Validating DNA Extraction Protocols for Bentonite Clay.

Bentonite clay is an integral component of the engineered barrier system of deep geological repositories (DGRs) that are planned for the long-term storage of high-level radioactive waste. Although nucleic acid extraction and analysis can provide powerful qualitative and quantitative data reflecting the presence, abundance, and functional potential of microorganisms within DGR materials, extraction of microbial DNA from bentonite clay is challenging due to the low biomass and adsorption of nucleic acids to the charged clay matrix. In this study, we used quantitative PCR, gel fingerprinting, and high-throughput sequencing of 16S rRNA gene amplicons to assess DNA extraction efficiency from natural MX-80 bentonite and the same material "spiked" with Escherichia coli genomic DNA. Extraction protocols were tested without additives and with casein and phosphate as blocking agents. Although we demonstrate improved DNA recovery by blocking agents at relatively high DNA spiking concentrations, at relatively low spiking concentrations, we detected a high proportion of contaminant nucleic acids from blocking agents that masked sample-specific microbial profile data. Because bacterial genomic DNA associated with casein preparations was insufficiently removed by UV treatment, casein is not recommended as an additive for DNA extractions from low-biomass samples. Instead, we recommend a kit-based extraction protocol for bentonite clay without additional blocking agents, as tested here and validated with multiple MX-80 bentonite samples, ensuring relatively high DNA recoveries with minimal contamination.IMPORTANCE Extraction of microbial DNA from MX-80 bentonite is challenging due to low biomass and adsorption of nucleic acid molecules to the charged clay matrix. Blocking agents improve DNA recovery, but their impact on microbial community profiles from low-biomass samples has not been characterized well. In this study, we evaluated the effect of casein and phosphate as blocking agents for quantitative recovery of nucleic acids from MX-80 bentonite. Our data justify a simplified framework for analyzing microbial community DNA associated with swelling MX-80 bentonite samples within the context of a deep geological repository for used nuclear fuel. This study is among the first to demonstrate successful extraction of DNA from Wyoming MX-80 bentonite.

Reduction in the earthworm metabolomic response after phenanthrene exposure in soils with high soil organic carbon content.

We evaluated the correlation between soil organic carbon (OC) content and metabolic responses of Eisenia fetida earthworms after exposure to phenanthrene (58 ± 3 mg/kg) spiked into seven artificial soils with OC contents ranging from 1 to 27% OC. Principal component analysis of (1)H nuclear magnetic resonance (NMR) spectra of aqueous extracts identified statistically significant differences in the metabolic profiles of control and phenanthrene-exposed E. fetida in the 1% OC soil only. Partial least squares analysis identified a metabolic response in the four soils with OC values ≤11% which was well correlated to estimated phenanthrene porewater concentrations. The results suggest that the higher sorption capability of high OC soils decreased the bioavailability of phenanthrene and the subsequent metabolic response of E. fetida.

Metabolic responses of Eisenia fetida after sub-lethal exposure to organic contaminants with different toxic modes of action.

Nuclear magnetic resonance (NMR)--based metabolomics has the potential to identify toxic responses of contaminants within a mixture in contaminated soil. This study evaluated the metabolic response of Eisenia fetida after exposure to an array of organic compounds to determine whether contaminant-specific responses could be identified. The compounds investigated in contact tests included: two pesticides (carbaryl and chlorpyrifos), three pharmaceuticals (carbamazephine, estrone and caffeine), two persistent organohalogens (Aroclor 1254 and PBDE 209) and two industrial compounds (nonylphenol and dimethyl phthalate). Control and contaminant-exposed metabolic profiles were distinguished using principal component analysis and potential contaminant-specific biomarkers of exposure were found for several contaminants. These results suggest that NMR-based metabolomics offers considerable promise for differentiating between the different toxic modes of action (MOA) associated with sub-lethal toxicity to earthworms.

Correlations of Eisenia fetida metabolic responses to extractable phenanthrene concentrations through time.

Eisenia fetida earthworms were exposed to phenanthrene for thirty days to compare hydroxypropyl-beta-cyclodextrin (HPCD) extraction of soil and 1H NMR earthworm metabolomics as indicators of bioavailability. The phenanthrene 28-d LC50 value was 750 mg/kg (632-891, 95% confidence intervals) for the peat soil tested. The initial phenanthrene concentration was 319 mg/kg, which biodegraded to 16 mg/kg within 15 days, at which time HPCD extraction suggested that phenanthrene was no longer bioavailable. Multivariate statistical analysis of 1H NMR spectra for E. fetida tissue extracts indicated that phenanthrene exposed and control earthworms differed throughout the 30 day experiment despite the low phenanthrene concentrations present after 15 days. This metabolic response was better correlated to total phenanthrene concentrations (Q2 = 0.59) than HPCD-extractable phenanthrene concentrations (Q2 = 0.46) suggesting that 1H NMR metabolomics offers considerable promise as a novel, molecular-level method to directly monitor the bioavailability of contaminants to earthworms in the environment.

1H NMR metabolomics of earthworm exposure to sub-lethal concentrations of phenanthrene in soil.

1H NMR metabolomics was used to monitor earthworm responses to sub-lethal (50-1500 mg/kg) phenanthrene exposure in soil. Total phenanthrene was analyzed via soxhlet extraction, bioavailable phenanthrene was estimated by hydroxypropyl-beta-cyclodextrin (HPCD) and 1-butanol extractions and sorption to soil was assessed by batch equilibration. Bioavailable phenanthrene (HPCD-extracted) comprised approximately 65-97% of total phenanthrene added to the soil. Principal component analysis (PCA) showed differences in responses between exposed earthworms and controls after 48 h exposure. The metabolites that varied with exposure included amino acids (isoleucine, alanine and glutamine) and maltose. PLS models indicated that earthworm response is positively correlated to both total phenanthrene concentration and bioavailable (HPCD-extracted) phenanthrene in a freshly spiked, unaged soil. These results show that metabolomics is a powerful, direct technique that may be used to monitor contaminant bioavailability and toxicity of sub-lethal concentrations of contaminants in the environment. These initial findings warrant further metabolomic studies with aged contaminated soils.

Quantitative site-specific (2)H NMR investigation of MTBE: potential for assessing contaminant sources and fate.

Site-specific isotopic values of methyl tertiary butyl ether (MTBE) were measured using quantitative site-specific (2)H nuclear magnetic resonance (NMR) spectroscopy for seven commercially available MTBE products. The delta(2)H values of the methoxy and tertiary butyl groups ranged from -103 per thousand to -171 per thousand, and from -76 per thousand to -104 per thousand, reflecting their production from methanol and isobutene, respectively. Several MTBE products whose whole-compound delta(13)C and delta(2)H MTBE values were within error of each other, as measured by isotope ratio mass spectrometry (IRMS), had demonstrably different delta(2)H values for their methoxy and tertiary butyl groups measured by (2)H NMR. Site-specific isotopic variations were large enough to provide proof of principle that quantitative site-specific (2)H NMR may provide an additional parameter for contaminant sourcing at field sites. Isotopic variations were small enough to not bias the comparability of degradation-associated isotopic enrichment factors determined using different MTBE products. Calculated delta(2)H values for MTBE, derived as weighted averages of (2)H NMR measurements of the two functional groups, showed good agreement with IRMS measurements. The ability to gain accurate information about the site-specific isotopic ratios of (2)H/(1)H within a molecule offers considerable promise as a new environmental tool to track the source and fate of environmental contaminants.

Isotopic fractionation of methyl tert-butyl ether suggests different initial reaction mechanisms during aerobic biodegradation.

Carbon isotopic enrichment factors (epsilonC) measured during cometabolic biodegradation of methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME) by Pseudonocardia tetrahydrofuranoxydans strain K1 were -2.3 +/- 0.2 per thousand, -1.7 +/- 0.2 per thousand, and -1.7 +/- 0.3 per thousand, respectively. The measured carbon apparent kinetic isotope effect was 1.01 for all compounds, consistent with the expected kinetic isotope effects for both oxidation of the methoxy (or ethoxy) group and enzymatic SN1 biodegradation mechanisms. Significantly, delta13C measurements of the tert-butyl alcohol and tert-amyl alcohol products indicated that the tert-butyl and tert-amyl groups do not participate in the reaction and confirmed that ether biodegradation by strain K1 involves oxidation of the methoxy (or ethoxy) group. Measured hydrogen isotopic enrichment factors (epsilonH) were -100 +/- 10 per thousand, -73 +/- 7 per thousand, and -72 +/- 20 per thousand for MTBE, ETBE, and TAME respectively. Previous results reported for aerobic biodegradation of MTBE by Methylibium petroleiphilum PM1 and Methylibium R8 showed smaller epsilonH values (-35 per thousand and -42 per thousand, respectively). Plots of Delta2H/Delta13C show different slopes for strain K1 compared with strains PM1 and R8, suggesting that different mechanisms are utilized by K1 and PM1/R8 during aerobic MTBE biodegradation.

Environmental metabolomics: new insights into earthworm ecotoxicity and contaminant bioavailability in soil.

Environmental metabolomics is a growing and emerging sub-discipline of metabolomics. Studies with earthworms have progressed from the initial stages of simple contact exposure tests to detailed studies of earthworm responses in soil. Over the past decade, a variety of endogenous metabolites have been identified as potential biomarkers of contaminant exposure. Furthermore, metabolomic methods have delineated responses from sub-lethal exposure of earthworms to polycyclic aromatic hydrocarbons and metals in soil suggesting that environmental metabolomics may be used as a direct measure of contaminant bioavailability in soil. Environmental metabolomics has the potential to fill knowledge gaps related to earthworm toxicity and contaminant bioavailability. However, challenges with metabolite quantification and limited systems-level models of metabolic data require improvement before detailed models of "normal" responses can be developed and used routinely in assessment of contaminated sites. Nonetheless, environmental metabolomics is poised to improve our fundamental understanding of earthworm responses and toxicity to contaminants in soil.

Comparative assessments of benzene, toluene, and xylene natural attenuation by quantitative polymerase chain reaction analysis of a catabolic gene, signature metabolites, and compound-specific isotope analysis.

A controlled-release study conducted at Vandenberg Air Force Base involved the injection of anaerobic groundwater amended with benzene, toluene, and o-xylene (BToX; 1-3 mg/L each) in two parallel lanes: lane A injectate contained no ethanol, whereas lane B injectate contained approximately 500 mg/L ethanol. As reported previously by Mackay and co-workers, ethanol led to slower BToX disappearance in lane B. Here, we report on assessments of BToX natural attenuation by three independent and specific monitoring approaches: signature metabolites diagnostic of anaerobic TX metabolism (benzysuccinates), compound-specific isotope analysis (CSIA), and quantitative polymerase chain reaction (qPCR) analysis of a catabolic gene involved in anaerobic TX degradation (bssA). In combination, the three monitoring methods provided strong evidence of in situ TX biodegradation in both lanes A and B; however, no single method provided strong evidence for TX biodegradation in both lanes. Benzylsuccinates were detected almost exclusively in lane B, where slower TX degradation and higher residual TX concentrations led to higher metabolite concentrations. In contrast, CSIA provided evidence of TX biodegradation almost exclusively in lane A, as greater degradation rates led to more pronounced isotopic enrichment. qPCR analyses of bssA were more complex. Evidence of increases in bssA copy number (up to 200-fold) after the release started was stronger in lane A, but higher absolute bssA copy number (and bacterial abundance, based on 16S rRNA genes) was observed in lane B, where bacteria genetically capable of anaerobic TX degradation may have been growing primarily on ethanol or its metabolites rather than TX.

Quantifying MTBE biodegradation in the Vandenberg Air Force Base ethanol release study using stable carbon isotopes.

Compound-specific isotope analysis (CSIA) was used to assess biodegradation of MTBE and TBA during an ethanol release study at Vandenberg Air Force Base. Two continuous side-by-side field releases were conducted within a preexisting MTBE plume to form two lanes. The first involved the continuous injection of site groundwater amended with benzene, toluene and o-xylene ("No ethanol lane"), while the other involved the continuous injection of site groundwater amended with benzene, toluene and o-xylene and ethanol ("With ethanol lane"). The delta(13)C of MTBE for all wells in the "No ethanol lane" remained constant during the experiment with a mean value of -31.3 +/- 0.5 per thousand (n=40), suggesting the absence of any substantial MTBE biodegradation in this lane. In contrast, substantial enrichment in (13)C of MTBE by 40.6 per thousand, was measured in the "With ethanol lane", consistent with the effects of biodegradation. A substantial amount of TBA (up to 1200 microg/L) was produced by the biodegradation of MTBE in the "With ethanol lane". The mean value of delta(13)C for TBA in groundwater samples in the "With ethanol lane" was -26.0 +/- 1.0 per thousand (n=32). Uniform delta(13)C TBA values through space and time in this lane suggest that substantial anaerobic biodegradation of TBA did not occur during the experiment. Using the reported range in isotopic enrichment factors for MTBE of -9.2 per thousand to -15.6 per thousand, and values of delta(13)C of MTBE in groundwater samples, MTBE first-order biodegradation rates in the "With ethanol lane" were 12.0 to 20.3 year(-1) (n=18). The isotope-derived rate constants are in good agreement with the previously published rate constant of 16.8 year(-1) calculated using contaminant mass-discharge for the "With ethanol lane".

Analysis of anaerobic BTX biodegradation in a subarctic aquifer using isotopes and benzylsuccinates.

In situ biodegradation of benzene, toluene, and xylenes in a petroleum hydrocarbon contaminated aquifer near Fairbanks, Alaska was assessed using carbon and hydrogen compound specific isotope analysis (CSIA) of benzene and toluene and analysis of signature metabolites for toluene (benzylsuccinate) and xylenes (methylbenzylsuccinates). Carbon and hydrogen isotope ratios of benzene were between -25.9 per thousand and -26.8 per thousand for delta13C and -119 per thousand and -136 per thousand for delta2H, suggesting that biodegradation of benzene is unlikely at this site. However, biodegradation of both xylenes and toluene were documented in this subarctic aquifer. Biodegradation of xylenes was indicated by the presence of methylbenzylsuccinates with concentrations of 17-50 microg/L in three wells. Anaerobic toluene biodegradation was also indicated by benzylsuccinate concentrations of 10-49 microg/L in the three wells with the highest toluene concentrations (1500-5000 microg/L toluene). Since benzylsuccinate typically accounts for a very small fraction of the toluene present in groundwater (generally <1 mol%), the signature metabolite approach works best at higher toluene concentrations when it is not constrained by detection limits. In wells with lower toluene concentrations (410-640 microg/L), carbon and hydrogen isotopic values were enriched by up to approximately 2 per thousand for delta13C and approximately 70 per thousand for delta2H. This evidence of isotopic fractionation verifies the effects of biodegradation in these low concentration wells where metabolites may already be below detection limits. The combined use of signature metabolite and CSIA data is particularly valuable given the challenge of verifying biodegradation in subarctic environments where degradation rates are typically much slower than in temperate environments.