Field trial demonstrating phytoremediation of the military explosive RDX by XplA/XplB-expressing switchgrass.

The explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), a major component of munitions, is used extensively on military training ranges. As a result, widespread RDX pollution in groundwater and aquifers in the United States is now well documented. RDX is toxic, but its removal from training ranges is logistically challenging, lacking cost-effective and sustainable solutions. Previously, we have shown that thale cress (Arabidopsis thaliana) engineered to express two genes, xplA and xplB, encoding RDX-degrading enzymes from the soil bacterium Rhodococcus rhodochrous 11Y can break down this xenobiotic in laboratory studies. Here, we report the results of a 3-year field trial of XplA/XplB-expressing switchgrass (Panicum virgatum) conducted on three locations in a military site. Our data suggest that XplA/XplB switchgrass has in situ efficacy, with potential utility for detoxifying RDX on live-fire training ranges, munitions dumps and minefields.

Nitrous oxide emissions associated with ammonia-oxidizing bacteria abundance in fields of switchgrass with and without intercropped alfalfa.

The nitrogen (N) fertilizer required to supply a bioenergy industry with sufficient feedstocks is associated with adverse environmental impacts, including loss of oxidized reactive nitrogen through leaching and the production of the greenhouse gas nitrous oxide (N2 O). We examined effects on crop yield, N fate and the response of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) to conventional fertilizer application or intercropping with N-fixing alfalfa, for N delivery to switchgrass (Panicum virgatum), a potential bioenergy crop. Replicated field plots in Prosser, WA, were sampled over two seasons for reactive nitrogen, N2 O gas emissions, and bacterial and archaeal ammonia monooxygenase gene (amoA) counts. Intercropping with alfalfa (70:30, switchgrass:alfalfa) resulted in reduced dry matter yields compared to fertilized plots, but three times lower N2 O fluxes (≤ 4 g N2 O-N ha-1 d-1 ) than fertilized plots (12.5 g N2 O-N ha-1 d-1 ). In the fertilized switchgrass plots, AOA abundance was greater than AOB abundance, but only AOB abundance was positively correlated with N2 O emissions, implicating AOB as the major producer of N2 O emissions. A life cycle analysis of N2 O emissions suggested the greenhouse gas emissions from cellulosic ethanol produced from switchgrass intercropped with alfalfa cultivation would be 94% lower than emissions from equivalent gasoline usage.

Greatly Enhanced Removal of Volatile Organic Carcinogens by a Genetically Modified Houseplant, Pothos Ivy ( Epipremnum aureum) Expressing the Mammalian Cytochrome P450 2e1 Gene.

The indoor air in urban homes of developed countries is usually contaminated with significant levels of volatile organic carcinogens (VOCs), such as formaldehyde, benzene, and chloroform. There is a need for a practical, sustainable technology for the removal of VOCs in homes. Here we show that a detoxifying transgene, mammalian cytochrome P450 2e1 can be expressed in a houseplant, Epipremnum aureum, pothos ivy, and that the resulting genetically modified plant has sufficient detoxifying activity against benzene and chloroform to suggest that biofilters using transgenic plants could remove VOCs from home air at useful rates.

Genetic modification of western wheatgrass (Pascopyrum smithii) for the phytoremediation of RDX and TNT.

MAIN CONCLUSION: Transgenic western wheatgrass degrades the explosive RDX and detoxifies TNT. Contamination, from the explosives, hexahydro-1, 3, 5-trinitro-1, 3, 5-triazine (RDX), and 2, 4, 6-trinitrotoluene (TNT), especially on live-fire training ranges, threatens environmental and human health. Phytoremediation is an approach that could be used to clean-up explosive pollution, but it is hindered by inherently low in planta RDX degradation rates, and the high phytotoxicity of TNT. The bacterial genes, xplA and xplB, confer the ability to degrade RDX in plants, and a bacterial nitroreductase gene nfsI enhances the capacity of plants to withstand and detoxify TNT. While the previous studies have used model plant species to demonstrate the efficacy of this technology, trials using plant species able to thrive in the challenging environments found on military training ranges are now urgently needed. Perennial western wheatgrass (Pascopyrum smithii) is a United States native species that is broadly distributed across North America, well-suited for phytoremediation, and used by the US military to re-vegetate military ranges. Here, we present the first report of the genetic transformation of western wheatgrass. Plant lines transformed with xplA, xplB, and nfsI removed significantly more RDX from hydroponic solutions and retained much lower, or undetectable, levels of RDX in their leaf tissues when compared to wild-type plants. Furthermore, these plants were also more resistant to TNT toxicity, and detoxified more TNT than wild-type plants. This is the first study to engineer a field-applicable grass species capable of both RDX degradation and TNT detoxification. Together, these findings present a promising biotechnological approach to sustainably contain, remove RDX and TNT from training range soil and prevent groundwater contamination.

Ammonia-oxidizing bacteria are the primary N2 O producers in an ammonia-oxidizing archaea dominated alkaline agricultural soil.

Most agricultural N2 O emissions are a consequence of microbial transformations of nitrogen (N) fertilizer, and mitigating increases in N2 O emission will depend on identifying microbial sources and variables influencing their activities. Here, using controlled microcosm and field studies, we found that synthetic N addition in any tested amount stimulated the production of N2 O from ammonia-oxidizing bacteria (AOB), but not archaea (AOA), from a bioenergy crop soil. The activities of these two populations were differentiated by N treatments, with abundance and activity of AOB increasing as nitrate and N2 O production increased. Moreover, as N2 O production increased, the isotopic composition of N2 O was consistent with an AOB source. Relative N2 O contributions by both populations were quantified using selective inhibitors and varying N availability. Complementary field analyses confirmed a positive correlation between N2 O flux and AOB abundance with N application. Collectively, our data indicate that AOB are the major N2 O producers, even with low N addition, and that better-metered N application, complemented by selective inhibitors, could reduce projected N2 O emissions from agricultural soils.

Phytodetoxification of TNT by transplastomic tobacco (Nicotiana tabacum) expressing a bacterial nitroreductase.

KEY MESSAGE: Expression of the bacterial nitroreductase gene, nfsI, in tobacco plastids conferred the ability to detoxify TNT. The toxic pollutant 2,4,6-trinitrotoluene (TNT) is recalcitrant to degradation in the environment. Phytoremediation is a potentially low cost remediation technique that could be applied to soil contaminated with TNT; however, progress is hindered by the phytotoxicity of this compound. Previous studies have demonstrated that plants transformed with the bacterial nitroreductase gene, nfsI have increased ability to tolerate and detoxify TNT. It has been proposed that plants engineered to express nfsI could be used to remediate TNT on military ranges, but this could require steps to mitigate transgene flow to wild populations. To address this, we have developed nfsI transplastomic tobacco (Nicotiana tabacum L.) to reduce pollen-borne transgene flow. Here we have shown that when grown on solid or liquid media, the transplastomic tobacco expressing nfsI were significantly more tolerant to TNT, produced increased biomass and removed more TNT from the media than untransformed plants. Additionally, transplastomic plants expressing nfsI regenerated with high efficiency when grown on medium containing TNT, suggesting that nfsI and TNT could together be used to provide a selectable screen for plastid transformation.

A Field Trial of TCE Phytoremediation by Genetically Modified Poplars Expressing Cytochrome P450 2E1.

A controlled field study was performed to evaluate the effectiveness of transgenic poplars for phytoremediation. Three hydraulically contained test beds were planted with 12 transgenic poplars, 12 wild type (WT) poplars, or left unplanted, and dosed with equivalent concentrations of trichloroethylene (TCE). Removal of TCE was enhanced in the transgenic tree bed, but not to the extent of the enhanced removal observed in laboratory studies. Total chlorinated ethene removal was 87% in the CYP2E1 bed, 85% in the WT bed, and 34% in the unplanted bed in 2012. Evapotranspiration of TCE from transgenic leaves was reduced by 80% and diffusion of TCE from transgenic stems was reduced by 90% compared to WT. Cis-dichloroethene and vinyl chloride levels were reduced in the transgenic tree bed. Chloride ion accumulated in the planted beds corresponding to the TCE loss, suggesting that contaminant dehalogenation was the primary loss fate.

Expression in grasses of multiple transgenes for degradation of munitions compounds on live-fire training ranges.

The deposition of toxic munitions compounds, such as hexahydro-1, 3, 5-trinitro-1, 3, 5-triazine (RDX), on soils around targets in live-fire training ranges is an important source of groundwater contamination. Plants take up RDX but do not significantly degrade it. Reported here is the transformation of two perennial grass species, switchgrass (Panicum virgatum) and creeping bentgrass (Agrostis stolonifera), with the genes for degradation of RDX. These species possess a number of agronomic traits making them well equipped for the uptake and removal of RDX from root zone leachates. Transformation vectors were constructed with xplA and xplB, which confer the ability to degrade RDX, and nfsI, which encodes a nitroreductase for the detoxification of the co-contaminating explosive 2, 4, 6-trinitrotoluene (TNT). The vectors were transformed into the grass species using Agrobacterium tumefaciens infection. All transformed grass lines showing high transgene expression levels removed significantly more RDX from hydroponic solutions and retained significantly less RDX in their leaf tissues than wild-type plants. Soil columns planted with the best-performing switchgrass line were able to prevent leaching of RDX through a 0.5-m root zone. These plants represent a promising plant biotechnology to sustainably remove RDX from training range soil, thus preventing contamination of groundwater.

Agricultural land usage transforms nitrifier population ecology.

Application of nitrogen fertilizer has altered terrestrial ecosystems. Ammonia is nitrified by ammonia and nitrite-oxidizing microorganisms, converting ammonia to highly mobile nitrate, contributing to the loss of nitrogen, soil nutrients and production of detrimental nitrogen oxides. Mitigating these costs is of critical importance to a growing bioenergy industry. To resolve the impact of management on nitrifying populations, amplicon sequencing of markers associated with ammonia and nitrite-oxidizing taxa (ammonia monooxygenase-amoA, nitrite oxidoreductase-nxrB, respectively) was conducted from long-term managed and nearby native soils in Eastern Washington, USA. Native nitrifier population structure was altered profoundly by management. The native ammonia-oxidizing archaeal community (comprised primarily by Nitrososphaera sister subclusters 1.1 and 2) was displaced by populations of Nitrosopumilus, Nitrosotalea and different assemblages of Nitrososphaera (subcluster 1.1, and unassociated lineages of Nitrososphaera). A displacement of ammonia-oxidizing bacterial taxa was associated with management, with native groups of Nitrosospira (cluster 2 related, cluster 3A.2) displaced by Nitrosospira clusters 8B and 3A.1. A shift in nitrite-oxidizing bacteria (NOB) was correlated with management, but distribution patterns could not be linked exclusively to management. Dominant nxrB sequences displayed only distant relationships to other NOB isolates and environmental clones.

Evaluation of revised polymerase chain reaction primers for more inclusive quantification of ammonia-oxidizing archaea and bacteria.

Ammonia-oxidizing archaea (AOA) and bacteria (AOB) fill key roles in the nitrogen cycle. Thus, well-vetted methods for characterizing their distribution are essential for framing studies of their significance in natural and managed systems. Quantification of the gene coding for one subunit of the ammonia monooxygenase (amoA) by polymerase chain reaction is frequently employed to enumerate the two groups. However, variable amplification of sequence variants comprising this conserved genetic marker for ammonia oxidizers potentially compromises within- and between-system comparisons. We compared the performance of newly designed non-degenerate quantitative polymerase chain reaction primer sets to existing primer sets commonly used to quantify the amoA of AOA and AOB using a collection of plasmids and soil DNA samples. The new AOA primer set provided improved quantification of model mixtures of different amoA sequence variants and increased detection of amoA in DNA recovered from soils. Although both primer sets for the AOB provided similar results for many comparisons, the new primers demonstrated increased detection in environmental application. Thus, the new primer sets should provide a useful complement to primers now commonly used to characterize the environmental distribution of AOA and AOB.

Influence of edaphic and management factors on the diversity and abundance of ammonia-oxidizing thaumarchaeota and bacteria in soils of bioenergy crop cultivars.

Ammonia-oxidizing thaumarcheota (AOA) and ammonia-oxidizing bacteria (AOB) differentially influence soil and atmospheric chemistry, but soil properties that control their distributions are poorly understood. In this study, the ammonia monooxygenase gene (amoA) was used to identify and quantify presumptive AOA and AOB and relate their distributions to soil properties in two experimental fields planted with different varieties of switchgrass (Panicum virgatum), a potential bioenergy feedstock. Differences in ammonia oxidizer diversity were associated primarily with soil properties of the two field sites, with pH displaying significant correlations with both AOA and AOB population structure. Percent nitrogen (%N), carbon to nitrogen ratios (C : N), and pH were also correlated with shifts nitrifier population structure. Nitrosotalea-like and Nitrosospira cluster II populations were more highly represented in acidic soils, whereas populations affiliated with Nitrososphaera and Nitrosospira cluster 3A.1 were relatively more abundant in alkaline soils. AOA were the dominant functional group in all plots based on quantitative polymerase chain reaction and high-throughput sequencing analyses. These data suggest that AOA contribute significantly to nitrification rates in carbon and nitrogen rich soils influenced by perennial grasses.

Analysis of the xplAB-containing gene cluster involved in the bacterial degradation of the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine.

Repeated use of the explosive compound hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) on military land has resulted in significant soil and groundwater pollution. Rates of degradation of RDX in the environment are low, and accumulated RDX, which the U.S. Environmental Protection Agency has determined is a possible human carcinogen, is now threatening drinking water supplies. RDX-degrading microorganisms have been isolated from RDX-contaminated land; however, despite the presence of these species in contaminated soils, RDX pollution persists. To further understand this problem, we studied RDX-degrading species belonging to four different genera (Rhodococcus, Microbacterium, Gordonia, and Williamsia) isolated from geographically distinct locations and established that the xplA and xplB (xplAB) genes, which encode a cytochrome P450 and a flavodoxin redox partner, respectively, are nearly identical in all these species. Together, the xplAB system catalyzes the reductive denitration of RDX and subsequent ring cleavage under aerobic and anaerobic conditions. In addition to xplAB, the Rhodococcus species studied here share a 14-kb region flanking xplAB; thus, it appears likely that the RDX-metabolizing ability was transferred as a genomic island within a transposable element. The conservation and transfer of xplAB-flanking genes suggest a role in RDX metabolism. We therefore independently knocked out genes within this cluster in the RDX-degrading species Rhodococcus rhodochrous 11Y. Analysis of the resulting mutants revealed that XplA is essential for RDX degradation and that XplB is not the sole contributor of reducing equivalents to XplA. While XplA expression is induced under nitrogen-limiting conditions and further enhanced by the presence of RDX, MarR is not regulated by RDX.

Influence of bioselector processes on 17α-ethinylestradiol biodegradation in activated sludge wastewater treatment systems.

The removal of the potent endocrine-disrupting estrogen hormone, 17α-ethinylestradiol (EE2), in municipal wastewater treatment plant (WWTP) activated sludge (AS) processes can occur through biodegradation by heterotrophic bacteria growing on other organic wastewater substrates. Different kinetic and metabolic substrate utilization conditions created with AS bioselector processes can affect the heterotrophic population composition in AS. The primary goal of this research was to determine if these changes also affect specific EE2 biodegradation kinetics. A series of experiments were conducted with parallel bench-scale AS reactors treating municipal wastewater with estrogens at 100-300 ng/L concentrations to evaluate the effect of bioselector designs on pseudo first-order EE2 biodegradation kinetics normalized to mixed liquor volatile suspended solids (VSS). Kinetic rate coefficient (kb) values for EE2 biodegradation ranged from 5.0 to 18.9 L/g VSS/d at temperatures of 18 °C to 24 °C. EE2 kb values for aerobic biomass growth at low initial food to mass ratio feeding conditions (F/Mf) were 1.4 to 2.2 times greater than that from growth at high initial F/Mf. Anoxic/aerobic and anaerobic/aerobic metabolic bioselector reactors achieving biological nutrient removal had similar EE2 kb values, which were lower than that in aerobic AS reactors with biomass growth at low initial F/Mf. These results provide evidence that population selection with growth at low organic substrate concentrations can lead to improved EE2 biodegradation kinetics in AS treatment.

Identification of microbial populations assimilating nitrogen from RDX in munitions contaminated military training range soils by high sensitivity stable isotope probing.

The leaching of RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) from particulates deposited in live-fire military training range soils contributes to significant pollution of groundwater. In situ microbial degradation has been proposed as a viable method for onsite containment of RDX. However, there is only a single report of RDX degradation in training range soils and the soil microbial communities involved in RDX degradation were not identified. Here we demonstrate aerobic RDX degradation in soils taken from a target area of an Eglin Air Force Base bombing range, C52N Cat's Eye, (Eglin, Florida U.S.A.). RDX-degradation activity was spatially heterogeneous (found in less than 30% of initial target area field samples) and dependent upon the addition of exogenous carbon sources to the soils. Therefore, biostimulation (with exogenous carbon sources) and bioaugmentation may be necessary to sustain timely and effective in situ microbial biodegradation of RDX. High sensitivity stable isotope probing analysis of extracted soils incubated with fully labeled (15)N-RDX revealed several organisms with (15)N-labeled DNA during RDX-degradation, including xplA-bearing organisms. Rhodococcus was the most prominent genus in the RDX-degrading soil slurries and was completely labeled with (15)N-nitrogen from the RDX. Rhodococcus and Williamsia species isolated from these soils were capable of using RDX as a sole nitrogen source and possessed the genes xplB and xplA associated with RDX-degradation, indicating these genes may be suitable genetic biomarkers for assessing RDX degradation potential in soils. Other highly labeled species were primarily Proteobacteria, including: Mesorhizobium sp., Variovorax sp., and Rhizobium sp.

Towards engineering degradation of the explosive pollutant hexahydro-1,3,5-trinitro-1,3,5-triazine in the rhizosphere.

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a serious environmental pollutant on military land. This compound is the most widely used explosive and pollution has arisen primarily as the result of military training, along with munition manufacturing and disassembly processes. This toxic explosive is recalcitrant to degradation in the environment and leaches rapidly into groundwater, where accumulation in aquifers is threatening drinking water supplies (Clausen, et al., 2004). While plants have only limited degradative activity towards RDX, microorganisms, including Rhodococcus rhodochrous 11Y, have been isolated from contaminated land. Despite the presence of microbial RDX-metabolising activity in contaminated soils, the persistence of RDX in leachate from contaminated soil indicates that this activity or biomass is insufficient, limiting its use to remediate polluted soils. Bacterial activity in the rhizosphere is of magnitudes greater than in the surrounding soil, and the roots of grass species on training ranges in the United States are known to penetrate deeply into the soil, producing a compact root system and providing an ideal environment to support the capture of RDX by microorganisms in the rhizosphere. Here, we have investigated the ability of the root-colonising bacterium Pseudomonas fluorescens, engineered to express XplA, to degrade RDX in the rhizosphere.

Phytoremediation of chlorpyrifos by Populus and Salix.

Chlorpyrifos is one of the commonly used organophosphorus insecticides that are implicated in serious environmental and human health problems. To evaluate plant potential for uptake of chlorpyrifos, several plant species of poplar (Populus sp.) and willow (Salix sp.) were investigated. Chlorpyrifos was taken up from nutrient solution by all seven plant species. Significant amounts of chlorpyrifos accumulated in plant tissues, and roots accumulated higher concentrations of chlorpyrifos than did shoots. Chlorpyrifos did not persist in the plant tissues, suggesting further metabolism of chlorpyrifos in plant tissue. To our knowledge, this work represents the first report for phytoremediation of chlorpyrifos using poplar and willow plants.

High-sensitivity stable-isotope probing by a quantitative terminal restriction fragment length polymorphism protocol.

Stable-isotope probing (SIP) has proved a valuable cultivation-independent tool for linking specific microbial populations to selected functions in various natural and engineered systems. However, application of SIP to microbial populations with relatively minor buoyant density increases, such as populations that utilize compounds as a nitrogen source, results in reduced resolution of labeled populations. We therefore developed a tandem quantitative PCR (qPCR)-TRFLP (terminal restriction fragment length polymorphism) protocol that improves resolution of detection by quantifying specific taxonomic groups in gradient fractions. This method combines well-controlled amplification with TRFLP analysis to quantify relative taxon abundance in amplicon pools of FAM-labeled PCR products, using the intercalating dye EvaGreen to monitor amplification. Method accuracy was evaluated using mixtures of cloned 16S rRNA genes, DNA extracted from low- and high-G+C bacterial isolates (Escherichia coli, Rhodococcus, Variovorax, and Microbacterium), and DNA from soil microcosms amended with known amounts of genomic DNA from bacterial isolates. Improved resolution of minor shifts in buoyant density relative to TRFLP analysis alone was confirmed using well-controlled SIP analyses.

Engineering plants for the phytoremediation of RDX in the presence of the co-contaminating explosive TNT.

The explosive compounds hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and 2,4,6-trinitrotoluene (TNT) are widespread environmental contaminants commonly found as co-pollutants on military training ranges. TNT is a toxic carcinogen which remains tightly bound to the soil, whereas RDX is highly mobile leaching into groundwater and threatening drinking water supplies. We have engineered Arabidopsis plants that are able to degrade RDX, whilst withstanding the phytotoxicity of TNT. Arabidopsis thaliana (Arabidopsis) was transformed with the bacterial RDX-degrading xplA, and associated reductase xplB, from Rhodococcus rhodochrous strain 11Y, in combination with the TNT-detoxifying nitroreductase (NR), nfsI, from Enterobacter cloacae. Plants expressing XplA, XplB and NR remove RDX from soil leachate and grow on soil contaminated with RDX and TNT at concentrations inhibitory to XplA-only expressing plants. This is the first study to demonstrate the use of transgenic plants to tackle two chemically diverse organic compounds at levels comparable with those found on contaminated training ranges, indicating that this technology is capable of remediating concentrations of RDX found in situ. In addition, plants expressing XplA and XplB have substantially less RDX available in aerial tissues for herbivory and potential bioaccumulation.

The explosive-degrading cytochrome P450 XplA: biochemistry, structural features and prospects for bioremediation.

XplA is a cytochrome P450 that mediates the microbial metabolism of the military explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). It has an unusual structural organisation comprising a heme domain that is fused to its flavodoxin redox partner. XplA along with its partnering reductase XplB are plasmid encoded and the gene xplA has now been found in divergent genera across the globe with near sequence identity. Importantly, it has only been detected at explosives contaminated sites suggesting rapid dissemination of this novel catabolic activity, possibly within the 50-year period since the introduction of RDX into the environment. The X-ray structure of XplA-heme has been solved, providing fundamental information on the heme binding site. Interestingly, oxygen is not required for the degradation of RDX, but its presence determines the final degradation products, demonstrating that the degradation chemistry is flexible with both anaerobic and aerobic pathways resulting in the release of nitrite from the substrate. Transgenic plants expressing xplA are able to remove saturating levels of RDX from soil leachate and may provide a low cost sustainable remediation strategy for contaminated military sites.

Biodegradation of RDX and MNX with Rhodococcus sp. strain DN22: new insights into the degradation pathway.

Previously we demonstrated that Rhodococcus sp. strain DN22 can degrade RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) aerobically via initial denitration. The present study describes the role of oxygen and water in the key denitration step leading to RDX decomposition using (18)O(2) and H(2)(18)O labeling experiments. We also investigated degradation of MNX (hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine) with DN22 under similar conditions. DN22 degraded RDX and MNX giving NO(2)(-), NO(3)(-), NDAB (4-nitro-diazabutanal), NH(3), N(2)O, and HCHO with NO(2)(-)/NO(3)(-) molar ratio reaching 17 and ca. 2, respectively. In the presence of (18)O(2), DN22 degraded RDX and produced NO(2)(-) with m/z at 46 Da that subsequently oxidized to NO(3)(-) containing one (18)O atom, but in the presence of H(2)(18)O we detected NO(3)(-) without (18)O. A control containing NO(2)(-), DN22, and (18)O(2) gave NO(3)(-) with one (18)O, confirming biotic oxidation of NO(2)(-) to NO(3)(-). Treatment of MNX with DN22 and (18)O(2) produced NO(3)(-) with two mass ions, one (66 Da) incorporating two (18)O atoms and another (64 Da) incorporating only one (18)O atom and we attributed their formation to bio-oxidation of the initially formed NO and NO(2)(-), respectively. In the presence of H(2)(18)O we detected NO(2)(-) with two different masses, one representing NO(2)(-) (46 Da) and another representing NO(2)(-) (48 Da) with the inclusion of one (18)O atom suggesting auto-oxidation of NO to NO(2)(-). Results indicated that denitration of either RDX or MNX and denitrosation of MNX by DN22 did not involve direct participation of either oxygen or water, but both played major roles in subsequent secondary chemical and biochemical reactions of NO and NO(2)(-).