A protocol for obtaining DNA barcodes from plant and insect fragments isolated from forensic-type soils.

Soil is often collected from a suspect's tire, vehicle, or shoes during a criminal investigation and subsequently submitted to a forensic laboratory for analysis. Plant and insect material recovered in such samples is rarely analyzed, as morphological identification is difficult. In this study, DNA barcoding was used for taxonomic identifications by targeting the gene regions known to permit discrimination in plants [maturase K (matK) and ribulose 1,5-biphosphate carboxylase (rbcL)] and insects [cytochrome oxidase subunit I (COI)]. A DNA barcode protocol suitable for processing forensic-type biological fragments was developed and its utility broadly tested with forensic-type fragments (e.g., seeds, leaves, bark, head, legs; n, 213) isolated from soils collected within Virginia, USA (n, 11). Difficulties with PCR inhibitors in plant extracts and obtaining clean Sanger sequence data from insect amplicons were encountered during protocol development; however, the final protocol produced sequences specific to the expected locus and taxa. The overall quantity and quality of DNA extracted from the 213 forensic-type biological fragments was low (< 15 ng/µL). For plant fragments, only the rbcL sequence data was deemed reliable; thus, taxonomic identifications were limited to the family level. The majority of insect sequences matched COI in both GenBank and Barcode of Life DataSystems; however, they were identified as an undescribed environmental contaminant. Although limited taxonomic information was gleaned from the forensic-type fragments processed in this study, the new protocol shows promise for obtaining reliable and specific identifications through DNA barcoding, which could ultimately enhance the information gleaned from soil examinations.

Gaseous byproducts from high-temperature thermal conversion elemental analysis of nitrogen- and sulfur-bearing compounds with considerations for δ2H and δ18O analyses.

RATIONALE: High-temperature, conversion-reduction (HTC) systems convert hydrogen and oxygen in materials into H2 and CO for δ(2)H and δ(18)O measurements by isotope ratio mass spectrometry. HTC of nitrogen- and sulfur-bearing materials produces unintended byproduct gases that could affect isotope analyses by: (1) allowing isotope exchange reactions downstream of the HTC reactor, (2) creating isobaric or co-elution interferences, and (3) causing deterioration of the chromatography. This study characterizes these HTC byproducts. METHODS: A HTC system (ThermoFinnigan TC/EA) was directly connected to a gas chromatograph/quadrupole mass spectrometer in scan mode (m/z 8 to 88) to identify the volatile products generated by HTC at conversion temperatures of 1350 °C and 1450 °C for a range of nitrogen- and sulfur-bearing solids [keratin powder, horse hair, caffeine, ammonium nitrate, potassium nitrate, ammonium sulfate, urea, and three nitrated organic explosives (PETN, RDX, and TNT)]. RESULTS: The prominent HTC byproduct gases include carbon dioxide, hydrogen cyanide, methane, acetylene, and water for all nitrogen-bearing compounds, as well as carbon disulfide, carbonyl sulfide, and hydrogen sulfide for sulfur-bearing compounds. The 1450 °C reactor temperature reduced the abundance of most byproduct gases, but increased the significant byproduct, hydrogen cyanide. Inclusion of a post-reactor chemical trap containing Ascarite II and Sicapent, in series, eliminated the majority of byproducts. CONCLUSIONS: This study identified numerous gaseous HTC byproducts. The potential adverse effects of these gases on isotope ratio analyses are unknown but may be mitigated by higher HTC reactor temperatures and purifying the products with a purge-and-trap system or with chemical traps.

Improved accuracy in high-temperature conversion elemental analyzer δ18O measurements of nitrogen-rich organics.

RATIONALE: The use of high-temperature conversion (HTC) reduction systems interfaced with isotope ratio mass spectrometers for δ(18)O measurements of nitrogen-containing organic materials is complicated by isobaric interference from (14)N(16)O(+). This ion is produced in the ion source when N(2) reacts with trace oxygen shifting the m/z 30 baseline prior to elution of CO. METHODS: We compared adaptations to a typical HTC system (TC/EA) to determine the best method to measure the δ(18)O values of nitrogen-rich organic substrates including: (1) 0.6 and 1.5 m 5 Šmolecular sieve GC columns; (2) reduction of N(2) peak via He dilution; and (3) diversion of N(2) to waste via an automated four-port valve. These methods were applied to caffeine (IAEA-600), glycine, 4-nitroacetanilide, pentaerythritol tetranitrate (PETN) and cyclotrimethylene trinitramine (RDX), as well as pure and sodium azide-doped benzoic acid (IAEA-601) and sucrose (IAEA-CH6). RESULTS: The efficiency of N(2) production in the HTC interface was highly variable among these compounds. Both the longer column and the dilutor improved, but did not eliminate, the adverse effects of nitrogen. CONCLUSIONS: The diversion of N(2) adequately addressed the nitrogen-induced problems as indicated by: (1) consistent m/z 30 background offset between reference and sample CO for both N-free and N-rich materials; (2) production of the highest δ(18)O values; and (3) high correlation between the increase in the δ(18)O values relative to the GC-only measurements and the N(2) peak area. Additional validation would require N-rich oxygen isotope standards for inter-laboratory comparisons. Further, more stringent methodology may improve the poor inter-laboratory δ(18)O reproducibility of IAEA-600.

Forensic utility of carbon isotope ratio variations in PVC tape backings.

Forensic interest in adhesive tapes with polyvinyl chloride (PVC) backings (electrical tape) derives from their use in a variety of illicit activities. Due to the range of physical characteristics, chemical compositions, and homogeneity within a single roll of tape, traditional microscopic and chemical analyses can offer a high degree of discrimination between tapes, permitting the assessment of potential associations between evidentiary tape samples. The carbon isotope ratios of tapes could provide additional discrimination among tape samples. To evaluate whether carbon isotope ratios may be able to increase discrimination of electrical tapes, particularly with regards to different rolls of tape of the same product, we assessed the δ(13)C values of backings from 87 rolls of PVC-based black electrical tape (~20 brands, >60 products) Prior to analysis, adhesives were removed to prevent contamination by adhering debris, and plasticizers were extracted because of concern over their potential mobility. This result is consistent with each of these tapes having approximately the same plasticizer δ(13)C value and proportion of carbon in these plasticizers. The δ(13)C values of the 87 PVC tape backings ranged between -23.5 and -41.3 (‰, V-PDB), with negligible carbon isotopic variation within single rolls of tape, yet large variations among tape brands and tape products. Within this tape population, carbon isotope ratios permitted an average exclusion power of 93.7%, using a window of +/-0.3‰; the combination of carbon isotope ratio measurement with additional chemical and physical analyses raises the discrimination power to over 98.9%, with only 41 out of a possible 3741 pairs of tape samples being indistinguishable. There was a linear relationship between the δ(13)C value of tape backings and the change in δ(13)C value with the extraction of plasticizers. Analyses of pre- and post-blast tape sample pairs show that carbon isotope signatures are within 0.3‰ of pre-blast values, indicating that carbon isotope values are largely preserved during an explosion.

Soil survey laboratory grain count data to substantiate the rarity of mineral grains in forensic soil reports of examination.

The Natural Resources Conservation Service-Kellogg Soil Survey Laboratory has a large publicly available database of laboratory analyses of soil horizons collected from soil profiles largely from the United States. Among these soil properties are mineral grain counts from selected sand and silt fractions of soil horizons, performed by polarized light microscopy (PLM). These grain counts of over 20,000 fractions from 7534 sites provide a substantial reference that a forensic soil examiner could use to substantiate the rarity or commonness of a mineral species. The statement of the rarity or commonness of various minerals provide juries with additional context for the interpreting the results of a forensic soil comparison within the framework of a trial. The grain count data at specific locations can also be assessed to aid in soil provenance investigations, for cases where there are grain-counted sites in relevant locations. Two examples of application of these to data to soil evidence are included, one relating soil the rarity of a mineral (andalusite) to provide context in a soil comparison and one to aid in narrowing target regions in a soil provenance investigation.

Strengths, Limitations, and Recommendations for Instrumental Color Measurement in Forensic Soil Characterization.

Color determination of soil evidence is often done by visual comparison to soil color charts. A handheld spectrophotometer was tested with representative materials for its suitability for forensic soil characterization. Instrumental colorimetry provides accurate colorimetry with ~10-fold better precision than a soil color chart. The minimum sample size for accurate color determination was between 0.02 and 0.04 mg of fine soil for the specific instrument tested. Reporting colors in the L*a*b* space permits quantification of ΔE00 , a measure of perceptible color difference, could enable objective quantification of small color differences and thresholds for forensic soil comparisons. A ΔE00 greater than ~ 3.5 to 6 likely indicates disparate soil sources in a forensic comparison, in the absence of confounding factors like sample alteration. Despite the superior precision of instrumental colorimetry, this approach is inappropriate for samples which are mottled at an inseparable scale, attached to a substrate, or too small for instrumental measurement.

Forensic utility of a nitrogen and oxygen isotope ratio time series of ammonium nitrate and its isolated ions.

Ammonium nitrate (AN) based fertilizers are inexpensive and easily obtained, characteristics that often lead to their use in homemade explosive devices. The stable nitrogen and oxygen isotope ratios (15N/14N and 18O/16O, expressed as δ15N and δ18O) of AN have the potential to aid in forensic investigations by providing supplemental properties for sample-to-sample comparison in materials which are otherwise chemically identical. The forensic utility of stable isotope analyses depends on demonstrated variation between different sources and minimal variation within a source. To test the variability within a single manufacturer (here considered a source), a total of 26 samples representing two production time periods and two product lines were analyzed for bulk δ15N and δ18O. Additionally, because AN is known to have a modest isotopic range, a potassium nitrate precipitation method was developed to separate the component ions (NO3- and NH4+) for individual δ15N analysis and increased discriminatory power. The average δ15N and δ18O of bulk AN (- 0.10‰ and + 22.8‰, respectively) is similar to the isotopic signature of atmospheric N2 and O2, the starting reactants in AN production. The bulk δ15N, δ18O, and NO3- δ15N show average values from both product lines that differ by 1.5‰, 2.0‰, and 2.6‰, respectively, between the production periods of June and November 2015. Conversely, the NH4+ δ15N remained relatively consistent over time. Furthermore, whereas samples in the two product lines produced on the same day in June are isotopically similar, there are isotopic differences between samples in the two product lines manufactured within 6h of each other in November. The observed variability could be useful in comparing AN from two or more bombs, or a bomb and a stash of AN in a suspect's possession, but the observed lot-to-lot differences within one manufacturer could complicate attribution efforts. In contrast, the NH4+ δ15N values, which appear to be the most consistent over time within this factory, need to be further explored as a potentially reliable signal.

Bacterial diversity and ecosystem function of filamentous microbial mats from aphotic (cave) sulfidic springs dominated by chemolithoautotrophic "Epsilonproteobacteria".

Filamentous microbial mats from three aphotic sulfidic springs in Lower Kane Cave, Wyoming, were assessed with regard to bacterial diversity, community structure, and ecosystem function using a 16S rDNA-based phylogenetic approach combined with elemental content and stable carbon isotope ratio analyses. The most prevalent mat morphotype consisted of white filament bundles, with low C:N ratios (3.5-5.4) and high sulfur content (16.1-51.2%). White filament bundles and two other mat morphotypes had organic carbon isotope values (mean delta13C=-34.7 per thousand, 1sigma=3.6) consistent with chemolithoautotrophic carbon fixation from a dissolved inorganic carbon reservoir (cave water, mean delta13C=-7.4 per thousand for two springs, n=8). Bacterial diversity was low overall in the clone libraries, and the most abundant taxonomic group was affiliated with the "Epsilonproteobacteria" (68%), with other bacterial sequences affiliated with Gammaproteobacteria (12.2%), Betaproteobacteria (11.7%), Deltaproteobacteria (0.8%), and the Acidobacterium (5.6%) and Bacteriodetes/Chlorobi (1.7%) divisions. Six distinct epsilonproteobacterial taxonomic groups were identified from the microbial mats. Epsilonproteobacterial and bacterial group abundances and community structure shifted from the spring orifices downstream, corresponding to changes in dissolved sulfide and oxygen concentrations and metabolic requirements of certain bacterial groups. Most of the clone sequences for epsilonproteobacterial groups were retrieved from areas with high sulfide and low oxygen concentrations, whereas Thiothrix spp. and Thiobacillus spp. had higher retrieved clone abundances where conditions of low sulfide and high oxygen concentrations were measured. Genetic and metabolic diversity among the "Epsilonproteobacteria" maximizes overall cave ecosystem function, and these organisms play a significant role in providing chemolithoautotrophic energy to the otherwise nutrient-poor cave habitat. Our results demonstrate that sulfur cycling supports subsurface ecosystems through chemolithoautotrophy and expand the evolutionary and ecological views of "Epsilonproteobacteria" in terrestrial habitats.

Forensic utility of isotope ratio analysis of the explosive urea nitrate and its precursors.

Urea nitrate (UN) is an improvised explosive made from readily available materials. The carbon and nitrogen isotope composition of UN and its component ions, urea and nitrate, could aid in a forensic investigation. A method was developed to separate UN into its component ions for δ(15)N measurements by dissolving the sample with KOH, drying the sample, followed by removal of the urea by dissolution into 100% methanol. UN was synthesized to assess for preservation of the carbon and nitrogen isotope compositions of reactants (urea and nitric acid) and product UN. Based on nitrogen isotope mass balance, all UN samples contained varying amounts of excess nitric acid, making the ionic separation an essential step in the nitrogen isotope analysis. During UN synthesis experiments, isotopic composition of the reactants is preserved in the product UN, but the urea in the product UN is slightly enriched in (15)N (<1‰) relative to the reactant urea. Published isotopic compositions of UN reactants, urea and nitric acid, have large ranges (urea δ(15)N = -10.8 to +3.3‰; urea δ(13)C = -18.2 to -50.6‰; and nitric acid δ(15)N = -1.8 to +4.0‰). The preservation of isotopic composition of reactants in UN, along with a significant variability in isotopic composition of reactants, indicates that isotope ratio analysis may be used to test if urea or nitric acid collected during an investigation is a possible reactant for a specific UN sample. The carbon and nitrogen isotope ratios differ significantly between two field-collected UN samples, as well as the lab-synthesized UN samples. These observed variations suggest that this approach is useful for discriminating between materials which are otherwise chemically identical.

Linking phylogenetic and functional diversity to nutrient spiraling in microbial mats from Lower Kane Cave (USA).

Microbial mats in sulfidic cave streams offer unique opportunities to study redox-based biogeochemical nutrient cycles. Previous work from Lower Kane Cave, Wyoming, USA, focused on the aerobic portion of microbial mats, dominated by putative chemolithoautotrophic, sulfur-oxidizing groups within the Epsilonproteobacteria and Gammaproteobacteria. To evaluate nutrient cycling and turnover within the whole mat system, a multidisciplinary strategy was used to characterize the anaerobic portion of the mats, including application of the full-cycle rRNA approach, the most probable number method, and geochemical and isotopic analyses. Seventeen major taxonomic bacterial groups and one archaeal group were retrieved from the anaerobic portions of the mats, dominated by Deltaproteobacteria and uncultured members of the Chloroflexi phylum. A nutrient spiraling model was applied to evaluate upstream to downstream changes in microbial diversity based on carbon and sulfur nutrient concentrations. Variability in dissolved sulfide concentrations was attributed to changes in the abundance of sulfide-oxidizing microbial groups and shifts in the occurrence and abundance of sulfate-reducing microbes. Gradients in carbon and sulfur isotopic composition indicated that released and recycled byproduct compounds from upstream microbial activities were incorporated by downstream communities. On the basis of the type of available chemical energy, the variability of nutrient species in a spiraling model may explain observed differences in microbial taxonomic affiliations and metabolic functions, thereby spatially linking microbial diversity to nutrient spiraling in the cave stream ecosystem.

Filamentous "Epsilonproteobacteria" dominate microbial mats from sulfidic cave springs.

Hydrogen sulfide-rich groundwater discharges from springs into Lower Kane Cave, Wyoming, where microbial mats dominated by filamentous morphotypes are found. The full-cycle rRNA approach, including 16S rRNA gene retrieval and fluorescence in situ hybridization (FISH), was used to identify these filaments. The majority of the obtained 16S rRNA gene clones from the mats were affiliated with the "Epsilonproteobacteria" and formed two distinct clusters, designated LKC group I and LKC group II, within this class. Group I was closely related to uncultured environmental clones from petroleum-contaminated groundwater, sulfidic springs, and sulfidic caves (97 to 99% sequence similarity), while group II formed a novel clade moderately related to deep-sea hydrothermal vent symbionts (90 to 94% sequence similarity). FISH with newly designed probes for both groups specifically stained filamentous bacteria within the mats. FISH-based quantification of the two filament groups in six different microbial mat samples from Lower Kane Cave showed that LKC group II dominated five of the six mat communities. This study further expands our perceptions of the diversity and geographic distribution of "Epsilonproteobacteria" in extreme environments and demonstrates their biogeochemical importance in subterranean ecosystems.