Fungal interactions with the explosive RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine)

The bacterially mediated, anaerobic biodegradation of the explosive RDX (hexahydro 1,3,5 trinitro-1,3,5-triazine) is well established. Reports of successful mineralization of RDX by white rot fungi, and the enhanced transformation of RDX in stirred as compared to static composts, led us to study the possible aerobic role of several filamentous fungi in RDX biodegradation.Cladosporium resinae, Cunninghamella echinulata varelegans, Cyathus pallidus andPhanerochaete chrysosporium were grown in the presence of 50 and 100 g ml^–1 of RDX on a vegetable juice agar. Little inhibition of radial growth was observed, while control cultures with TNT exhibited substantial inhibition. When 100 g ml^–1 of RDX was added to pre-grown mycelia in a nonlignolytic liquid medium, between 12 and 31% was lost after 3 days. In similar experiments using¹⁴C-RDX, most of the label remained in the organic fraction, and little or none was found in the aqueous fraction, the volatile fraction or incorporated into cell walls. Although disappearance of RDX was observed for all four species tested, there was no evidence of mineralization. Mixed cultures of microorganisms, including both bacteria and fungi, merit further study as agents for the decontamination of munitions-contaminated soils.     

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).     

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.     

Prediction of growth and biotransformation rates of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in the presence of barium

The biotransformation of explosives has been investigated by many researchers. Bioremediation of soil and water contaminated with hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is becoming the method of choice for clean-up of a variety of sites. In this study, we investigated biotransformation of RDX in the presence of barium. Ba is a metal commonly found in combination with RDX at sites requiring remediation. RDX was biotransformed by both a consortium of bacteria and an isolate from the consortium under anoxic conditions using a rich medium. However, Ba inhibited cell growth under both aerobic and anoxic conditions and slowed biotransformation rates by 40%. RDX and Ba inhibited growth of the isolate more than growth of the consortium. An additive inhibition model is proposed that accurately predicts the reduced growth rates observed.     

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.     

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.     

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.     

Biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by a rabbit liver cytochrome P450: insight into the mechanism of RDX biodegradation by Rhodococcus sp. strain DN22

A unique metabolite with a molecular mass of 119 Da (C(2)H(5)N(3)O(3)) accumulated during biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by Rhodococcus sp. strain DN22 (D. Fournier, A. Halasz, J. C. Spain, P. Fiurasek, and J. Hawari, Appl. Environ. Microbiol. 68:166-172, 2002). The structure of the molecule and the reactions that led to its synthesis were not known. In the present study, we produced and purified the unknown metabolite by biotransformation of RDX with Rhodococcus sp. strain DN22 and identified the molecule as 4-nitro-2,4-diazabutanal using nuclear magnetic resonance and elemental analyses. Furthermore, we tested the hypothesis that a cytochrome P450 enzyme was responsible for RDX biotransformation by strain DN22. A cytochrome P450 2B4 from rabbit liver catalyzed a very similar biotransformation of RDX to 4-nitro-2,4-diazabutanal. Both the cytochrome P450 2B4 and intact cells of Rhodococcus sp. strain DN22 catalyzed the release of two nitrite ions from each reacted RDX molecule. A comparative study of cytochrome P450 2B4 and Rhodococcus sp. strain DN22 revealed substantial similarities in the product distribution and inhibition by cytochrome P450 inhibitors. The experimental evidence led us to propose that cytochrome P450 2B4 can catalyze two single electron transfers to RDX, thereby causing double denitration, which leads to spontaneous hydrolytic ring cleavage and decomposition to produce 4-nitro-2,4-diazabutanal. Our results provide strong evidence that a cytochrome P450 enzyme is the key enzyme responsible for RDX biotransformation by Rhodococcus sp. strain DN22.     

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.     

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.     

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.     

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.     

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.     

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.     

A DFT study of adsorption and decomposition of hexahydro-1,3,5-trinitro-1,3,5-triazine on Mg(0001) surface

The adsorption and decomposition of hexogen (RDX) molecule on the Mg(0001) surface were investigated by the generalized gradient approximation (GGA) of density functional theory (DFT). The calculations employed a supercell (4?×?4?×?4) slab model and three-dimensional periodic boundary conditions. The strong attractive forces between RDX molecule and magnesium atoms induce the RDX’s N???O bond breaking. Subsequently, the dissociated oxygen atoms and radical fragment of RDX oxidize the Mg surface. The largest adsorption energy is ?2104.0 kJ mol^-1. We also investigated the decomposition mechanism of RDX molecule on the Mg(0001) surface. The activation energy for the dissociation step of configuration V4 is as small as 2.5 kJ mol^-1, while activation energies of other configurations are much larger, in the range of 964.9–1375.1 kJ mol^-1. Mg powder is more active than Al powder, and Mg powder performs better in increasing the combustion exothermicity of RDX as well.     

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.     

Cosubstrate independent mineralization of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by a <Emphasis Type="Italic">Desulfovibrio</Emphasis> species under anaerobic conditions

Past handling practices associated with the manufacturing and processing of the high explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) has resulted in extensive environmental contamination. In-situ biodegradation is a promising technology for remediating RDX contaminated sites but often relies on the addition of a cosubstrate. A sulfate-reducing bacterium isolated from an RDX-degrading enrichment culture was studied for its ability to grow on RDX as a sole source of carbon and nitrogen and for its ability to mineralize RDX in the absence of a cosubstrate. The results showed the isolate degraded 140 μM RDX in 63 days when grown on RDX as a carbon source. Biomass within the carbon limited culture increased 9-fold compared to the RDX unamended controls. When the isolate was incubated with RDX as sole source of nitrogen it degraded 160 μM RDX in 41 days and exhibited a 4-fold increase in biomass compared to RDX unamended controls. Radiolabeled studies under carbon limiting conditions with ¹⁴C-hexahydro-1,3,5-trinitro-1,3,5-triazine confirmed mineralization of the cyclic nitramine. After 60 days incubation 26% of the radiolabel was recovered as ¹⁴CO₂, while in the control bottles less than 1% of the radiolabel was recovered as ¹⁴CO₂. Additionally, ~2% of the radiolabeled carbon was found to be associated with the biomass. The 16S rDNA gene was sequenced and identified the isolate as a novel species of Desulfovibrio, having a 95.1% sequence similarity to Desulfovibrio desulfuricans. This is the first known anaerobic bacterium capable of mineralizing RDX when using it as a carbon and energy source for growth.