magnetic resonance (NMR) and liquid chromatography with a mass spectrometer (LC-MS). 13C-NMR spectra and LC-MS data indicated that DBN can be labeled on these organochloride compounds by chlorine–nitrogen interaction. Furthermore, to demonstrate the organochloride sensing capability, the labeling yield and limit of detection were determined by a colorimetric assay as well as micellar electrokinetic chromatography (MEKC). The interaction with DBN was most appreciable for TCE, among other organochlorides. TCE was detected at picomolar levels, which is two orders of magnitude lower than the maximum contaminant level set by the United States Environmental Protection Agency. MEKC, in conjunction with this DBN-labeling method, enables us to develop a field-deployable sensing platform for detecting toxic organochlorides with high sensitivity.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Progress and Challenges in Ocean Metaproteomics and Proposed Best Practices for Data Sharing

Ocean metaproteomics is an emerging field enabling discoveries about marine microbial communities and their impact on global biogeochemical processes. Recent ocean metaproteomic studies have provided insight into microbial nutrient transport, colimitation of carbon fixation, the metabolism of microbial biofilms, and dynamics of carbon flux in marine ecosystems. Future methodological developments could provide new capabilities such as characterizing long-term ecosystem changes, biogeochemical reaction rates, and in situ stoichiometries. Yet challenges remain for ocean metaproteomics due to the great biological diversity that produces highly complex mass spectra, as well as the difficulty in obtaining and working with environmental samples. This review summarizes the progress and challenges facing ocean metaproteomic scientists and proposes best practices for data sharing of ocean metaproteomic data sets, including the data types and metadata needed to enable intercomparisons of protein distributions and annotations that could foster global ocean metaproteomic capabilities.

54 ENVIRONMENTAL SCIENCES↗

Genus-wide assessment of lignocellulose utilization in the extremely thermophilic Caldicellulosiruptor by genomic, pan-genomic and metagenomic analysis

Metagenomic data from Obsidian Pool (Yellowstone National Park, USA) and thirteen genome sequences were used to re-assess genus-wide biodiversity for the extremely thermophilic Caldicellulosiruptor . The updated core-genome contains 1,401 ortholog groups (average genome size for thirteen species = 2,516 genes). The pan-genome, which remains open with a revised total of 3,493 ortholog groups, encodes a variety of multi-domain glycoside hydrolases (GH). These include three cellulases with GH48 domains that are co-located in the Glucan Degradation Locus (GDL) and are specific determinants for microcrystalline cellulose utilization. Three recently sequenced species, Caldicellulosiruptor sp. str. Rt8.B8 (re-named here Caldicellulosiruptor morganii ), Thermoanaerobacter cellulolyticus str. NA10 (re-named here Caldicellulosiruptor naganoensis NA10), and Caldicellulosiruptor sp. str. Wai35.B1 (re-named here Caldicellulosiruptor danielii ) degraded Avicel and lignocellulose (switchgrass). C. morganii was more efficient than C. bescii in this regard and differed from the other twelve species examined here, both based on genome content and organization and in the specific domain features of conserved GHs. Metagenomic analysis of lignocellulose-enriched samples from Obsidian Pool revealed limited new information on genus biodiversity. Enrichments yielded genomic signatures closely related to Caldicellulosiruptor obsidiansis , but there was also evidence for other thermophilic fermentative anaerobes ( Caldanaerobacter , Fervidobacterium , Caloramator , and Clostridium ). One enrichment, containing 89.7% Caldicellulosiruptor and 9.7% Caloramator , had a capacity for switchgrass solubilization comparable to C. bescii . These results refine the known biodiversity of Caldicellulosiruptor and indicate that microcrystalline cellulose degradation at temperatures above 70°C, based on current information, is limited to certain members of this genus that produce GH48 domain-containing enzymes. The genus Caldicellulosiruptor contains the most thermophilic bacteria capable of lignocellulose deconstruction and are promising candidates for consolidated bioprocessing for the production of biofuels and bio-based chemicals. The focus here is on the extant capability of this genus for plant biomass degradation and the extent to which this can be inferred from the core and pan-genomes, based on analysis of thirteen species and metagenomic sequence information from environmental samples. Key to microcrystalline hydrolysis is the content of the Glucan Degradation Locus (GDL), a set of genes encoding glycoside hydrolases (GH), several of which have GH48 and family 3 carbohydrate binding module domains, that function as primary cellulases. Resolving the relationship between the GDL and lignocellulose degradation will inform efforts to identify more prolific members of the genus and to develop metabolic engineering strategies to improve this characteristic

59 BASIC BIOLOGICAL SCIENCES↗

Canary

CANARY is a wearable environmental sampling device that is small, lightweight, and low-power. CANARY integrates a novel mechanism that uses magnets rather than powered pumps to mimic human breathing. In its current form, CANARY breathes, but sensor integration can easily turn this into a standalone device for use in airborne detection of chemicals, biologicals, viruses, etc. CANARY has a small footprint and requires little power to operate enabling it to be used in various environments. Los Alamos researcher have developed a working prototype of the air intake system and are seeking licensees for the current air intake, validation outside the lab, or further development to integrate biosensors as desired.

47 OTHER INSTRUMENTATION↗

Geochemistry, RNA radiocarbon signature, and metatranscriptome data from Rifle, Colorado groundwater well

Understanding the different sources of carbon subsurface microbes use is highly informative yet remains challenging. The goal of this work was to develop and test a cultivation-independent method that analyzes both the radiocarbon and ribosomal RNA (rRNA) signature of unamplified RNA from the same environmental sample, and that collectively characterizes community carbon utilization under in situ conditions. The method was applied to a filtered groundwater sample from a geochemically reduced zone of a shallow alluvial aquifer near Rife, Colorado (CO), where multiple potential carbon sources co-occur. The radiocarbon and RNA-Seq results, while addressing diverse sample properties, indicated a strikingly similar conclusion about the quantitative role of autotrophy in the subsurface microbial community.This data package provides (1) ribosomal RNA (rRNA) sequences for groundwater microbial populations, (2) the taxonomic identity and relative abundance of groundwater microbial populations based on these rRNA sequences, (3) radiocarbon signatures of the rRNA indicating the source of carbon used by the groundwater microbial populations (provided in both Word docx file and pdf file), (4) geochemical measurements for the monitoring well corresponding to these measurements.

54 ENVIRONMENTAL SCIENCES↗

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 cm2). 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 LOD95 was lowest for glass (0.429 CFU/cm2 with BAS and 0.341 CFU/cm2 with BG) and highest for vinyl tile (0.919 CFU/cm2 with BAS and 0.917 CFU/cm2 with BG). These mRV-PCR LOD95 values were lower than the culture values (BAS: 0.678 to 1.023 CFU/cm2 and BG: 0.820 to 1.489 CFU/cm2). The FNR and LOD95 values reported in this work provide guidance for environmental sampling of Bacillus spores at low concentrations.

59 BASIC BIOLOGICAL SCIENCES↗

Comprehensive analysis of common polymers using hyphenated TGA-FTIR-GC/MS and Raman spectroscopy towards a database for micro- and nanoplastics identification, characterization, and quantitation

Environmental contamination by micro- and nanoplastics (MNPs) is well documented with potential for their increased accumulation globally. Growing public concern over environmental, ecological, and human exposure to MNPs has led to exponential increase in publications, news articles, and reports. Significant knowledge gap exists in standardized analytical methods for the identification and quantification of MNPs from real world environmental samples. Here, in this study, we report comprehensive datasets utilizing thermogravimetric analyzer (TGA) coupled to a Fourier transformed infrared spectrometer (FTIR) and a gas chromatography/mass spectrometer (GC/MS) with corresponding Raman spectral data for the most common polymers documented to be present in the environment (35 plastics of 12 polymer types), to serve as a base line reference for the identification and quantitation of MNPs. Various parameters for TGA-FTIR-GC/MS data acquisition were optimized. Commercial consumer plastic product compositions were identified using this analytical database. Case studies to showcase the utility of the method for polymer mixtures analysis is included. This dataset would serve towards the development of a collaborative, global, comprehensive, and curated public database for the identification of various MNPs and mixtures.

77 NANOSCIENCE AND NANOTECHNOLOGY↗