Carbon and Nitrogen Isotope Fractionations During Biotic and Abiotic Transformations of 2,4-Dinitroanisole (DNAN).

2,4-Dinitroanisole (DNAN) is a main constituent in various new insensitive munition formulations. Although DNAN is susceptible to biotic and abiotic transformations, in many environmental instances, transformation mechanisms are difficult to resolve, distinguish, or apportion on the basis solely of analysis of concentrations. We used compound-specific isotope analysis (CSIA) to investigate the characteristic isotope fractionations of the biotic (by three microbial consortia and three pure cultures) and abiotic (by 9,10-anthrahydroquinone-2-sulfonic acid [AHQS]) transformations of DNAN. The correlations of isotope enrichment factors (ΛN/C) for biotic transformations had a range of values from 4.93 ± 0.53 to 12.19 ± 1.23, which is entirely distinct from ΛN/C values reported previously for alkaline hydrolysis, enzymatic hydrolysis, reduction by Fe2+-bearing minerals and iron-oxide-bound Fe2+, and UV-driven phototransformations. The ΛN/C value associated with the abiotic reduction by AHQS was 38.76 ± 2.23, within the range of previously reported values for DNAN reduction by Fe2+-bearing minerals and iron-oxide-bound Fe2+, albeit the mean ΛN/C was lower. These results enhance the database of isotope effects accompanying DNAN transformations under environmentally relevant conditions, allowing better evaluation of the extents of biotic and abiotic transformations of DNAN that occur in soils, groundwaters, surface waters, and the marine environment.

Integrated Advanced Molecular Tools Predict In Situ cVOC Degradation Rates: Field Demonstration.

Chlorinated volatile organic compound (cVOC) degradation rate constants are crucial information for site management. Conventional approaches generate rate estimates from the monitoring and modeling of cVOC concentrations. This requires time series data collected along the flow path of the plume. The estimates of rate constants are often plagued by confounding issues, making predictions cumbersome and unreliable. Laboratory data suggest that targeted quantitative analysis of Dehalococcoides mccartyi (Dhc) biomarker genes (qPCR) and proteins (qProt) can be directly correlated with reductive dechlorination activity. To assess the potential of qPCR and qProt measurements to predict rates, we collected data from cVOC-contaminated aquifers. At the benchmark study site, the rate constant for degradation of cis-dichloroethene (cDCE) extracted from monitoring data was 11.0 ± 3.4 yr-1, and the rate constant predicted from the abundance of TceA peptides was 6.9 yr-1. The rate constant for degradation of vinyl chloride (VC) from monitoring data was 8.4 ± 5.7 yr-1, and the rate constant predicted from the abundance of TceA peptides was 5.2 yr-1. At the other study sites, the rate constants for cDCE degradation predicted from qPCR and qProt measurements agreed within a factor of 4. Under the right circumstances, qPCR and qProt measurements can be useful to rapidly predict rates of cDCE and VC biodegradation, providing a major advance in effective site management.

Metagenome-Guided Proteomic Quantification of Reductive Dehalogenases in the Dehalococcoides mccartyi-Containing Consortium SDC-9.

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.

Evaluation of methanotrophic bacterial communities capable of biodegrading trichloroethene (TCE) in acidic aquifers.

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 < 5.5). Acidophilic methanotrophs have been detected in several low pH environments, but their presence and potential role in TCE degradation in acidic aquifers is unknown. This study applied a stable isotope probing-based technique to identify active methanotrophs that are capable of degrading TCE in microcosms prepared from two low pH aquifers. A total of thirty-five clones of methanotrophs were derived from low pH microcosms in which methane and TCE degradation had been observed, with 29 clustered in γ-Proteobacteria and 6 clustered in α-Proteobacteria. None of the clones has a high similarity to known acidophilic methanotrophs from other environments. The presence and diversity of particulate MMO and soluble MMO were also investigated. The pmoA gene was detected predominantly at one site, and the presence of a specific form of mmoX in numerous samples suggested that Methylocella spp. may be common in acidic aquifers. Finally, a methane-grown culture at pH 4 was enriched from an acidic aquifer and its ability to biodegrade various chlorinated ethenes was tested. Interestingly, the mixed culture rapidly degraded TCE and vinyl chloride, but not cis-dichloroethene after growth on methane. The data suggest that aerobic biodegradation of TCE and other chlorinated solvents in low pH groundwater may be facilitated by methanotrophic bacteria, and that there are potentially a wide variety of different strains that inhabit acidic aquifers.

Carbon Isotope Fractionation of 1,2-Dibromoethane by Biological and Abiotic Processes.

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 (εbulk) associated with biological degradation covered a large range, with mixed anaerobic cultures fractionating more (εbulk from -8 to -20‰) than aerobic cultures (εbulk from -3 to -6‰). εbulk magnitudes associated with the abiotic processes (dihaloelimination by FeS/FeS2 and hydrolysis) were large but fairly well constrained (εbulk from -19 to -29‰). As expected, oxidative mechanisms fractionated EDB less than dihaloelimination and substitution mechanisms, and biological systems exhibited a larger range of fractionation, potentially due to isotope masking effects. In addition to quantifying and discussing εbulk values, which are highly relevant for quantifying in situ EDB degradation, an innovative approach for constraining the age of EDB in the aqueous phase, based on fractionation during hydrolysis, is described.

Abundance of Chlorinated Solvent and 1,4-Dioxane Degrading Microorganisms at Five Chlorinated Solvent Contaminated Sites Determined via Shotgun Sequencing.

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.

Stable isotope analyses of oxygen (18 O:17 O:16 O) and chlorine (37 Cl:35 Cl) in perchlorate: reference materials, calibrations, methods, and interferences.

RATIONALE: Perchlorate (ClO4- ) is a common trace constituent of water, soils, and plants; it has both natural and synthetic sources and is subject to biodegradation. The stable isotope ratios of Cl and O provide three independent quantities for ClO4- source attribution and natural attenuation studies: δ37 Cl, δ18 O, and δ17 O (or Δ17 O or 17 Δ) values. Documented reference materials, calibration schemes, methods, and interferences will improve the reliability of such studies. METHODS: Three large batches of KClO4 with contrasting isotopic compositions were synthesized and analyzed against VSMOW-SLAP, atmospheric O2 , and international nitrate and chloride reference materials. Three analytical methods were tested for O isotopes: conversion of ClO4- to CO for continuous-flow IRMS (CO-CFIRMS), decomposition to O2 for dual-inlet IRMS (O2-DIIRMS), and decomposition to O2 with molecular-sieve trap (O2-DIIRMS+T). For Cl isotopes, KCl produced by thermal decomposition of KClO4 was reprecipitated as AgCl and converted into CH3 Cl for DIIRMS. RESULTS: KClO4 isotopic reference materials (USGS37, USGS38, USGS39) represent a wide range of Cl and O isotopic compositions, including non-mass-dependent O isotopic variation. Isotopic fractionation and exchange can affect O isotope analyses of ClO4- depending on the decomposition method. Routine analyses can be adjusted for such effects by normalization, using reference materials prepared and analyzed as samples. Analytical errors caused by SO42- , NO3- , ReO42- , and C-bearing contaminants include isotope mixing and fractionation effects on CO and O2 , plus direct interference from CO2 in the mass spectrometer. The results highlight the importance of effective purification of ClO4- from environmental samples. CONCLUSIONS: KClO4 reference materials are available for testing methods and calibrating isotopic data for ClO4- and other substances with widely varying Cl or O isotopic compositions. Current ClO4- extraction, purification, and analysis techniques provide relative isotope-ratio measurements with uncertainties much smaller than the range of values in environmental ClO4- , permitting isotopic evaluation of environmental ClO4- sources and natural attenuation. Copyright © 2016 John Wiley & Sons, Ltd.

Development and application of a rapid, user-friendly, and inexpensive method to detect Dehalococcoides sp. reductive dehalogenase genes from groundwater.

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.

RDX degradation in bioaugmented model aquifer columns under aerobic and low oxygen conditions.

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 <2 mg/L, good RDX degradation was observed in the KTR9-bioaugmented column, and some degradation was also observed in the Ps I-C-bioaugmented column. After DO levels were kept below 1 mg/L for more than a month, columns bioaugmented with KTR9 became unresponsive to fructose addition, while RDX degradation was still observed in the Ps I-C-bioaugmented columns. These results indicate that bioaugmentation with the aerobic RDX degrader KTR9 could be effective at sites where site geology or geochemistry allow higher DO levels to be maintained. Further, inclusion of strains capable of anoxic RDX degradation such as Ps I-C may facilitate bimodal RDX removal when DO levels decrease.

Potential for cometabolic biodegradation of 1,4-dioxane in aquifers with methane or ethane as primary substrates.

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.

Relating Carbon and Nitrogen Isotope Effects to Reaction Mechanisms during Aerobic or Anaerobic Degradation of RDX (Hexahydro-1,3,5-Trinitro-1,3,5-Triazine) by Pure Bacterial Cultures.

UNLABELLED: 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 NO2 (-) 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 NO2 (-) production were much larger than, but systematically related to, the bulk RDX N isotope effects with different bacteria. Apparent intrinsic ε(15)N-NO2 (-) values were consistent with an initial denitration pathway in the aerobic experiments and more complex processes of NO2 (-) 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., NOx) 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.

Evaluation of Biostimulation and Bioaugmentation To Stimulate Hexahydro-1,3,5-trinitro-1,3,5,-triazine Degradation in an Aerobic Groundwater Aquifer.

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.

Evaluation of microbial transport during aerobic bioaugmentation of an RDX-contaminated aquifer.

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.

Laboratory evaluation of bioaugmentation for aerobic treatment of RDX in groundwater.

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.

Perchlorate in the Great Lakes: isotopic composition and origin.

Perchlorate is a persistent and mobile contaminant in the environment with both natural and anthropogenic sources. Stable isotope ratios of oxygen (δ(18)O, Δ(17)O) and chlorine (δ(37)Cl) along with the abundance of the radioactive isotope (36)Cl were used to trace perchlorate sources and behavior in the Laurentian Great Lakes. These lakes were selected for study as a likely repository of recent atmospheric perchlorate deposition. Perchlorate concentrations in the Great Lakes range from 0.05 to 0.13 µg per liter. δ(37)Cl values of perchlorate from the Great Lakes range from +3.0‰ (Lake Ontario) to +4.0‰ (Lake Superior), whereas δ(18)O values range from -4.1‰ (Lake Superior) to +4.0‰ (Lake Erie). Great Lakes perchlorate has mass-independent oxygen isotopic variations with positive Δ(17)O values (+1.6‰ to +2.7‰) divided into two distinct groups: Lake Superior (+2.7‰) and the other four lakes (∼+1.7‰). The stable isotopic results indicate that perchlorate in the Great Lakes is dominantly of natural origin, having isotopic composition resembling that measured for indigenous perchlorate from preindustrial groundwaters of the western USA. The (36)Cl/Cl ratio of perchlorate varies widely from 7.4 × 10(-12) (Lake Ontario) to 6.7 × 10(-11) (Lake Superior). These (36)ClO4(-) abundances are consistent with an atmospheric origin of perchlorate in the Great Lakes. The relatively high (36)ClO4(-) abundances in the larger lakes (Lakes Superior and Michigan) could be explained by the presence of (36)Cl-enriched perchlorate deposited during the period of elevated atmospheric (36)Cl activity following thermonuclear bomb tests in the Pacific Ocean.

Isolation and characterization of nitroguanidine-degrading microorganisms.

Nitroguanidine (NQ) is a component of newly developed insensitive munition (IM) formulations which are more resistant to impact, friction, heat, or sparks than conventional explosives. NQ is also used to synthesize various organic compounds and herbicides, and has both human and environmental health impacts. Despite the wide application and associated health concerns, limited information is known regarding NQ biodegradation, and only one NQ-degrading pure culture identified as Variovorax strain VC1 has been characterized. Here, we present results for three new NQ-degrading bacterial strains isolated from soil, sediment, and a lab-scale aerobic membrane bioreactor (MBR), respectively. Each of these strains -utilizes NQ as a nitrogen (N) source rather than as a source of carbon or energy. The MBR strain, identified as Pseudomonas extremaustralis strain NQ5, is capable of degrading NQ at a rate of approximately 150 µmole L-1 h-1 under aerobic conditions with glucose as a sole carbon source - and NQ as a sole N source. The addition of NH4+ to strain NQ5 during active growth with NQ as a sole N source slowed the growth rate for several hours, and the strain released NH4+, presumably from NQ. When NO3- was added as an alternate N source under similar conditions, the NO3- was not consumed, but NH4+ release into the culture medium was again observed. Strain NQ5 was also able to utilize guanylurea, guanidine, and ethyl allophanate as N sources, and - tolerate salt concentrations as high as 4 % (as NaCl). The other two stains, NQ4 and NQ7, both identified as Arthrobacter spp., grew significantly slower than strain NQ5 under similar culture conditions and tolerated only ∼1 % NaCl. In addition, neither strain NQ4 nor strain NQ7 was able to degrade guanlyurea or ethyl allophanate, but each degraded guanidine. These strains, particularly strain NQ5, may have practical applications for in-situ and ex-situ NQ bioremediation.

Formation of volatile chlorinated and brominated products during low temperature thermal decomposition of the representative PFAS perfluorohexane sulfonate (PFHxS) in the presence of NaCl and NaBr.

Per- and polyfluoroalkyl substances (PFAS) are synthetic organofluorine compounds known for their chemical and physical stability as well as their wide range of uses. Some PFAS are widely distributed in the environment, leading to concerns related to both environmental and human health. High temperature thermal treatment (i.e., incineration) has been utilized for PFAS treatment, but this requires significant infrastructure and energy, prompting interest in lower temperature approaches that may still lead to efficient destruction. Lower treatment temperatures, however, increase the potential for incomplete PFAS mineralization and formation of volatile organofluorine (VOF) products. Herein, we report the formation of novel VOF products that include chlorinated and brominated compounds during the thermal treatment of potassium perfluorohexane sulfonate (PFHxS), a representative perfluoroalkyl acid (PFAA). By comparing the gas chromatography-mass spectrometry (GC-MS) results of known VOF stocks to evolved VOF during thermal treatment of PFAS, the formation of perfluorohexyl chloride and perfluorohexyl bromide was observed when PFHxS was heated at temperatures between 275 and 475 °C in the presence of NaCl and NaBr, respectively. To our knowledge, this is the first report of chlorinated or brominated VOF products during thermal treatment of a PFAA. These findings suggest that a range of mixed halogenated VOF may form during thermal treatment of PFAS at relatively low temperature (e.g., 500 °C) and that these can be a function of salts present in the matrix.

Acidophilic methanotrophs: Occurrence, diversity, and possible bioremediation applications.

Methanotrophs have been identified and isolated from acidic environments such as wetlands, acidic soils, peat bogs, and groundwater aquifers. Due to their methane (CH4 ) utilization as a carbon and energy source, acidophilic methanotrophs are important in controlling the release of atmospheric CH4 , an important greenhouse gas, from acidic wetlands and other environments. Methanotrophs have also played an important role in the biodegradation and bioremediation of a variety of pollutants including chlorinated volatile organic compounds (CVOCs) using CH4 monooxygenases via a process known as cometabolism. Under neutral pH conditions, anaerobic bioremediation via carbon source addition is a commonly used and highly effective approach to treat CVOCs in groundwater. However, complete dechlorination of CVOCs is typically inhibited at low pH. Acidophilic methanotrophs have recently been observed to degrade a range of CVOCs at pH < 5.5, suggesting that cometabolic treatment may be an option for CVOCs and other contaminants in acidic aquifers. This paper provides an overview of the occurrence, diversity, and physiological activities of methanotrophs in acidic environments and highlights the potential application of these organisms for enhancing contaminant biodegradation and bioremediation.

Draft genomes of three nitroguanidine-degrading bacteria: Pseudomonas extremaustralis NQ5, Arthrobacter strain NQ4, and Arthrobacter strain NQ7.

We report the draft genome sequences of Pseudomonas extremaustralis NQ5, Arthrobacter strain NQ4, and Arthrobacter strain NQ7 isolated from a laboratory-scale membrane bioreactor, soils from San Antonio, TX, USA and sediments from Galveston Bay, TX, USA, respectively. These bacteria degrade the explosive compound nitroguanidine, which is present in some insensitive munitions.

Evaluation of a sequential anaerobic-aerobic membrane bioreactor system for treatment of traditional and insensitive munitions constituents.

New energetic formulations containing insensitive high explosives (IHE), such as 2,4-dinitroanisole (DNAN), 3-nitro-1,2,4-triazole-5-one (NTO), and nitroguanidine (NQ) are being developed to provide safer munitions. The addition of IHE to munitions formulations results in complex wastewaters from explosives manufacturing, load and pour operations and demilitarization activities. New technologies are required to treat those wastewaters. The core objective of this research effort was to develop and optimize a dual anaerobic-aerobic membrane bioreactor (MBR) system for treatment of wastewater containing variable mixtures of traditional energetics, IHE, and anions. The combined system proved highly effective for treatment of traditional explosives (TNT, RDX, HMX), IHE (DNAN, NTO, NQ) and anions commonly used as military oxidants (ClO4-, NO3-). The anaerobic MBR, which was operated for more than 500 d, was observed to completely degrade mg L-1 concentrations of TNT, DNAN, ClO4- and NO3- under all operational conditions, including at the lowest hydraulic residence time (HRT) tested (2.2 d). The combined system generally resulted in complete treatment of mg L-1 concentrations of RDX and HMX to <20 µg L-1, with most of the degradation occurring in the anaerobic MBR and polishing in the aerobic system. No common daughter products of DNAN, TNT, RDX, or HMX were detected in the effluent. NTO was completely transformed in the anaerobic MBR, but residual 3-amino-1,2,4-triazole-5-one (ATO) was detected in system effluent. The ATO rapidly decomposed when bleach solution was added to the final effluent. NQ was initially recalcitrant in the system, but microbial populations eventually developed that could degrade >90% of the ∼10 mg L-1 NQ entering the anaerobic MBR, with the remainder degraded to <50 µg L-1 in the aerobic system. The dual MBR system proved to be capable of complete degradation of a wide mixture of munitions constituents and was resilient to changing influent composition.