Biodegradation of nitro-substituted explosives 2,4,6-trinitrotoluene, hexahydro-1,3,5-trinitro-1,3,5-triazine, and octahydro-1,3,5,7-tetranitro-1,3,5-tetrazocine by a phytosymbiotic Methylobacterium sp. associated with poplar tissues (Populus deltoides x nigra DN34)

A pink-pigmented symbiotic bacterium was isolated from hybrid poplar tissues (Populus deltoides x nigra DN34). The bacterium was identified by 16S and 16S-23S intergenic spacer ribosomal DNA analysis as a Methylobacterium sp. (strain BJ001). The isolated bacterium was able to use methanol as the sole source of carbon and energy, which is a specific attribute of the genus Methylobacterium. The bacterium in pure culture was shown to degrade the toxic explosives 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazene (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5-tetrazocine (HMX). [U-ring-(14)C]TNT (25 mg liter(-1)) was fully transformed in less than 10 days. Metabolites included the reduction derivatives amino-dinitrotoluenes and diamino-nitrotoluenes. No significant release of (14)CO(2) was recorded from [(14)C]TNT. In addition, the isolated methylotroph was shown to transform [U-(14)C]RDX (20 mg liter(-1)) and [U-(14)C]HMX (2.5 mg liter(-1)) in less than 40 days. After 55 days of incubation, 58.0% of initial [(14)C]RDX and 61.4% of initial [(14)C]HMX were mineralized into (14)CO(2). The radioactivity remaining in solution accounted for 12.8 and 12.7% of initial [(14)C]RDX and [(14)C]HMX, respectively. Metabolites detected from RDX transformation included a mononitroso RDX derivative and a polar compound tentatively identified as methylenedinitramine. Since members of the genus Methylobacterium are distributed in a wide diversity of natural environments and are very often associated with plants, Methylobacterium sp. strain BJ001 may be involved in natural attenuation or in situ biodegradation (including phytoremediation) of explosive-contaminated sites.     

Development of a Relative Source Contribution Factor for Drinking Water Criteria: The Case of Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)

The consideration of multiple or cumulative sources of exposure to a chemical is important for adequately protecting human health. This assessment demonstrates one way to consider multiple or cumulative sources through the development of a relative source contribution (RSC) factor for the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), using the Exposure Decision Tree approach (subtraction method) recommended by the U.S. Environmental Protection Agency. The RSC factor is used to ensure that the concentration of a chemical allowed by a regulatory criterion or multiple criteria, when combined with other identified sources of exposure common to the population of concern, will not result in unacceptable exposures. An exposure model was used to identify relevant potential sources for receptors. Potential exposure pathways include ingestion of soil, water, contaminated local crops and fish, and dermal contact with soil and water. These pathways are applicable only to areas that are in close proximity to current or former military bases where RDX may have been released into the environment. Given the physical/chemical properties and the available environmental occurrence data on RDX, there are adequate data to support a chemical-specific RSC factor for RDX of 50% for drinking water ingestion.     

Use of pressurized liquid extraction (PLE)/gas chromatography-electron capture detection (GC-ECD) for the determination of biodegradation intermediates of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in soils

A rapid, sensitive, and reproducible method was developed for quantitative determination of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and its biodegradation intermediates, hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX), and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX) in soils. RDX, MNX, DNX, or TNX was extracted from soil by pressurized liquid extraction (PLE), followed by cleanup using florisil. Instrumental analysis was performed using gas chromatography with electron capture detection (GC-ECD), which was highly sensitive to the parent explosive and its metabolites. The method detection limits (MDLs) were 0.243, 0.095, 0.138, and 0.057 ng/g for RDX, MNX, DNX, and TNX, respectively. The method gave high recovery (98-102%), good precision (0.22-5.14%), and reproducibility, and proved to be suitable for real world sample analysis.     

Characterization of Metabolites during Biodegradation of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine (RDX) with Municipal Anaerobic Sludge

The biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in liquid cultures with municipal anaerobic sludge showed that at least two degradation routes were involved in the disappearance of the cyclic nitramine. In one route, RDX was reduced to give the familiar nitroso derivatives hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) and hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX). In the second route, two novel metabolites, methylenedinitramine [(O₂NNH)₂CH₂] and bis(hydroxymethyl)nitramine [(HOCH₂)₂NNO₂], formed and were presumed to be ring cleavage products produced by enzymatic hydrolysis of the inner C—N bonds of RDX. None of the above metabolites accumulated in the system, and they disappeared to produce nitrous oxide (N₂O) as a nitrogen-containing end product and formaldehyde (HCHO), methanol (MeOH), and formic acid (HCOOH) that in turn disappeared to produce CH₄ and CO₂ as carbon-containing end products.     

Evaluating Biodegradation as a Primary and Secondary Treatment for Removing RDX (Hexahydro-1,3,5-trinitro-1,3,5-triazine) from a Perched Aquifer

Ground water beneath the U.S. Department of Energy (USDOE) Pantex Plant is contaminated with the high explosive RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine). The authors evaluated biodegradation as a remedial option by measuring RDX mineralization in Pantex aquifer microcosms spiked with ¹⁴C-labeled RDX (75 g soil, 15 ml of 5 mg RDX/L). Under anaerobic conditions and constant temperature (16°C), cumulative ¹⁴CO₂ production ranged between 52% and 70% after 49 days, with nutrient-amended (C, N, P) microcosms yielding the greatest mineralization (70%). The authors also evaluated biodegradation as a secondary treatment for removing RDX degradates following oxidation by permanganate (KMnO₄) or reduction by dithionite-reduced aquifer solids (i.e., redox barriers). Under this coupled abiotic/biotic scenario, we found that although unconsumed permanganate initially inhibited biodegradation, > 48% of the initial ¹⁴C-RDX was recovered as ¹⁴CO₂ within 77 days. Following exposure to dithionite-reduced solids, RDX transformation products were also readily mineralized (> 47% in 98 days). When we seeded Pantex aquifer material into Ottawa Sand that had no prior exposure to RDX, mineralization increased 100%, indicating that the Pantex aquifer may have an adapted microbial community that could be exploited for remediation purposes. These results indicate that biodegradation effectively transformed and mineralized RDX in Pantex aquifer microcosms. Additionally, biodegradation may be an excellent secondary treatment for RDX degradates produced from in situ treatment with permanganate or redox barriers.     

Biodegradation of Nitro-Substituted Explosives 2,4,6-Trinitrotoluene, Hexahydro-1,3,5-Trinitro-1,3,5-Triazine, and Octahydro-1,3,5,7-Tetranitro-1,3,5-Tetrazocine by a Phytosymbiotic Methylobacterium sp. Associated with Poplar Tissues (Populus deltoides × nigra DN34)

A pink-pigmented symbiotic bacterium was isolated from hybrid poplar tissues (Populus deltoides × nigra DN34). The bacterium was identified by 16S and 16S-23S intergenic spacer ribosomal DNA analysis as a Methylobacterium sp. (strain BJ001). The isolated bacterium was able to use methanol as the sole source of carbon and energy, which is a specific attribute of the genus Methylobacterium. The bacterium in pure culture was shown to degrade the toxic explosives 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazene (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5-tetrazocine (HMX). [U-ring-¹⁴C]TNT (25 mg liter⁻¹) was fully transformed in less than 10 days. Metabolites included the reduction derivatives amino-dinitrotoluenes and diamino-nitrotoluenes. No significant release of ¹⁴CO₂ was recorded from [¹⁴C]TNT. In addition, the isolated methylotroph was shown to transform [U-¹⁴C]RDX (20 mg liter⁻¹) and [U-¹⁴C]HMX (2.5 mg liter⁻¹) in less than 40 days. After 55 days of incubation, 58.0% of initial [¹⁴C]RDX and 61.4% of initial [¹⁴C]HMX were mineralized into ¹⁴CO₂. The radioactivity remaining in solution accounted for 12.8 and 12.7% of initial [¹⁴C]RDX and [¹⁴C]HMX, respectively. Metabolites detected from RDX transformation included a mononitroso RDX derivative and a polar compound tentatively identified as methylenedinitramine. Since members of the genus Methylobacterium are distributed in a wide diversity of natural environments and are very often associated with plants, Methylobacterium sp. strain BJ001 may be involved in natural attenuation or in situ biodegradation (including phytoremediation) of explosive-contaminated sites.     

An explosive-degrading cytochrome P450 activity and its targeted application for the phytoremediation of RDX

The widespread presence in the environment of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), one of the most widely used military explosives, has raised concern owing to its toxicity and recalcitrance to degradation. To investigate the potential of plants to remove RDX from contaminated soil and water, we engineered Arabidopsis thaliana to express a bacterial gene xplA encoding an RDX-degrading cytochrome P450 (ref. 1). We demonstrate that the P450 domain of XplA is fused to a flavodoxin redox partner and catalyzes the degradation of RDX in the absence of oxygen. Transgenic A. thaliana expressing xplA removed and detoxified RDX from liquid media. As a model system for RDX phytoremediation, A. thaliana expressing xplA was grown in RDX-contaminated soil and found to be resistant to RDX phytotoxicity, producing shoot and root biomasses greater than those of wild-type plants. Our work suggests that expression of xplA in landscape plants may provide a suitable remediation strategy for sites contaminated by this class of explosives.     

Ovine Ruminal Microbes Are Capable of Biotransforming Hexahydro-1,3,5-Trinitro-1,3,5-Triazine (RDX)

Bioremediation is of great interest in the detoxification of soil contaminated with residues from explosives such as hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). Although there are numerous forms of in situ and ex situ bioremediation, ruminants would provide the option of an in situ bioreactor that could be transported to the site of contamination. Bovine rumen fluid has been previously shown to transform 2,4,6-trinitrotoluene (TNT), a similar compound, in 4 h. In this study, RDX incubated in whole ovine rumen fluid was nearly eliminated within 4 h. Whole ovine rumen fluid was then inoculated into five different types of media to select for archaeal and bacterial organisms capable of RDX biotransformation. Cultures containing 30 μg mL⁻¹ RDX were transferred each time the RDX concentration decreased to 5 μg mL⁻¹ or less. Time point samples were analyzed for RDX biotransformation by HPLC. The two fastest transforming enrichments were in methanogenic and low nitrogen basal media. After 21 days, DNA was extracted from all enrichments able to partially or completely transform RDX in 7 days or less. To understand microbial diversity, 16S rRNA-gene-targeted denaturing gradient gel electrophoresis (DGGE) fingerprinting was conducted. Cloning and sequencing of partial 16S rRNA fragments were performed on both low nitrogen basal and methanogenic media enrichments. Phylogenetic analysis revealed similar homologies to eight different bacterial and one archaeal genera classified under the phyla Firmicutes, Actinobacteria, and Euryarchaeota. After continuing enrichment for RDX degraders for 1 year, two consortia remained: one that transformed RDX in 4 days and one which had slowed after 2 months of transfers without RDX. DGGE comparison of the slower transforming consortium to the faster one showed identical banding patterns except one band. Homology matches to clones from the two consortia identified the same uncultured Clostridia genus in both; Sporanaerobacter acetigenes was identified only in the consortia able to completely transform RDX. This is the first study to examine the rumen as a potential bioremediation tool for soils contaminated with RDX, as well as to discover S. acetigenes in the rumen and its potential ability to metabolize this energetic compound.     

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) Biodegradation in Liquid and Solid-State Matrices by Phanerochaete chrysosporium

Extensive biodegradation of hexahydro-1,3,5 -trinitro-1,3,5 -triazine (RDX) by the white-rot fungus Phanerochaete chrysosporium in liquid and solid matrices was observed. Some degradation in liquid occurred under nonligninolytic conditions, but was approximately 10 times higher under ligninolytic conditions. Moreover, elimination was accounted for almost completely as carbon dioxide. No RDX metabolites were detected. The degradation rates in liquid appeared to be limited to RDX concentration in solution (approximately 80 mg/L), but degradation rates in soil were nonsaturable to 250 mg/kg. Manganese-dependent peroxidase (MnP) and cellobiose dehydrogenase (CDH) from P. chrysosporium, but not lignin peroxidase, were able to degrade RDX. MnP degradation of RDX required addition of manganese, but CDH degraded RDX anaerobically without addition of mediators. Attempts to improve biodegradation by supplementing cultures with micronutrients showed that addition of manganese and oxalate stimulated degradation rates in liquid, sawdust, and sand by the fungus, but not in loam soil. RDX degradation by P. chrysosporium in sawdust and sand was better than observed in liquid. However, degradation in solid matrices by the fungus only began after a lag period of 2 to 3 weeks, during which time extractable metabolites from wood were degraded.     

Sulfate-Mediated Bacterial Population Shift in a Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)-Degrading Anaerobic Enrichment Culture

The effects of sulfate on the population dynamics of an anaerobic hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)-degrading consortium were studied using terminal restriction fragment length polymorphism (T-RFLP) analysis. One hundred percent of the initial RDX was degraded in the sulfate-amended culture within 3 days of incubation. In the sulfate-unamended cultures, 35% of the initial RDX remained after 3 days and 8% after 7 days of incubation. Based on the T-RFLP distribution of the community 16S rDNA genes, the microcosm consisted predominantly of two organisms, a Geobacter sp. (78%) and an Acetobacterium sp. (14%). However, in the presence of sulfate, both species decreased to less than 3% of the total population within 3 days and an unclassified Clostridiaceae became the dominant organism at 40% the total fragment distribution. This indicated the explosive-degrading consortium had greater diversity than initially perceived and rapidly adapted to a readily available electron acceptor, which in turn stimulated RDX degradation.     

Characterization of metabolites during biodegradation of hexahydro-1, 3,5-trinitro-1,3,5-triazine (RDX) with municipal anaerobic sludge

The biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in liquid cultures with municipal anaerobic sludge showed that at least two degradation routes were involved in the disappearance of the cyclic nitramine. In one route, RDX was reduced to give the familiar nitroso derivatives hexahydro-1-nitroso-3,5-dinitro-1,3, 5-triazine (MNX) and hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX). In the second route, two novel metabolites, methylenedinitramine [(O(2)NNH)(2)CH(2)] and bis(hydroxymethyl)nitramine [(HOCH(2))(2)NNO(2)], formed and were presumed to be ring cleavage products produced by enzymatic hydrolysis of the inner C---N bonds of RDX. None of the above metabolites accumulated in the system, and they disappeared to produce nitrous oxide (N(2)O) as a nitrogen-containing end product and formaldehyde (HCHO), methanol (MeOH), and formic acid (HCOOH) that in turn disappeared to produce CH(4) and CO(2) as carbon-containing end products.     

Microaerophilic degradation of hexahydro‐1,3,5‐trinitro‐1,3,5‐triazine (RDX) by three Rhodococcus strains

Aim: The goal of this study was to compare the degradation of hexahydro‐1,3,5‐trinitro‐1,3,5‐triazine (RDX) by three Rhodococcus strains under anaerobic, microaerophilic (<0·04 mg l^?1 dissolved oxygen) and aerobic (dissolved oxygen (DO) maintained at 8 mg l^?1) conditions. Methods and Results: Three Rhodococcus strains were incubated with no, low and ambient concentrations of oxygen in minimal media with succinate as the carbon source and RDX as the sole nitrogen source. RDX and RDX metabolite concentrations were measured over time. Under microaerophilic conditions, the bacteria degraded RDX, albeit about 60‐fold slower than under fully aerobic conditions. Only the breakdown product, 4‐nitro‐2,4‐diazabutanal (NDAB) accumulated to measurable concentrations under microaerophilic conditions. RDX degraded quickly under both aerated and static aerobic conditions (DO allowed to drop below 1 mg l^?1) with the accumulation of both NDAB and methylenedinitramine (MEDINA). No RDX degradation was observed under strict anaerobic conditions. Conclusions: The Rhodococcus strains did not degrade RDX under strict anaerobic conditions, while slow degradation was observed under microaerophilic conditions. The RDX metabolite NDAB was detected under both microaerophilic and aerobic conditions, while MEDINA was detected only under aerobic conditions. Impact and Significance of the Study: This work confirmed the production of MEDINA under aerobic conditions, which has not been previously associated with aerobic RDX degradation by these organisms. More importantly, it demonstrated that aerobic rhodococci are able to degrade RDX under a broader range of oxygen concentrations than previously reported.     

Trace level detection of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by microimmunosensor.

Reported in this paper is the development and characterization of a highly sensitive microcapillary immunosensor for the detection of the explosive, hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). The immunosensor exploits antibodies as recognition elements for target antigens, fluorescence dye conjugates for reporter molecules and fused silica microcapillaries for its high surface-to-volume ratio. Detection of RDX with the microcapillary immunosensor requires covalent immobilization of anti-RDX antibodies on the inner core of the microcapillaries via heterobifunctional cross-linker chemistry. Subsequent saturation of all antibody binding domains follows with a synthetically prepared fluorescent analog of RDX. Displacement immunoassays were performed with the microcapillary immunosensor with the injection of unlabeled RDX at concentration levels from 1 part-per-trillion (pptr) to 1000 part-per-billion (ppb). As unlabeled RDX reaches the binding domain of the antibody, fluorescent RDX analog is displaced from the antibody, flows downstream and is measured by a spectrofluorometer. Fluorescence measurements of the displaced fluorescent RDX analog were equated to a standard calibration curve to quantify sample concentration. Complete evaluation of the RDX microcapillary immunosensor for selectivity and sensitivity was performed based on the following criteria: variable flow rates, antibody cross-reactivity, reproducibility and cross-linker (carbon spacer) comparison. Results indicate the lowest detectable limit (LDL) for RDX is 10 pptr (ng/l) with a linear dynamic range from 0.1 to 1000 ppb (ug/l).     

RDX (Hexahydro-1,3,5-trinitro-1,3,5-triazine) Biodegradation in Aquifer Sediments under Manganese-Reducing Conditions

A shallow, RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine)-contaminated aquifer at Naval Submarine Base Bangor has been characterized as predominantly manganese-reducing, anoxic with local pockets of oxic conditions. The potential contribution of microbial RDX degradation to localized decreases observed in aquifer RDX concentrations was assessed in sediment microcosms amended with [U-¹⁴C] RDX. Greater than 85% mineralization of ¹⁴C-RDX to ¹⁴CO₂ was observed in aquifer sediment microcosms under native, manganese-reducing, anoxic conditions. Significant increases in the mineralization of ¹⁴C-RDX to ¹⁴CO₂ were observed in anoxic microcosms under NO₃-amended or Mn(IV)-amended conditions. No evidence of ¹⁴C-RDX biodegradation was observed under oxic conditions. These results indicate that microbial degradation of RDX may contribute to natural attenuation of RDX in manganese-reducing aquifer systems.     

Biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by a prospective consortium and its most effective isolate Serratia marcescens

The biotransformation of hexahydro-1,3,5-trinitro-1,3,5 triazine (RDX) has been observed in liquid culture by a consortium of bacteria found in horse manure. Five types of bacteria were found to predominate in the consortium and were isolated. The most effective of these isolates at transforming RDX was Serratia marcescens. The biotransformation of RDX by all of these bacteria was found to occur only in the anoxic stationary phase. The process of bacterial growth and RDX biotransformation was quantified for the purpose of developing a predictive type model. Cell growth was assumed to follow Monod kinetics. All of the aerobic and anoxic growth parameters were determined: mu(max), K(s), and Y(x/s). RDX was found to competitively inhibit cell growth in both atmospheres. Degradation of RDX by Serratia marcescens was found to proceed through the stepwise reduction of the three nitro groups to nitroso groups. Each of these reductions was found to be first order in both component and cell concentrations. The degradation rate constant for the first step in this reduction process by the consortium was 0.022 L/g cells . h compared to 0.033 L/g cells . h for the most efficient isolate. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 515-522, 1997.     

Growth changes of eighteen herbaceous angiosperms induced by Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in soil

Study objectives were to describe and quantify growth responses (tolerance as shoot and root biomass accumulation) to soil-applied Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) treatments of eighteen terrestrial, herbaceous, angiospermous species and also; to determine how much of RDX, RDX transformation products, total N and RDX-derived N accumulated in the foliage. RDX altered growth of eighteen plant species or cultivars at levels of 100, 500, and 1,000 mg kg^?1dry soil in a 75-d greenhouse study. Sixteen species or cultivars exhibited growth inhibition while two were stimulated in growth by RDX. A maximum amount of foliar RDX in a subset of three plant species was 36.0 mg per plant in Coronilla varia. Foliar concentrations of transformation products of RDX were low relative to RDX in the subset of three species. The proportion of RDX-N with respect to total N was constant, suggesting that foliar RDX transformation did not explain differences in tolerance. There was a δ ¹⁵N shift towards that of synthetic RDX in foliage of the three species at a level of 1,000 mg kg^?1 RDX, proportional in magnitude to uptake of N from RDX and tolerance ranking.Reddened leaf margins for treated Sida spinosa indicate the potential of this species as a biosensor for RDX.     

Biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine by novel fungi isolated from unexploded ordnance contaminated marine sediment

Undersea deposition of unexploded ordnance (UXO) constitutes a potential source of contamination of marine environments by hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX). Using sediment from a coastal UXO field, Oahu Island, Hawaii, we isolated four novel aerobic RDX-degrading fungi HAW-OCF1, HAW-OCF2, HAW-OCF3 and HAW-OCF5, tentatively identified as members of Rhodotorula, Bullera, Acremonium and Penicillium, respectively. The four isolates mineralized 15–34% of RDX in 58 days as determined by liberated ¹⁴CO₂. Subsequently we selected Acremonium to determine biotransformation pathway(s) of RDX in more details. When RDX (100 μM) was incubated with resting cells of Acremonium we detected methylenedinitramine (MEDINA), N₂O and HCHO. Also we detected hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) together with trace amounts of hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX) and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX). Under the same conditions MNX produced N₂O and HCHO together with trace amounts of DNX and TNX, but we were unable to detect MEDINA. TNX did not degrade with Acremonium. These experimental findings suggested that RDX degraded via at least two major initial routes; one route involved direct ring cleavage to MEDINA and another involved reduction to MNX prior to ring cleavage. Nitrite was only detected in trace amounts suggesting that degradation via initial denitration did take place but not significantly. Aerobic incubation of Acremonium in sediment contaminated with RDX led to enhanced removal of the nitramine.     

Degradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by Stenotrophomonas maltophilia PB1.

A mixed microbial culture capable of metabolizing the explosive RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) was obtained from soil enrichments under aerobic and nitrogen-limiting conditions. A bacterium, Stenotrophomonas maltophilia PB1, isolated from the culture used RDX as a sole source of nitrogen for growth. Three moles of nitrogen was used per mole of RDX, yielding a metabolite identified by mass spectroscopy and 1H nuclear magnetic resonance analysis as methylene-N-(hydroxymethyl)-hydroxylamine-N'-(hydroxymethyl)nitroamin e. The bacterium also used s-triazine as a sole source of nitrogen but not the structurally similar compounds octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine, cyanuric acid, and melamine. An inducible RDX-degrading activity was present in crude cell extracts.     

Type I nitroreductases in soil enterobacteria reduce TNT (2,4,6,-trinitrotoluene) and RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine)

Many enteric bacteria express a type I oxygen-insensitive nitroreductase, which reduces nitro groups on many different nitroaromatic compounds under aerobic conditions. Enzymatic reduction of nitramines was also documented in enteric bacteria under anaerobic conditions. This study indicates that nitramine reduction in enteric bacteria is carried out by the type I, or oxygen-insensitive nitroreductase, rather than a type II enzyme. The enteric bacterium Morganella morganii strain B2 with documented hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) nitroreductase activity, and Enterobacter cloacae strain 96-3 with documented 2,4,6-trinitrotoluene (TNT) nitroreductase activity, were used here to show that the explosives TNT and RDX were both reduced by a type I nitroreductase. Morganella morganii and E. cloacae exhibited RDX and TNT nitroreductase activities in whole cell assays. Type I nitroreductase, purified from E. cloacae, oxidized NADPH with TNT or RDX as substrate. When expression of the E. cloacae type I nitroreductase gene was induced in an Escherichia coli strain carrying a plasmid, a simultaneous increase in TNT and RDX nitroreductase activities was observed. In addition, neither TNT nor RDX nitroreductase activity was detected in nitrofurazone-resistant mutants of M. morganii. We conclude that a type I nitroreductase present in these two enteric bacteria was responsible for the nitroreduction of both types of explosive.     

Enhanced Biodegradation and Fate of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine (RDX) and Octahydro-1,3,5,7-Tetranitro-1,3,5,7-Tetrazocine (HMX) in Anaerobic Soil Slurry Bioprocess

Native soil microbial populations and unadapted municipal anaerobic sludges were compared for nitramine explosive degradation in microcosm assays under various conditions. Microbial populations from an explosive-contaminated soil were only able to mineralize 12% hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) (at a concentration of 800 mg/kg slurry) or 4% octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) (at a concentration of 267 mg/kg slurry). In contrast, municipal anaerobic sludges were able to mineralize them to carbon dioxide, with efficiencies of up to 65%. Reduction of RDX and HMX into their corresponding nitroso-derivatives was notably faster than their mineralization. The biodegradation of HMX was typically delayed by the presence of RDX in the microcosm, confirming RDX is used as an electron acceptor preferentially to HMX. The laboratory-scale bioslurry reactor reproduced the results of the microcosm assays, yet with much higher RDX and HMX degradation rates. A radiolabel-based mass balance in the soil slurry indicated that, besides a significant mineralization to carbon dioxide, 25% and 31% of RDX and HMX, respectively, appeared as acetonitrile-extractable metabolites, while the remaining part was incorporated into biomass and irreversibly bound to the soil matrix. About 10% of the HMX derivatives were estimated to be chemically bound to the soil matrix, while for RDX the estimation was nil.