Biodegradation Of RDX Research Articles

A sketch of microbiological remediation of explosives-contaminated soil focused on state of art and the impact of technological advancement on hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) degradation

When high-energy explosives such as hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), 2,4,6-trinitrotoluene (TNT) are discharged into the surrounding soil and water during production, testing, open dumping, military, or civil activities, they leave a toxic footprint. The US Environmental Protection Agency has labeled RDX as a potential human carcinogen that must be degraded from contaminated sites quickly. Bioremediation of RDX is an exciting prospect that has received much attention in recent years. However, a lack of understanding of RDX biodegradation and the limitations of current approaches have hampered the widespread use of biodegradation-based strategies for RDX remediation at contamination sites. Consequently, new bioremediation technologies are required to enhance performance. In this review, we explore the requirements for in-silico analysis for producing biological models of microbial remediation of RDX in soil. On the other hand, potential gene editing methods for getting the host with target gene sequences responsible for the breakdown of RDX are also reported. Microbial formulations and biosensors for detection and bioremediation are also briefly described. The biodegradation of RDX offers an alternative remediation method that is both cost-effective and ecologically acceptable. It has the potential to be used in conjunction with other cutting-edge technologies to further increase the efficiency of RDX degradation.

Preliminary investigation on ultrasound frequency determination in TNT, RDX and HMX degradation

The wastewater of the munitions industry varies in terms of the recalcitrant compounds depending on the products and the manufacturing process. The most commonly encountered energetic nitro-aromatic compounds in ammunition wastewater are trinitrotoluene (TNT), nitro-heterocyclic hexahydro-trinitrotriazine (RDX), and cyclotetramethylene-tetranitramine (HMX). These compounds are known to be very toxic and resistant to biodegradation. The aim of this study was to determine the appropriate ultrasound frequency to assist the anaerobic biodegradation of TNT, RDX, and HMX Therefore, the optimum ultrasound frequencies were investigated to degrade TNT, RDX, and HMX without destroying anaerobic microorganisms. The results showed that ultrasound irradiation was effective for the degradation of all the explosive compounds used. However, the maximum removal efficiencies and minimum microorganism lysis were achieved by the frequencies of 20 and 800 kHz.

Enhancing the Potential for in situ Bioremediation of RDX Contaminated Soil from a Former Military Demolition Range

ABSTRACTHexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a toxic, mobile groundwater contaminant common to military sites. Biodegradation of RDX is an alternative, cost effective and environmentally friendly remediation approach. The effects of carbon amendments (waste glycerol and cheese whey) used alone or with a potential electron shuttle (ammonium lignosulfonate) on RDX biodegradation were assessed. These substrates are readily available waste materials that can be used as nutrients to promote oxygen consumption, creating a more reducing environment. Nutrient amended batch assays were conducted using RDX spiked contaminated demolition range soil under anaerobic conditions. The amendments that improved RDX mineralization the most were subsequently tested in a scaled up repacked soil column study to verify if this strategy could be effectively implemented on-site. Microcosm results indicated that RDX mineralization by indigenous anaerobic microorganisms was enhanced the most by the low carbon amendment concentration. The use of ammonium lignosulfonate was not effective, exhibiting an inhibitory effect on RDX biodegradation that was stronger at higher concentrations. The soil column study showed that the low concentration of waste was the most promising treatment scenario. These results offer good prospects for the use of waste glycerol for in situ treatment of soils contaminated with energetic-materials, such as RDX.

Impact of glycerin and lignosulfonate on biodegradation of high explosives in soil

Soil microcosms were constructed and monitored to evaluate the impact of substrate addition and transient aerobic and anaerobic conditions on TNT, RDX and HMX biodegradation in grenade range soils. While TNT was rapidly biodegraded under both aerobic and anaerobic conditions with and without organic substrate, substantial biodegradation of RDX, HMX, and RDX daughter products was not observed under aerobic conditions. However, RDX and HMX were significantly biodegraded under anaerobic conditions, without accumulation of TNT or RDX daughter products (2-ADNT, 4-ADNT, MNX, DNX, and TNX). In separate microcosms containing grenade range soil, glycerin and lignosulfonate addition enhanced oxygen consumption, increasing the consumption rate >200% compared to untreated soils. Mathematical model simulations indicate that oxygen consumption rates of 5 to 20g/m3/d can be achieved with reasonable amendment loading rates. These results indicate that glycerin and lignosulfonate can be potentially used to stimulate RDX and HMX biodegradation by increasing oxygen consumption rates in soil.

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.

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.

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. 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.

Anaerobic biodegradation of RDX and HMX with different co-substrates

Simulated wastewater of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) was treated under anaerobic conditions with co-substrates such as ammonium chloride, dextrose, sodium acetic, sodium nitrate and sulfate. The results showed that with nitrogen compounds such as ammonium chloride added as co-substrate, no significant change was observed, indicating that the molar ratio of N/C for RDX and HMX is sufficient for biodegradation. With the addition of dextrose and acetate to the system, biodegradation efficiency was enhanced greatly. For example, with dextrose as the co-substrate, degradation efficiency of 99.1% and 98.5% was achieved for RDX and HMX, respectively, after treatment for 7days. When sodium acetic was used as the co-substrate, the enhancement of degradation percentage was similar, but was not as high as that with dextrose, indicating the selectivity of RDX and HMX to co-substrate during anaerobic degradation. With sodium nitrate as the co-substrate, the degradation efficiency of RDX or HMX decreased with the increase of salt concentration. Sodium sulfate has no significant effect on the biodegradation of RDX and HMX. A well-selected co-substrate should be employed in applications for degradation of RDX and HMX wastewaters.

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.

Biodegradation of explosives mixture in soil under different water-content conditions

Soil redox potential plays a key role in the rates and pathways of explosives degradation, and is highly influenced by water content and microbial activity. Soil redox potential can vary significantly both temporally and spatially in micro-sites. In this study, when soil water content increased, the redox potential decreased, and there was significant enhancement in the biodegradation of a mixture of three explosives. Whereas TNT degradation occurred under both aerobic and anaerobic conditions, RDX and HMX degradation occurred only when water content conditions resulted in a prolonged period of negative redox potential. Moreover, under unsaturated conditions, which are more representative of real environmental conditions, the low redox potential, even when measured for temporary periods, was sufficient to facilitate anaerobic degradation. Our results clearly indicate a negative influence of TNT on the biodegradation of RDX and HMX, but this effect was less pronounced than that found in previous slurry batch experiments: this can be explained by a masking effect of the soil in the canisters. Fully or partially saturated soils can promote the existence of micro-niches that differ considerably in their explosives concentration, microbial community and redox conditions.

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)(-).

Quantifying RDX biodegradation in groundwater using δ15N isotope analysis

Isotope analysis was used to examine the extent of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) biodegradation in groundwater along a ca. 1.35-km contamination plume. Biodegradation was proposed as a natural attenuating remediation method for the contaminated aquifer. By isotope analysis of RDX, the extent of biodegradation was found to reach up to 99.5% of the initial mass at a distance of 1.15–1.35 km down gradient from the contamination sources. A range of first-order biodegradation rates was calculated based on the degradation extents, with average half-life values ranging between 4.4 and 12.8 years for RDX biodegradation in the upper 15 m of the aquifer, assuming purely aerobic biodegradation, and between 10.9 and 31.2 years, assuming purely anaerobic biodegradation. Based on the geochemical data, an aerobic biodegradation pathway was suggested as the dominant attenuation process at the site. The calculated biodegradation rate was correlated with depth, showing decreasing degradation rates in deeper groundwater layers. Exceptionally low first-order kinetic constants were found in a borehole penetrating the bottom of the aquifer, with half life ranging between 85.0 to 161.5 years, assuming purely aerobic biodegradation, and between 207.5 and 394.3 years, assuming purely anaerobic biodegradation. The study showed that stable isotope fractionation analysis is a suitable tool to detect biodegradation of RDX in the environment. Our findings clearly indicated that RDX is naturally biodegraded in the contaminated aquifer. To the best of our knowledge, this is the first reported use of RDX isotope analysis to quantify its biodegradation in contaminated aquifers.

Sequential biodegradation of TNT, RDX and HMX in a mixture

We describe TNT's inhibition of RDX and HMX anaerobic degradation in contaminated soil containing indigenous microbial populations. Biodegradation of RDX or HMX alone was markedly faster than their degradation in a mixture with TNT, implying biodegradation inhibition by the latter. The delay caused by the presence of TNT continued even after its disappearance and was linked to the presence of its intermediate, tetranitroazoxytoluene. PCR–DGGE analysis of cultures derived from the soil indicated a clear reduction in microbial biomass and diversity with increasing TNT concentration. At high-TNT concentrations (30 and 90 mg/L), only a single band, related to Clostridium nitrophenolicum, was observed after 3 days of incubation. We propose that the mechanism of TNT inhibition involves a cytotoxic effect on the RDX- and HMX-degrading microbial population. TNT inhibition in the top active soil can therefore initiate rapid transport of RDX and HMX to the less active subsurface and groundwater.

Uptake, bioaccumulation, and biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and its reduced metabolites (MNX and TNX) by the earthworm ( Eisenia fetida)

Uptake and accumulation kinetics of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and its two major N-nitroso metabolites, hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX), in earthworms was investigated. Results indicated that RDX and its N-nitroso metabolites were rapidly absorbed into earthworms ( Eisenia fetida), reaching the highest concentrations within a few days. Accumulation of RDX was greater than its N-nitroso metabolites, as evidenced by a higher bioconcentration factor (BCF); BCFs were 1.86, 0.39, and 0.05 for RDX, MNX, and TNX, respectively. RDX and its N-nitroso metabolites were also rapidly eliminated from the earthworm and/or transformed to other metabolites, as evidenced by the rapid decrease of test compounds in earthworms after reaching their highest concentrations. The uptake of MNX and TNX increased as exposure concentration increased. Although these earthworms might (anaerobically) degrade RDX to MNX and MNX to TNX, it is hypothesized that this process would be slow. Other biotransformation pathways may be involved in biodegradation of RDX and its N-nitroso metabolites due to the fact that concentrations of tested compounds decreased in both soil and earthworms. It is hoped that these data can be used to refine environmental management strategies for RDX and for performing specific risk assessments of RDX and its N-nitroso metabolites.

Anaerobic biotransformation of explosives in aquifer slurries amended with ethanol and propylene glycol

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), and 2,4,6-trinitrotoluene (TNT) are explosives that are frequently found as environmental contaminants on military installations. Hydrogen has been shown to support the anaerobic transformation of these explosives. We investigated ethanol and propylene glycol as electron donors for providing syntrophically produced H 2 for stimulating the anaerobic biodegradation of explosives in contaminated soil. The study was conducted using anoxic microcosms constructed with slurries of the contaminated soil and groundwater. The addition of 5 mM ethanol and propylene glycol enhanced the biodegradation of RDX and HMX relative to the control bottles. Ethanol was depleted within about 20 days, resulting in the transient formation of hydrogen, acetate, and methane. The hydrogen headspace concentration increased from 8 ppm to 1838 ppm before decreasing to background concentrations. Propylene glycol was completely degraded after 15 days, forming hydrogen, propionate, and acetate as end-products. The hydrogen headspace concentrations increased from 56 ppm to 628 ppm before decreasing to background concentrations. No methane formation was observed during the incubation period of 48 days. Our findings indicate the addition of ethanol and propylene to the aquifer slurries increased the hydrogen concentrations and enhanced the biotransformation of RDX and HMX in the explosive-contaminated soil.

Anaerobic Biodegradation of RDX and TCE: Single- and Dual-Contaminant Batch Tests

Several sites in the United States have groundwater contaminated with mixtures of high explosives and chlorinated solvents. This research examined the ability of two microbial cultures (anaerobic sludge and a facultative enrichment culture) to biodegrade single- and dual-contaminant mixtures of trichloroethene (TCE) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) under anaerobic conditions. In single component batch tests, both cultures degraded 0.6–1mgRDX∕L and its nitroso metabolites to below detection limits in <7days. During initial 9-day TCE biodegradation tests, the anaerobic sludge did not transform TCE, whereas the facultative culture transformed approximately 10% of the initial 1.4mgTCE∕L. Prior to dual-contaminant batch tests, both cultures were grown in the presence of TCE. Subsequently, both acclimated cultures rapidly biodegraded mixtures of RDX and TCE. Both cultures degraded RDX and RDX-nitroso compounds to below detection limits in <4days. In the same tests, TCE-acclimated anaerobic sludge converted TCE primarily to cis-dichloroethene (cis-DCE), while the acclimated facultative culture produced cis-DCE and other chlorinated metabolites. These preliminary results demonstrate that anaerobic bioremediation may be part of a feasible groundwater remediation alternative for mixtures of TCE and RDX.

Effect of iron(III), humic acids and anthraquinone-2,6-disulfonate on biodegradation of cyclic nitramines by Clostridium sp. EDB2

To determine the biodegradation of cyclic nitramines by an anaerobic marine bacterium, Clostridium sp. EDB2, in the presence of Fe(III), humic acids (HA) and anthraquinone-2,6-disulfonate (AQDS). An obligate anaerobic bacterium, Clostridium sp. EDB2, degraded RDX and HMX, and produced similar product distribution including nitrite, methylenedinitramine, nitrous oxide, ammonium, formaldehyde, formic acid and carbon dioxide. Carbon (C) and nitrogen (N) mass balance for RDX products were 87% and 82%, respectively, and for HMX were 88% and 74%, respectively. Bacterial growth and biodegradation of RDX and HMX were stimulated in the presence of Fe(III), HA and AQDS suggesting that strain EDB2 utilized Fe(III), HA and AQDS as redox mediators to transfer electrons to cyclic nitramines. Strain EDB2 demonstrated a multidimensional approach to degrade RDX and HMX: first, direct degradation of the chemicals; second, indirect degradation by reducing Fe(III) to produce reactive-Fe(II); third, indirect degradation by reducing HA and AQDS which act as electron shuttles to transfer electrons to the cyclic nitramines. The present study could be helpful in determining the fate of cyclic nitramine energetic chemicals in the environments rich in Fe(III) and HA.

Biodegradation of RDX and HMX Mixtures: Batch Screening Experiments and Sequencing Batch Reactors

Biodegradation is a potential treatment method for the high explosives RDX and HMX. In batch biodegradation tests, a facultative microbial consortium consumed the dissolved oxygen (DO) or reduced the oxidation-reduction potential (ORP) of the culture medium and biodegraded mixtures of RDX and HMX when supplied with various carbon and nitrogen sources. In batch tests with a mixture of carbon sources (glucose, glycerol, and succinic acid), RDX was reduced from 1.1 mg/L to below detectable levels within 4 days in the presence of 0.6 mg HMX/L. With ethanol as the carbon source, the culture degraded 90% of the initial 2.5 mg RDX/L over 8 days in the presence of 0.5 mg HMX/L. In batch mineralization tests, the culture converted 8 and 30% of the initial 14C-RDX to 14CO2 when supplied with ethanol and the mixed carbon sources, respectively. In both cases, a significant fraction of the initial 14C-RDX was converted to water-soluble 14C compounds, indicating triazine ring cleavage. However, mineralization was limit...

Biodegradation of RDX within soil-water slurries using a combination of differing redox incubation conditions

Biodegradation of 14C-tagged hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) was studied in aerobic, anaerobic, and anaerobic/aerobic slurries to identify the conditions maximizing RDX-mineralization in Cornhusker Army Ammunition Plant (CAAP, NE) groundwater. Supplementation with phosphate and adequate quantities of acetate caused 25% mineralization of RDX in 3 weeks by microorganisms native to CAAP. Under anaerobic conditions, the same supplementation resulted in 20% mineralization in 3 weeks and 30% mineralization in 6 weeks. The highest degree of mineralization (50%) was obtained under aerobic conditions when the contaminated groundwater was augmented with a consortium of three microbes isolated from another RDX contaminated soil (Hastings, NE) in addition to supplemented with phosphate and acetic acid. Use of complex organic sources (potato or corn starch) slowed down the rates of mineralization under anaerobic conditions, but rapid mineralization ensued as soon as the aerobic conditions were created. Final RDX concentrations in aqueous phase were below detection limit under most conditions. Assimilation of RDX by the cells was negligible.

Determination of key metabolites during biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine with Rhodococcus sp. strain DN22.

Rhodococcus sp. strain DN22 can convert hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) to nitrite, but information on degradation products or the fate of carbon is not known. The present study describes aerobic biodegradation of RDX (175 microM) when used as an N source for strain DN22. RDX was converted to nitrite (NO(2)(-)) (30%), nitrous oxide (N(2)O) (3.2%), ammonia (10%), and formaldehyde (HCHO) (27%), which later converted to carbon dioxide. In experiments with ring-labeled [(15)N]-RDX, gas chromatographic/mass spectrophotometric (GC/MS) analysis revealed N(2)O with two molecular mass ions: one at 44 Da, corresponding to (14)N(14)NO, and the second at 45 Da, corresponding to (15)N(14)NO. The nonlabeled N(2)O could be formed only from -NO(2), whereas the (15)N-labeled one was presumed to originate from a nitramine group ((15)N-(14)NO(2)) in RDX. Liquid chromatographic (LC)-MS electrospray analyses indicated the formation of a dead end product with a deprotonated molecular mass ion [M-H] at 118 Da. High-resolution MS indicated a molecular formula of C(2)H(5)N(3)O(3). When the experiment was repeated with ring-labeled [(15)N]-RDX, the [M-H] appeared at 120 Da, indicating that two of the three N atoms in the metabolite originated from the ring in RDX. When [U-(14)C]-RDX was used in the experiment, 64% of the original radioactivity in RDX incorporated into the metabolite with a molecular weight (MW) of 119 (high-pressure LC/radioactivity) and 30% in (14)CO(2) (mineralization) after 4 days of incubation, suggesting that one of the carbon atoms in RDX was converted to CO(2) and the other two were incorporated in the ring cleavage product with an MW of 119. Based on the above stoichiometry, we propose a degradation pathway for RDX based on initial denitration followed by ring cleavage to formaldehyde and the dead end product with an MW of 119.

Identification of hydroxylamino‐dinitroso‐1,3,5‐triazine as a transient intermediate formed during the anaerobic biodegradation of hexahydro‐1,3,5‐trinitro‐1,3,5‐triazine

The metabolic fate of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in a mixed culture incubated under methanogenic conditions was studied. Analysis by high-performance liquid chromatography (HPLC) confirmed the loss of RDX and the formation of mono-, di-, and trinitroso-RDX as transient biodegradation intermediates. An additional peak observed in the HPLC chromatograms was identified by liquid chromatography-mass spectrometry as hydroxylamino-dinitroso-1,3,5-triazine. This is the first report identifying hydroxylamino-dinitroso-1,3,5-triazine as a transient intermediate produced during the anaerobic biodegradation of RDX.