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Quantitative measurement of process-specific biomarker genes of Dehalococcoides mccartyi (Dhc) supports monitoring at chlorinated ethene contaminated sites. In this study, we varied Dhc cell abunda...

Natural perchlorate (ClO4−) exists in many places on Earth, in lunar regolith, meteorites, and on the surface of Mars. Terrestrial natural ClO4− has widely variable Cl and O stable isotopic compositions (δ37Cl, δ18O, Δ17O). The δ18O and Δ17O values of ClO4− from the most hyper-arid locations co-vary. ClO4− from less arid areas has relatively little 17O excess and poor Δ17O-δ18O correlation. ClO4− from the Atacama Desert has unusually low δ37Cl (

At groundwater sites contaminated with chlorinated ethenes, fermentable substrates are often added to promote reductive dehalogenation by indigenous or augmented microorganisms. Contemporary bioremediation performance monitoring relies on nucleic acid biomarkers of key organohalide-respiring bacteria, such as Dehalococcoides mccartyi (Dhc). Metagenome sequencing of the commercial, Dhc-containing consortium, SDC-9, identified 12 reductive dehalogenase (RDase) genes, including pceA (two copies), vcrA, and tceA, and allowed for specific detection and quantification of RDase peptides using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Shotgun (i.e., untargeted) proteomics applied to the SDC-9 consortium grown with tetrachloroethene (PCE) and lactate identified 143 RDase peptides, and 36 distinct peptides that covered greater than 99% of the protein-coding sequences of the PceA, TceA, and VcrA RDases. Quantification of RDase peptides using multiple reaction monitoring (MRM) assays with 13C-/15N-labeled peptides determined 1.8 × 103 TceA and 1.2 × 102 VcrA RDase molecules per Dhc cell. The MRM mass spectrometry approach allowed for sensitive detection and accurate quantification of relevant Dhc RDases and has potential utility in bioremediation monitoring regimes.

This study contrasts the use of high-resolution passive sampling and traditional groundwater monitoring wells (GWMW) to characterize a chlorinated solvent site and assess the effectiveness of a biowall (mulch, compost and sand) that was installed to remediate trichloroethene (TCE), the primary contaminant of concern. High-resolution passive profilers (HRPPs) were direct driven hydraulically upgradient, within, and hydraulically downgradient of the biowall and in close proximity to existing GWMWs. Compared with hydraulically upgradient locations, the biowall was highly reducing, there were higher densities of bacteria/genes capable of reductive dechlorination, and TCE was being reductively transformed, but not completely, as cis-1,2-dichloroethene (cis-DCE) was detected within and hydraulically downgradient of the biowall. However, based on the high-resolution data, there were a number of important findings which were not discoverable using data from GWMWs alone. Data from the HRPPs indicate that the biowall was completely transforming TCE to ethene (C

While bioremediation technologies for trichloroethene (TCE), a suspected carcinogen, have been successfully demonstrated in neutral pH aquifers, these technologies are often ineffective for remediating TCE contamination in acidic aquifers (i.e., pH

Compound-specific isotope analysis (CSIA), position-specific isotope analysis (PSIA), and computational modeling (e.g., quantum mechanical models; reactive-transport models) are increasingly being used to monitor and predict biotic and abiotic transformations of organic contaminants in the field. However, identifying the isotope effect(s) associated with a specific transformation remains challenging in many cases. Here, we describe and interpret the position-specific isotope effects of C and N associated with a S

Natural chlorate (ClO

At groundwater sites contaminated with chlorinated ethenes, fermentable substrates are often added to promote reductive dehalogenation by indigenous or augmented microorganisms. Contemporary bioremediation performance monitoring relies on nucleic acid biomarkers of key organohalide-respiring bacteria, such as

Shotgun sequencing was used for the quantification of taxonomic and functional biomarkers associated with chlorinated solvent bioremediation in 20 groundwater samples (five sites), following bioaugmentation with SDC-9. The analysis determined the abundance of (1) genera associated with chlorinated solvent degradation, (2) reductive dehalogenase (RDases) genes, (3) genes associated with 1,4-dioxane removal, (4) genes associated with aerobic chlorinated solvent degradation, and (5) D. mccartyi genes associated with hydrogen and corrinoid metabolism. The taxonomic analysis revealed numerous genera previously linked to chlorinated solvent degradation, including Dehalococcoides, Desulfitobacterium, and Dehalogenimonas. The functional gene analysis indicated vcrA and tceA from D. mccartyi were the RDases with the highest relative abundance. Reads aligning with both aerobic and anaerobic biomarkers were observed across all sites. Aerobic solvent degradation genes, etnC or etnE, were detected in at least one sample from each site, as were pmoA and mmoX. The most abundant 1,4-dioxane biomarker detected was Methylosinus trichosporium OB3b mmoX. Reads aligning to thmA or Pseudonocardia were not found. The work illustrates the importance of shotgun sequencing to provide a more complete picture of the functional abilities of microbial communities. The approach is advantageous over current methods because an unlimited number of functional genes can be quantified.

1,2-Dibromoethane (ethylene dibromide; EDB) is a probable human carcinogen that was historically added to leaded gasoline as a scavenger to prevent the build-up of lead oxide deposits in engines. Studies indicate that EDB is present at thousands of past fuel spill sites above its stringent EPA Maximum Contaminant Level (MCL) of 0.05 μg/L. There are currently no proven in situ options to enhance EDB degradation in groundwater to meet this requirement. Based on successful laboratory studies showing that ethane can be used as a primary substrate to stimulate the aerobic, cometabolic biodegradation of EDB to0.015 μg/L (Hatzinger et al., 2015), a groundwater recirculation system was installed at the FS-12 EDB plume on Joint Base Cape Cod (JBCC), MA to facilitate in situ treatment. Groundwater was taken from an existing extraction well, amended with ethane, oxygen, and inorganic nutrients and then recharged into the aquifer upgradient of the extraction well creating an in situ reactive zone. The concentrations of EDB, ethane, oxygen, and anions in groundwater were measured with time in a series of nested monitoring wells installed between the extraction and injection well. EDB concentrations in the six monitoring wells that were hydraulically well-connected to the pumping system declined from ~ 0.3 μg/L (the average concentration in the recirculation cell after 3 months of operation without amendment addition) to0.02 μg/L during the 4-month amendment period, meeting both the federal MCL and the more stringent Massachusetts MCL (0.02 μg/L). The data indicate that cometabolic treatment is a promising in situ technology for EDB, and that low regulatory levels can be achieved with this biological approach.

Perchlorate (ClO4−) in groundwater can be from synthetic or natural sources. Natural sources include ClO4− associated with historical application of imported natural nitrate fertilizer from the Atacama Desert of Chile, and indigenous ClO4− that accumulates locally in arid regions from atmospheric deposition. The Rialto-Colton groundwater subbasin, 80 km east of Los Angeles, California, includes two mapped ClO4− plumes from known military/industrial sources. Larger areas downgradient from those plumes, and in the Chino subbasin to the southwest, also contain ClO4−. Perchlorate from wells was analyzed for chlorine and oxygen stable isotope ratios (δ37Cl, δ18O, Δ17O) and radioactive chlorine-36 (36Cl) isotopic abundance, along with other geochemical, isotopic, and hydrogeologic data. Isotopic data show that synthetic ClO4− was the dominant source within the mapped plumes. Downgradient from the mapped plumes, and in the Chino subbasin, the dominant source of ClO4− was related to past agricultural use of Chilean (Atacama) nitrate fertilizer. The 36Cl and δ18O data indicate that wells having predominantly synthetic or Atacama ClO4− also contained small fractions of indigenous ClO4−. Little or no differences were observed in isotopic composition or ClO4− source with depth in depth-dependent data from selected wells. Indigenous ClO4− was most evident in upgradient wells having ClO4− concentrations

This paper describes an offline low-temperature derivatization method combined with gas chromatography-isotope ratio mass spectrometry (GC-IRMS) to enable compound-specific isotope analysis (CSIA) of C and N in the explosive compound 3-nitro-1,2,4-triazole-5-one (NTO). NTO is being used widely as a component of insensitive munition (IM) composites such as IMX-101, a formulation that is being used to replace the more shock sensitive TNT in artillery shells. The alkylation of NTO with methyl iodide (MeI) as the derivatization reagent was performed in acetone solution at room temperature using triethylamine (Et3N) as a base catalyst. The methylated product of NTO derivatization was identified as 4-methyl-3-nitro-1,2,4-triazol-5-one (MNTO). Accurate and reproducible results were achieved by systematic optimization of MeI and Et3N concentrations and reaction time. Isotopic values of MNTO obtained by GC-IRMS were normalized against those of two 2,4-dinitroanisole (DNAN) in-house reference materials that had been calibrated with isotopic standard reference materials, USGS40 and USGS41a. The resulting method detection limit for derivatization/GC-IRMS of NTO was 788 ng of NTO, yielding a precision of ±0.3‰ for both δ13C and δ15N values in good agreement with δ13C and δ15N values for NTO determined by elemental analyzer-IRMS.

Compound-specific isotope analysis (CSIA), position-specific isotope analysis (PSIA), and computational modeling (e.g., quantum mechanical models; reactive-transport models) are increasingly being used to monitor and predict biotic and abiotic transformations of organic contaminants in the field. However, identifying the isotope effect(s) associated with a specific transformation remains challenging in many cases. Here, we describe and interpret the position-specific isotope effects of C and N associated with a SN2Ar reaction mechanism by a combination of CSIA and PSIA using quantitative 13C nuclear magnetic resonance spectrometry, and density-functional theory, using 2,4-dinitroanisole (DNAN) as a model compound. The position-specific 13C enrichment factor of O-C1 bond at the methoxy group attachment site (eC1) was found to be approximately -41‰, a diagnostic value for transformation of DNAN to its reaction products 2,4-dinitrophenol and methanol. Theoretical kinetic isotope effects calculated for DNAN isotopologues agreed well with the position-specific isotope effects measured by CSIA and PSIA. This combination of measurements and theoretical predictions demonstrates a useful tool for evaluating degradation efficiencies and/or mechanisms of organic contaminants and may promote new and improved applications of isotope analysis in laboratory and field investigations.

Natural chlorate (ClO3−) is widely distributed in terrestrial and extraterrestrial environments. To improve understanding of the origins and distribution of ClO3−, we developed and tested methods to determine the multi-dimensional isotopic compositions (δ18O, Δ17O, δ37Cl, 36Cl/Cl) of ClO3− and then applied the methods to samples of natural nitrate-rich caliche-type salt deposits in the Atacama Desert, Chile, and Death Valley, USA. Tests with reagents and artificial mixed samples indicate stable-isotope ratios were minimally affected by the purification processes. Chlorate extracted from Atacama samples had δ18O = +7.0 to +11.1‰, Δ17O = +5.7 to +6.4‰, δ37Cl = −1.4 to +1.3‰, and 36Cl/Cl = 48 × 10−15 to 104 × 10−15. Chlorate from Death Valley samples had δ18O = −6.9 to +1.6‰, Δ17O = +0.4 to +2.6‰, δ37Cl = +0.8 to +1.0‰, and 36Cl/Cl = 14 × 10−15 to 44 × 10−15. Positive Δ17O values of natural ClO3− indicate that its production involved reaction with O3, while its Cl isotopic composition is consistent with a tropospheric or near-surface source of Cl. The Δ17O and δ18O values of natural ClO3− are positively correlated, as are those of ClO4− and NO3− from the same localities, possibly indicating variation in the relative contributions of O3 as a source of O in the formation of the oxyanions. Additional isotopic analyses of ClO3− could provide stronger constraints on its production mechanisms and/or post-formational alterations, with applications for environmental forensics, global biogeochemical cycling of Cl, and the origins of oxyanions detected on Mars.

TaqMan probe-based quantitative polymerase chain reaction (qPCR) specific to the biomarker reductive dehalogenase (RDase) genes is a widely accepted molecular biological tool (MBT) for determining the abundance of Dehalococcoides sp. in groundwater samples from chlorinated solvent-contaminated sites. However, there are significant costs associated with this MBT. In this study, we describe an approach that requires only low-cost laboratory equipment (a bench top centrifuge and a water bath) and requires less time and resources compared to qPCR. The method involves the concentration of biomass from groundwater, without DNA extraction, and loop-mediated isothermal amplification (LAMP) of the cell templates. The amplification products are detected by a simple visual color change (orange/green). The detection limits of the assay were determined using groundwater from a contaminated site. In addition, the assay was tested with groundwater from three additional contaminated sites. The final approach to detect RDase genes, without DNA extraction or a thermal cycler, was successful to 1.8 × 105 gene copies per L for vcrA and 1.3 × 105 gene copies per L for tceA. Both values are below the threshold recommended for effective in situ dechlorination.

Perchlorate (ClO4−) contamination has received increased attention the past two decades because of health concerns from its impact on thyroid function coupled with its environmental ubiquity and persistence. Here we demonstrate the detection of ClO4− below micromolar (

We present the first report of biotransformation of 3-nitro-1,2,4-triazol-5-one (NTO) and 2,4-dinitroanisole (DNAN), replacements for the explosives 1,3,5-trinitro-1,3,5-triazine (RDX) and 2,4,6-trinitrotoluene (TNT), respectively, by methane-oxidizing cultures under aerobic conditions. Two consortia, dominated by Methylosinus spp., degraded both compounds with transient production of reduced NTO products, and non-stoichiometric production of reduced DNAN products. No release of inorganic nitrogen was observed with either compound, indicating that NTO and DNAN may be utilized as nitrogen sources by these consortia. The pure culture Methylosinus trichosporium OB3b also degraded both compounds. Degradation was observed in the presence of acetylene (a known inhibitor of methane monooxygenase; MMO) when methanol was supplied, indicating that MMO was not involved. Furthermore, studies with purified soluble MMO (sMMO) from OB3b indicated that neither compound was a substrate for sMMO. Degradation was inhibited by 2-iodosobenzoic acid, but not by dicoumarol, suggesting involvement of an oxygen- and dicoumarol-insensitive (nitro)reductase. These results indicate methanotrophs can aerobically degrade NTO and DNAN via one or more (nitro)reductases, with sMMO serving a supporting role deriving reducing equivalents from methane. This finding is important because methanotrophic bacteria are widely dispersed, and may represent a previously unrecognized route of NTO and DNAN biotransformation in aerobic environments.

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), a nitroamine explosive, is commonly detected in groundwater at military testing and training sites. The objective of this study was to characterize the microbial community capable of using nitrogen derived from the RDX or RDX intermediates during in situ bioremediation. Active groundwater microorganisms capable of utilizing nitro-, ring- or fully-labeled (15)N-RDX as a nitrogen source were identified using stable isotope probing (SIP) in groundwater microcosms prepared from two wells in an aquifer previously amended with cheese whey to promote RDX biodegradation. A total of fifteen 16S rRNA gene sequences, clustered in Clostridia, β-Proteobacteria, and Spirochaetes, were derived from the (15)N-labeled DNA fractions, suggesting the presence of metabolically active bacteria capable of using RDX and/or RDX intermediates as a nitrogen source. None of the derived sequences matched RDX-degrading cultures commonly studied in the laboratory, but some of these genera have previously been linked to RDX degradation in site groundwater via (13)C-SIP. When additional cheese whey was added to the groundwater samples, 28 sequences grouped into Bacteroidia, Bacilli, and α-, β-, and γ-Proteobacteria were identified. The data suggest that numerous bacteria are capable of incorporating N from ring- and nitro-groups in RDX during anaerobic bioremediation, and that some genera may be involved in both C and N incorporation from RDX.

Hexahydro-1,3,5-trinitro-1,3,5,-triazine (RDX) is a toxic and mobile groundwater contaminant common to military sites. This study compared in situ RDX degradation rates following bioaugmentation with Gordonia sp. strain KTR9 (henceforth KTR9) to rates under biostimulation conditions in an RDX-contaminated aquifer in Umatilla, OR. Bioaugmentation was achieved by injecting site groundwater (6000 L) amended with KTR9 cells (10(8) cells mL(-1)) and low carbon substrate concentrations (1 mM fructose) into site wells. Biostimulation (no added cells) was performed by injecting groundwater amended with low (1 mM fructose) or high (15 mM fructose) carbon substrate concentrations in an effort to stimulate aerobic or anaerobic microbial activity, respectively. Single-well push-pull tests were conducted to measure RDX degradation rates for each treatment. Average rate coefficients were 1.2 day(-1) for bioaugmentation and 0.7 day(-1) for high carbon biostimulation; rate coefficients for low carbon biostimulation were not significantly different from zero (p values ≥0.060). Our results suggest that bioaugmentation with KTR9 is a feasible strategy for in situ biodegradation of RDX and, at this site, is capable of achieving RDX concentration reductions comparable to those obtained by high carbon biostimulation while requiring ~97% less fructose. Bioaugmentation has potential to minimize substrate quantities and associated costs, as well as secondary groundwater quality impacts associated with anaerobic biostimulation processes (e.g., hydrogen sulfide, methane production) during full-scale RDX remediation.

Kinetic isotopic fractionation of carbon and nitrogen during RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) biodegradation was investigated with pure bacterial cultures under aerobic and anaerobic conditions. Relatively large bulk enrichments in 15 N were observed during biodegradation of RDX via anaerobic ring cleavage (ε 15 N = −12.7‰ ± 0.8‰) and anaerobic nitro reduction (ε 15 N = −9.9‰ ± 0.7‰), in comparison to smaller effects during biodegradation via aerobic denitration (ε 15 N = −2.4‰ ± 0.2‰). 13 C enrichment was negligible during aerobic RDX biodegradation (ε 13 C = −0.8‰ ± 0.5‰) but larger during anaerobic degradation (ε 13 C = −4.0‰ ± 0.8‰), with modest variability among genera. Dual-isotope ε 13 C/ε 15 N analyses indicated that the three biodegradation pathways could be distinguished isotopically from each other and from abiotic degradation mechanisms. Compared to the initial RDX bulk δ 15 N value of +9‰, δ 15 N values of the NO 2 − released from RDX ranged from −7‰ to +2‰ during aerobic biodegradation and from −42‰ to −24‰ during anaerobic biodegradation. Numerical reaction models indicated that N isotope effects of NO 2 − production were much larger than, but systematically related to, the bulk RDX N isotope effects with different bacteria. Apparent intrinsic ε 15 N-NO 2 − values were consistent with an initial denitration pathway in the aerobic experiments and more complex processes of NO 2 − formation associated with anaerobic ring cleavage. These results indicate the potential for isotopic analysis of residual RDX for the differentiation of degradation pathways and indicate that further efforts to examine the isotopic composition of potential RDX degradation products (e.g., NO x ) in the environment are warranted. IMPORTANCE This work provides the first systematic evaluation of the isotopic fractionation of carbon and nitrogen in the organic explosive RDX during degradation by different pathways. It also provides data on the isotopic effects observed in the nitrite produced during RDX biodegradation. Both of these results could lead to better understanding of the fate of RDX in the environment and help improve monitoring and remediation technologies.

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), and perchlorate (ClO4−) are common, and often co-mingled, contaminants at military ranges worldwide. This project investigated the feasibility of using a passive emulsified oil biobarrier plus a slow release pH buffering reagent to remediate RDX, HMX, and ClO4− in a low pH aquifer at an active range. A 33 m biobarrier was emplaced perpendicular to the contaminant plumes, and dissolved explosives, perchlorate, and other relevant parameters were monitored. The pH increased and the DO and ORP decreased after emulsified oil injection, leading to >90% reductions in perchlorate, RDX, and HMX compared to upgradient groundwater. Some nitroso breakdown products were observed immediately downstream of the barrier, but generally decreased to below detection limits farther downgradient. First-order rate constants of approximately 0.1/d were obtained for all three contaminants. Dissolved metals (including As) also increased in the wells immediately adjacent to the barrier, but attenuated as the plume re-aerated in downgradient areas. Biobarrier installation and sampling were performed during scheduled range downtime and had no impacts to ongoing range activities. The field trial suggests that an emulsified oil biobarrier with pH buffering can be a viable alternative to remove explosives and perchlorate from shallow groundwater on active ranges.

The fate and transport of groundwater contaminants depends partially on groundwater velocity, which can vary appreciably in highly stratified aquifers. A high-resolution passive profiler (HRPP) was developed to evaluate groundwater velocity, contaminant concentrations, and microbial community structure at ∼20 cm vertical depth resolution in shallow heterogeneous aquifers. The objective of this study was to use mass transfer of bromide (Br- ), a conservative tracer released from cells in the HRPP, to estimate interstitial velocity. Laboratory experiments were conducted to empirically relate velocity and the mass transfer coefficient of Br- based on the relative loss of Br- from HRPP cells. Laboratory-scale HRPPs were deployed in flow boxes containing saturated soils with differing porosities, and the mass transfer coefficient of Br- was measured at multiple interstitial velocities (0 to 100 cm/day). A two-dimensional (2D) quasi-steady-state model was used to relate velocity to mass transfer of Br- for a range of soil porosities (0.2-0.5). The laboratory data indicate that the mass transfer coefficient of Br- , which was directly-but non-linearly-related to velocity, can be determined with a single 3-week deployment of the HRPP. The mass transfer coefficient was relatively unaffected by sampler orientation, length of deployment time, or porosity. The model closely simulated the experimental results. The data suggest that the HRPP will be applicable for estimating groundwater velocity ranging from 1 to 100 cm/day in the field at a minimum depth resolution of 10 cm, depending on sampler design.

1,2-Dibromethane (EDB) is a toxic fuel additive that likely occurs at many sites where leaded fuels have impacted groundwater. This study quantified carbon (C) isotope fractionation of EDB associated with anaerobic and aerobic biodegradation, abiotic degradation by iron sulfides, and abiotic hydrolysis. These processes likely contribute to EDB degradation in source zones (biodegradation) and in more dilute plumes (hydrolysis). Mixed anaerobic cultures containing dehalogenating organisms (e.g., Dehaloccoides spp.) were examined, as were aerobic cultures that degrade EDB cometabolically. Bulk C isotope enrichment factors (ebulk) associated with biological degradation covered a large range, with mixed anaerobic cultures fractionating more (ebulk from −8 to −20‰) than aerobic cultures (ebulk from −3 to −6‰). ebulk magnitudes associated with the abiotic processes (dihaloelimination by FeS/FeS2 and hydrolysis) were large but fairly well constrained (ebulk from −19 to −29‰). As expected, oxidative mechanisms fra...

In situ bioaugmentation with aerobic hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)-degrading bacteria is being considered for treatment of explosives-contaminated groundwater at Umatilla Chemical Depot, Oregon (UMCD). Two forced-gradient bacterial transport tests of site groundwater containing chloride or bromide tracer and either a mixed culture of Gordonia sp. KTR9 (xplA (+)Km(R)), Rhodococcus jostii RHA1 (pGKT2 transconjugant; xplA (+)Km(R)) and Pseudomonas fluorescens I-C (xenB (+)), or a single culture of Gordonia sp. KTR9 (xplA (+); i.e. wild-type) were conducted at UMCD. Groundwater monitoring evaluated cell viability and migration in the injection well and downgradient monitoring wells. Enhanced degradation of RDX was not evaluated in these demonstrations. Quantitative PCR analysis of xplA, the kanamycin resistance gene (aph), and xenB indicated that the mixed culture was transported at least 3 m within 2 h of injection. During a subsequent field injection of bioaugmented groundwater, strain KTR9 (wild-type) migrated up to 23-m downgradient of the injection well within 3 days. Thus, the three RDX-degrading strains were effectively introduced and transported within the UMCD aquifer. This demonstration represents an innovative application of bioaugmentation to potentially enhance RDX biodegradation in aerobic aquifers.

Natural perchlorate (ClO 4 − ) is of increasing interest due to its wide-spread occurrence on Earth and Mars, yet little information exists on the relative abundance of ClO 4 − compared to other major anions, its stability, or long-term variations in production that may impact the observed distributions. Our objectives were to evaluate the occurrence and fate of ClO 4 − in groundwater and soils/caliche in arid and semi-arid environments (southwestern United States, southern Africa, United Arab Emirates, China, Antarctica, and Chile) and the relationship of ClO 4 − to the more well-studied atmospherically deposited anions NO 3 − and Cl − as a means to understand the prevalent processes that affect the accumulation of these species over various time scales. ClO 4 − is globally distributed in soil and groundwater in arid and semi-arid regions on Earth at concentrations ranging from 10 −1 to 10 6 μg/kg. Generally, the ClO 4 − concentration in these regions increases with aridity index, but also depends on the duration of arid conditions. In many arid and semi-arid areas, NO 3 − and ClO 4 − co-occur at molar ratios (NO 3 − /ClO 4 − ) that vary between ∼10 4 and 10 5 . We hypothesize that atmospheric deposition ratios are largely preserved in hyper-arid areas that support little or no biological activity (e.g. plants or bacteria), but can be altered in areas with more active biological processes including N 2 fixation, N mineralization, nitrification, denitrification, and microbial ClO 4 − reduction, as indicated in part by NO 3 − isotope data. In contrast, much larger ranges of Cl − /ClO 4 − and Cl − /NO 3 − ratios indicate Cl − varies independently from both ClO 4 − and NO 3 − . The general lack of correlation between Cl − and ClO 4 − or NO 3 − implies that Cl − is not a good indicator of co-deposition and should be used with care when interpreting oxyanion cycling in arid systems. The Atacama Desert appears to be unique compared to all other terrestrial locations having a NO 3 − /ClO 4 − molar ratio ∼10 3 . The relative enrichment in ClO 4 − compared to Cl − or NO 3 − and unique isotopic composition of Atacama ClO 4 − may reflect either additional in-situ production mechanism(s) or higher relative atmospheric production rates in that specific region or in the geological past. Elevated concentrations of ClO 4 − reported on the surface of Mars, and its enrichment with respect to Cl − and NO 3 − , could reveal important clues regarding the climatic, hydrologic, and potentially biologic evolution of that planet. Given the highly conserved ratio of NO 3 − /ClO 4 − in non-biologically active areas on Earth, it may be possible to use alterations of this ratio as a biomarker on Mars and for interpreting major anion cycles and processes on both Mars and Earth, particularly with respect to the less-conserved NO 3 − pool terrestrially.

Perchlorate from military, industrial, and legacy agricultural sources is present within an alluvial aquifer in the Rialto-Colton groundwater subbasin, 80 km east of Los Angeles, California (USA). The area is extensively faulted, with water-level differences exceeding 60 m across parts of the Rialto-Colton Fault separating the Rialto-Colton and Chino groundwater subbasins. Coupled well-bore flow and depth-dependent water-quality data show decreases in well yield and changes in water chemistry and isotopic composition, reflecting changing aquifer properties and groundwater recharge sources with depth. Perchlorate movement through some wells under unpumped conditions from shallower to deeper layers underlying mapped plumes was as high as 13 kg/year. Water-level maps suggest potential groundwater movement across the Rialto-Colton Fault through an overlying perched aquifer. Upward flow through a well in the Chino subbasin near the Rialto-Colton Fault suggests potential groundwater movement across the fault through permeable layers within partly consolidated deposits at depth. Although potentially important locally, movement of groundwater from the Rialto-Colton subbasin has not resulted in widespread occurrence of perchlorate within the Chino subbasin. Nitrate and perchlorate concentrations at the water table, associated with legacy agricultural fertilizer use, may be underestimated by data from long-screened wells that mix water from different depths within the aquifer.

The potential for bioaugmentation with aerobic explosive degrading bacteria to remediate hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) contaminated aquifers was demonstrated. Repacked aquifer sediment columns were used to examine the transport and RDX degradation capacity of the known RDX degrading bacterial strains Gordonia sp. KTR9 (modified with a kanamycin resistance gene) Pseudomonas fluorescens I-C, and a kanamycin resistant transconjugate Rhodococcus jostii RHA1 pGKT2:Km+. All three strains were transported through the columns and eluted ahead of the conservative bromide tracer, although the total breakthrough varied by strain. The introduced cells responded to biostimulation with fructose (18 mg L(-1), 0.1 mM) by degrading dissolved RDX (0.5 mg L(-1), 2.3 µM). The strains retained RDX-degrading activity for at least 6 months following periods of starvation when no fructose was supplied to the column. Post-experiment analysis of the soil indicated that the residual cells were distributed along the length of the column. When the strains were grown to densities relevant for field-scale application, the cells remained viable and able to degrade RDX for at least 3 months when stored at 4 °C. These results indicate that bioaugmentation may be a viable option for treating RDX in large dilute aerobic plumes.

The remediation of chlorinated solvent contaminated sites frequently involves bioaugmentation with mixed cultures containing Dehalococcoides mccartyi. Their activity is then examined by quantifying reductive dehalogenase (RDase) genes. Recently, we described a rapid, low cost approach, based on loop mediated isothermal amplification (LAMP), which allowed for the visual detection of RDase genes from groundwater. In that study, samples were concentrated (without DNA extraction), incubated in a water bath (avoiding the use of a thermal cycler) and amplification was visualized by the addition of SYBR green (post incubation). Despite having a detection limit less than the threshold recommended for effective remediation, the application of the assay was limited because of the semi-quantitative nature of the data. Moreover, the assay was prone to false positives due to the aerosolization of amplicons. In this study, deoxyuridine triphosphate (dUTP) and uracil DNA glycosylase (UNG) were incorporated into the assay to reduce the probability of false positives. Optimization experiments revealed a UNG concentration of 0.2units per reaction was adequate for degrading trace levels of AUGC based contamination (~1.4×104 gene copies/reaction) without significant changes to the detection limit (~100 gene copies/reaction). Additionally, the optimized assay was used with the most probable number (MPN) method to quantify RDase genes (vcrA and tceA) in multiple groundwater samples from a chlorinated solvent contaminated site. Using this approach, gene concentrations were significantly correlated to concentrations obtained using traditional methods (qPCR and DNA templates). Although the assay underestimated RDase genes concentrations, a strong correlation (R2=0.78 and 0.94) was observed between the two data sets. The regression equations obtained will be valuable to determine gene copies in groundwater using the newly developed, low cost and time saving method.

The objective of this research was to evaluate the potential for two gases, methane and ethane, to stimulate the biological degradation of 1,4-dioxane (1,4-D) in groundwater aquifers via aerobic cometabolism. Experiments with aquifer microcosms, enrichment cultures from aquifers, mesophilic pure cultures, and purified enzyme (soluble methane monooxygenase; sMMO) were conducted. During an aquifer microcosm study, ethane was observed to stimulate the aerobic biodegradation of 1,4-D. An ethane-oxidizing enrichment culture from these samples, and a pure culture capable of growing on ethane (Mycobacterium sphagni ENV482) that was isolated from a different aquifer also biodegraded 1,4-D. Unlike ethane, methane was not observed to appreciably stimulate the biodegradation of 1,4-D in aquifer microcosms or in methane-oxidizing mixed cultures enriched from two different aquifers. Three different pure cultures of mesophilic methanotrophs also did not degrade 1,4-D, although each rapidly oxidized 1,1,2-trichloroethene (TCE). Subsequent studies showed that 1,4-D is not a substrate for purified sMMO enzyme from Methylosinus trichosporium OB3b, at least not at the concentrations evaluated, which significantly exceeded those typically observed at contaminated sites. Thus, our data indicate that ethane, which is a common daughter product of the biotic or abiotic reductive dechlorination of chlorinated ethanes and ethenes, may serve as a substrate to enhance 1,4-D degradation in aquifers, particularly in zones where these products mix with aerobic groundwater. It may also be possible to stimulate 1,4-D biodegradation in an aerobic aquifer through addition of ethane gas. Conversely, our results suggest that methane may have limited importance in natural attenuation or for enhancing biodegradation of 1,4-D in groundwater environments.

The ex situ treatment of N-nitrosodimethylamine (NDMA) and N-nitrodimethylamine (NTDMA) in groundwater was evaluated in a field-scale fluidized bed bioreactor (FBR). Both of these compounds, which originally entered groundwater at the test site from the use of liquid rocket propellant, are suspected human carcinogens. The objective of this research was to examine the application of a novel field-scale propane-fed fluidized bed bioreactor as an alternative to ultraviolet irradiation (UV) for treating NDMA and NTDMA to low part-per-trillion (ng/L) concentrations. Previous laboratory studies have shown that the bacterium Rhodococcus ruber ENV425 can biodegrade NDMA and NTDMA during growth on propane as a primary substrate and that the strain can effectively reduce NDMA concentrations in propane-fed bench-scale bioreactors of different design. R. ruber ENV425 was used as a seed culture for the FBR, which operated at a fluidization flow of ∼19 L-per-min (LPM) and received propane, oxygen, and inorganic nutrients in the feed. The reactor effectively treated ∼1 μg/L of influent NDMA to effluent concentrations of less than 10 ng/L at a hydraulic residence time (HRT) of only 10 min. At a 20 min HRT, the FBR reduced NDMA to 3 of expanded bed/hr of operation and 1.1 mg NTDMA treated/m 3 of expanded bed/hr of operation, respectively. The FBR system was highly resilient to upsets including power outages. Treatment of NDMA, but not NTDMA, was marginally affected when trace co-contaminants including trichloroethene (TCE) and trichlorofluoromethane (Freon 11) were initially added to feed groundwater, but performance recovered over a few weeks in the continued presence of these compounds. Strain ENV425 appeared to be replaced by native propanotrophs over time based on qPCR analysis, but contaminant treatment was not diminished. The results suggest that a FBR can be a viable alternative to UV treatment for removing NDMA from groundwater.

Degradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in laboratory columns following biostimulation and bioaugmentation was investigated using sediment and groundwater from a contaminated aquifer at a US Navy facility. No RDX degradation was observed following aerobic biostimulation with either fructose or lactate (both 0.1 mM) prior to bioaugmentation. Replicate columns were then bioaugmented with either Gordonia sp. KTR9, Pseudomonas fluorescens I-C (Ps I-C), or both strains. Under aerobic conditions (influent dissolved oxygen (DO) >6 mg/L), RDX was degraded following the addition of fructose, and to a lesser extent with lactate, in columns bioaugmented with KTR9. No degradation was observed in columns bioaugmented with only Ps I-C under aerobic conditions, consistent with the known anaerobic RDX degradation pathway for this strain. When influent DO was reduced to

Natural perchlorate (ClO4−) in soil and groundwater exhibits a wide range in stable isotopic compositions (δ37Cl, δ18O, and Δ17O), indicating that ClO4− may be formed through more than one pathway and/or undergoes post-depositional isotopic alteration. Plants are known to accumulate ClO4−, but little is known about their ability to alter its isotopic composition. We examined the potential for plants to alter the isotopic composition of ClO4− in hydroponic and field experiments conducted with snap beans (Phaseolus vulgaris L.). In hydroponic studies, anion ratios indicated that ClO4− was transported from solutions into plants similarly to NO3− but preferentially to Cl− (4-fold). The ClO4− isotopic compositions of initial ClO4− reagents, final growth solutions, and aqueous extracts from plant tissues were essentially indistinguishable, indicating no significant isotope effects during ClO4− uptake or accumulation. The ClO4− isotopic composition of field-grown snap beans was also consistent with that of ClO4− in varying proportions from irrigation water and precipitation. NO3− uptake had little or no effect on NO3− isotopic compositions in hydroponic solutions. However, a large fractionation effect with an apparent e (15N/18O) ratio of 1.05 was observed between NO3− in hydroponic solutions and leaf extracts, consistent with partial NO3− reduction during assimilation within plant tissue. We also explored the feasibility of evaluating sources of ClO4− in commercial produce, as illustrated by spinach, for which the ClO4− isotopic composition was similar to that of indigenous natural ClO4−. Our results indicate that some types of plants can accumulate and (presumably) release ClO4− to soil and groundwater without altering its isotopic characteristics. Concentrations and isotopic compositions of ClO4− and NO3− in plants may be useful for determining sources of fertilizers and sources of ClO4− in their growth environments and consequently in food supplies.

N-Nitrosodimethylamine (NDMA) is found in groundwater and drinking water from industrial, agricultural, water treatment, and military/aerospace sources, and it must often be treated to part-per-trillion (ng/L) concentrations. The most effective remedial technology for NDMA in groundwater is pump-and-treat with ultraviolet irradiation (UV), but this approach is expensive because it requires ex situ infrastructure and high energy input. The objective of this project was to evaluate an in situ biological treatment approach for NDMA. Previous laboratory studies have revealed that propane-oxidizing bacteria are capable of biodegrading NDMA from μg/L to low ng/L concentrations ( Fournier et al., 2009 ; Webster et al., 2013 ). During this field study, air and propane gas were sparged into an NDMA-contaminated aquifer for more than 1 year. Groundwater samples were collected throughout the study from a series of monitoring wells within, downgradient, and sidegradient of the zone of influence of the biosparge system. Over the course of the study, NDMA concentrations declined by 99.7% to >99.9% in the four monitoring wells within the zone of influence of the biosparge system, reaching low ng/L concentrations whereas the control well declined by only 14%. Pseudo first-order degradation rate constants for NDMA in system monitoring wells ranged from ∼0.019 day −1 to 0.037 day −1 equating to half-lives ranging from 19 to 36 days. Native propanotrophs increased by more than one order of magnitude in the propane-impacted wells but not in the control well. The field data show for the first time that propane biosparging can be an effective in situ approach to reduce the concentrations of NDMA in a groundwater to ng/L concentrations.

Perchlorate (ClOThree large batches of KClOKClOKClO

Nucleic acid amplification of biomarkers is increasingly used to monitor microbial activity and assess remedial performance in contaminated aquifers. Previous studies described the use of filtration, elution, and direct isothermal amplification (i.e. no DNA extraction and purification) as a field-able means to quantify Dehalococcoides spp. in groundwater. This study expands previous work with direct loop mediated isothermal amplification (LAMP) for the detection and quantification of Dehalobacter spp. in groundwater. Experiments tested amplification of DNA with and without crude lysis and varying concentrations of humic acid. Three separate field-able methods of biomass concentration with eight aquifer samples were also tested, comparing direct LAMP with traditional DNA extraction and quantitative PCR (qPCR). A new technique was developed where filters were amplified directly within disposable Gene-Z chips. The direct filter amplification (DFA) method eliminated an elution step and provided a detection limit of 102 Dehalobacter cells per 100 mL. LAMP with crudely lysed Dehalobacter had a negligible effect on threshold time and sensitivity compared to lysed samples. The LAMP assay was more resilient than traditional qPCR to humic acid in sample, amplifying with up to 100 mg per L of humic acid per reaction compared to 1 mg per L for qPCR. Of the tested field-able concentrations methods, DFA had the lowest coefficient of variation among Dehalobacter spiked groundwater samples and lowest threshold time indicating high capture efficiency and low inhibition. While demonstrated with Dehalobacter, the DFA method can potentially be used for a number of applications requiring field-able, rapid (

N-Nitrosodimethylamine (NDMA) is a suspected human carcinogen that has recently been detected in wastewater, groundwater and drinking water. Treatment of this compound to low part-per-trillion (ng/L) concentrations is required to mitigate cancer risk. Current treatment generally entails UV irradiation, which while effective, is also expensive. The objective of this research was to explore potential bioremediation strategies as alternatives for treating NDMA to ng/L concentrations. Batch studies revealed that the propanotroph Rhodococcus ruber ENV425 was capable of metabolizing NDMA from 8 μg/L to2 ng/L after growth on propane, and that the strain produced metabolites that do not pose a significant risk at the concentrations generated (Fournier et al., 2009). A laboratory-scale membrane bioreactor (MBR) was subsequently constructed to evaluate the potential for long-term ex situ treatment of NDMA. The MBR was seeded with ENV425 and received propane as the primary growth substrate and oxygen as an electron acceptor. At an average influent NDMA concentration of 7.4 μg/L and a 28.5 h hydraulic residence time, the reactor effluent concentration was 3.0 ± 2.3 ng/L (99.95% removal) over more than 70 days of operation. The addition of trichloroethene (TCE) to the reactor resulted in a significant increase in effluent NDMA concentrations, most likely due to cell toxicity from TCE-epoxide produced during its cometabolic oxidation by ENV425. The data suggest that an MBR system can be a viable treatment option for NDMA in groundwater provided that high concentrations of TCE are not present.

A new ion chromatography electrospray tandem mass spectrometry (IC-ESI/MS/MS) method has been developed for quantification and confirmation of chlorate (ClO3−) in environmental samples. The method ...

Biodegradation of explosives in groundwater represents a promising remedial approach for these compounds. Although a range of bacteria capable of degrading the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in pure culture have been described, the role of these known strains (and the genera they represent) during RDX degradation in groundwater has not been established. RDX-contaminated groundwater was collected from the Pueblo Chemical Depot (CO, USA) and the Picatinny Arsenal (NJ, USA) where bioremediation technologies are being tested. Soil columns and enrichment cultures were derived from Picatinny Arsenal groundwater. Bacteria-specific primers were used to amplify the 16S rRNA genes that were used for phylogenetic analysis. The species detected ranged across multiple genera, many of which have not been previously associated with RDX biodegradation. None of the retrieved sequences were exact matches to previously described RDX-degrading strains, although multiple sequences that groupe...

Techniques for rapid and sensitive detection of energetics such as cyclotrimethylenetrinitramine (RDX) are needed both for environmental and security screening applications. Here we report the use of surface enhanced Raman spectroscopy (SERS) to detect traces of RDX with good sensitivity and reproducibility. Using gold (Au) nanoparticles (~90-100 nm in diameter) as SERS substrates, RDX was detectable at concentrations as low as 0.15 mg/L in a contaminated groundwater sample. This detection limit is about two orders of magnitude lower than those reported previously using SERS techniques. A surface enhancement factor of ~6x104 was obtained. This research further demonstrates the potential for using SERS as a rapid, in-situ field screening tool for energetics detection when coupled with a portable Raman spectrometer.

Perchlorate (ClO4(-)) is ubiquitous in the environment. It is produced naturally by atmospheric photochemical reactions, and also is synthesized in large quantities for military, aerospace, and industrial applications. Nitrate-enriched salt deposits of the Atacama Desert (Chile) contain high concentrations of natural ClO4(-), and have been exported worldwide since the mid-1800s for use in agriculture. The widespread introduction of synthetic and agricultural ClO4(-) into the environment has contaminated numerous municipal water supplies. Stable isotope ratio measurements of Cl and O have been applied for discrimination of different ClO4(-) sources in the environment. This study explores the potential of 36Cl measurements for further improving the discrimination of ClO4(-) sources. Groundwater and desert soil samples from the southwestern United States (U.S.) contain ClO4(-) having high 36Cl abundances (36Cl/Cl = 3100 x 10(-15) to 28,800 x 10(-15)), compared with those from the Atacama Desert (36Cl/Cl = 0.9 x 10(-15) to 590 x 10(-15)) and synthetic ClO4(-) reagents and products (36Cl/Cl = 0.0 x 10(-15) to 40 x 10(-15)). In conjunction with stable Cl and O isotope ratios, 36Cl data provide a clear distinction among three principal ClO4(-) source types in the environment of the southwestern U.S.

The propanotroph Rhodococcus ruber ENV425 was observed to rapidly biodegrade N -nitrosodimethylamine (NDMA) after growth on propane, tryptic soy broth, or glucose. The key degradation intermediates were methylamine, nitric oxide, nitrite, nitrate, and formate. Small quantities of formaldehyde and dimethylamine were also detected. A denitrosation reaction, initiated by hydrogen atom abstraction from one of the two methyl groups, is hypothesized to result in the formation of n -methylformaldimine and nitric oxide, the former of which decomposes in water to methylamine and formaldehyde and the latter of which is then oxidized further to nitrite and then nitrate. Although the strain mineralized more than 60% of the carbon in [ 14 C]NDMA to 14 CO 2 , growth of strain ENV425 on NDMA as a sole carbon and energy source could not be confirmed. The bacterium was capable of utilizing NDMA, as well as the degradation intermediates methylamine and nitrate, as sources of nitrogen during growth on propane. In addition, ENV425 reduced environmentally relevant microgram/liter concentrations of NDMA to