Determination of Key Metabolites during Biodegradation of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine with Rhodococcus sp. Strain DN22

Abstract

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.The two cyclic nitramine explosives hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) are powerful energetic compounds that are commonly used in conventional munitions and various military applications. Activities associated with manufacturing, training, waste disposal, and closures of bases have resulted in severe soil and groundwater contamination with the two explosives (9, 14, 21). RDX and HMX are both toxic (25, 29), thus necessitating their removal from the environment.Previous studies have focused on the degradation of RDX by microorganisms under anaerobic conditions (1,17,18,20,24,30). McCormick et al. (20) reported the formation of the three nitroso derivatives, 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), which after further reduction to the corresponding hydroxylamines undergo ring cleavage to produce hydrazine, dimethyl hydrazine, and methanol, respectively. No microorganisms or enzymes were identified in the study by McCormick et al. (20). Recently, Kitts et al. (19) reported the involvement of a type I nitroreductase in the degradation of RDX, but no products were identified. Using anaerobic sludge, Hawari et al.(11) reported that in addition to the occurrence of a ring cleavage via the nitroso route, other ring cleavage pathways, such as direct ring cleavage and/or denitration followed by ring cleavage, might be possible. In the latter study, several key intermediate ring cleavage products, including bis(hydroxymethyl)nitramine, methylenedinitramine (MEDINA), nitrous oxide, and formaldehyde, accumulated, but hydrazines were not detected (11, 12).Several groups described RDX biodegradation under aerobic conditions, but little information was provided on the degradation pathway (5,6,16,27). Products from biodegradation of cyclic nitramine explosives under aerobic conditions are poorly understood, particularly ring cleavage products (5, 10). Jones et al. (16) isolated a Rhodococcus sp. strain A from explosives-contaminated soil and demonstrated its potential for the degradation of RDX but did not report any products. Coleman et al. (6) reported the isolation and characterization of another Rhodococcus sp. strain DN22,...

Biodegradation of RDX and MNX with Rhodococcus sp. Strain DN22: New Insights into the Degradation Pathway

“…strain DN22 produced the key ring cleavage product 4-nitro-2,4-diazabutanal (NDAB, NO 2 NHCH 2 NHCHO) representing 60% of C-content in RDX, carbon dioxide (30% detected as 14 CO 2 ), nitrous oxide (N 2 O) together with nitrite and ammonia. The C mass balance was approximately 90% (5). In the present study we incubated RDX with DN22 under aerobic conditions and found that the missing 10% C was due to the formation of methylenedinitramine (MEDINA, NO 2 NHCH 2 NHNO 2 ) which decomposed to HCHO and 2N 2 O (Figure 1).…”

“…Rhodococcus sp. strain DN22, provided by N. V. Coleman, (University of Sydney, Sydney, Australia) was grown in mineral salt medium (MSM) supplemented with succinate (2.4 mM) as the carbon source (5). RDX (220 µM) or MNX (220 µM) was added as the sole nitrogen source from an acetone stock solution.…”

“…In some cases NH 4 Cl (1.0 mM) was added to prevent the uptake of NO 2 -. The assay bottles were then incubated at 25°C with agitation (250 rpm) under aerobic conditions (5). Chemical controls containing either RDX or MNX or NO 2 -were prepared in MSM without DN22 cells.…”

“…Cell suspensions were then added to the substrate solutions in an anaerobic glovebox. 18 O 2 was then added as described by Fournier et al (5). Controls containing either RDX or MNX or nitrite were prepared in water in the presence of DN22 cells and air.…”

Bioremediation Approaches for Treating Low Concentrations of N-Nitrosodimethylamine in Groundwater

“…Methylamine and dimethylamine were analyzed as described by Gui et al (2000). The capture and quantification of 14 CO 2 were performed as described previously (Fournier et al, 2002).…”