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.

The nitroaromatic explosive 2,4,6-trinitrotoluene (TNT) is a reactive molecule that biotransforms readily under both aerobic and anaerobic conditions to give aminodinitrotoluenes. The resulting amines biotransform to give several other products, including azo, azoxy, acetyl and phenolic derivatives, leaving the aromatic ring intact. Although some Meisenheimer complexes, initiated by hydride ion attack on the ring, can be formed during TNT biodegradation, little or no mineralization is encountered during bacterial treatment. Also, although the ligninolytic physiological phase and manganese peroxidase system of fungi can cause some TNT mineralization in liquid cultures, little to no mineralization is observed in soil. Therefore, despite more than two decades of intensive research to biodegrade TNT, no biomineralization-based technologies have been successful to date. The non-aromatic 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) lack the electronic stability enjoyed by TNT or its transformed products. Predictably, a successful enzymatic change on one of the N-NO2 or C-H bonds of the cyclic nitramine would lead to a ring cleavage because the inner C-N bonds in RDX become very weak (<2 kcal/mol). Recently this hypothesis was tested and proved feasible, when RDX produced high amounts of carbon dioxide and nitrous oxide following its treatment with either municipal anaerobic sludge or the fungus Phanaerocheate chrysosporium. Research aimed at the discovery of new microorganisms and enzymes capable of mineralizing energetic chemicals and/or enhancing irreversible binding (immobilization) of their products to soil is presently receiving considerable attention from the scientific community.

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,...

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In an earlier study, we reported that hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) biodegraded with domestic anaerobic sludge to produce a key RDX ring cleavage intermediate that was tentatively identified as methylenedinitramine (O2NNHCH2NHNO2) using LC/MS with negative electrospray ionization (ES-). Recently, we obtained a standard material of methylenedinitramine and thus were able to confirm its formation as the key initial RDX intermediate. In water alone or in the presence of sludge, methylenedinitramine decomposed to N20 and HCHO. Only in the presence of sludge HCHO converted further to carbon dioxide. To test our hypothesis that water was involved in the formation of methylenedinitramine during incubation of RDX with sludge, we allowed the energetic compound to biodegrade in several D2O/H2O solutions (90, 50, and 0% v/v). We observed three distinctive deprotonated or dedeuterated mass ions at 135, 136, and 137 Da that were attributed to the formation of nondeuterated (H-methylenedinitramine), monodeuterated (D1-methylenedinitramine), and dideuterated methylenedinitramine (D2-methylenedinitramine), respectively. Two controls were prepared in D2O both in the absence of sludge; the first contained methylenedinitramine, and the second contained RDX. Neither control produced any deuterated methylenedinitramine, thus excluding the occurrence of any abiotic D/H exchange between D2O and either methylenedinitramine or RDX. The results supported the occurrence of an initial enzymatic reaction on RDX, yet they did not provide compelling evidence on whether methylenedinitramine was an initial RDX enzymatic hydrolysis product or simply formed via the spontaneous hydrolysis of an anonymous initial RDX enzymatic product.

In previous work, we found that an anaerobic sludge efficiently degraded hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), but the role of isolates in the degradation process was unknown. Recently, we isolated a facultatively anaerobic bacterium, identified as Klebsiella pneumoniae strain SCZ-1, using MIDI and the 16S rRNA method from this sludge and employed it to degrade RDX. Strain SCZ-1 degraded RDX to formaldehyde (HCHO), methanol (CH 3 OH) (12% of total C), carbon dioxide (CO 2 ) (72% of total C), and nitrous oxide (N 2 O) (60% of total N) through intermediary formation of methylenedinitramine (O 2 NNHCH 2 NHNO 2 ). Likewise, hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) was degraded to HCHO, CH 3 ). These findings suggested the possible involvement of a common initial reaction, possibly denitration, followed by ring cleavage and decomposition in water. The trace amounts of MNX detected during RDX degradation and the trace amounts of hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine detected during MNX degradation suggested that another minor degradation pathway was also present that reduced ONO 2 groups to the corresponding ONO groups.Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a widely used explosive that severely contaminates soil and groundwater (13,22). RDX has been found to be toxic in various terrestrial and aquatic environments (26,29). The toxicity of cyclic nitramines means that contaminated soil and groundwater must be remediated, preferably by bioremediation. Biodegradation of RDX with anaerobic sludge was extensively studied by McCormick et al. (21) and Kaplan (19), who proposed a pathway based on the sequential reduction of RDX to 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). It was proposed that the nitroso compounds undergo further transformation to hydroxylamino derivatives (HOHN-RDX), which are subsequently cleaved to produce formaldehyde (HCHO), methanol (CH 3 OH), hydrazine (NH 2 NH 2 ), and dimethyl hydrazine [(H 3 C) 2 NNH 2 ].Other research groups have employed mixed anaerobic microbial cultures that have included methanogens (1, 2), acetogens (4), nitrate reducers (12), and individual isolates such as isolates of Desulfovibrio sp. and Serratia marcescens (31) to biotransform RDX, but in most cases no clear details concerning ring cleavage products or mineralization were provided in the descriptions. Subsequent work in our laboratory (14,15,16) showed that RDX could be degraded in anaerobic sludge via intermediary formation of methylenedinitramine (O 2 NNHCH 2 NHNO 2 ), which decomposes in water to nitrous oxide (N 2 O) and HCHO. Recently, Oh et al. (23) confirmed the formation of O 2 NNHCH 2 NHNO 2 during RDX degradation with anaerobic sludge. Despite these previous efforts, there is still insufficient information regarding the roles of individual anaerobic isolates and their products during RDX degradation. Without these details, particularly details concerning...

The sorption-desorption behavior and long-term fate of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) was examined in sterilized and nonsterilized topsoil. Results of this study indicate that although RDX is not extensively sorbed by the topsoil (Ks(d) of 0.83 L/kg), sorption is nearly irreversible. Furthermore, there was no difference in the sorption behavior for sterile and nonsterile topsoil. However, over the longterm, RDX completely disappeared within 5 weeks in nonsterile topsoil, and 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) metabolites formed in the aqueous phase. Over the same period, recovery of RDX from sterile topsoil was high (55-99%), and the nitroso metabolites were not detected. Only traces of RDX were mineralized to CO2 and N2O by the indigenous microorganisms in nonsterile topsoil. Of the RDX that was mineralized to N2O, one N originated from the ring and the other from the nitro group substituent, as determined using N15 ring-labeled RDX. However, N2O from RDX represented only 3% of the total N2O that formed from the process of nitrification/denitrification.

). 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.Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a cyclic nitramine explosive commonly used for military and commercial purposes worldwide. The extensive manufacturing, use, and disposal of RDX have resulted in severe environmental contamination (7,14). RDX is toxic, mutagenic, and carcinogenic for humans and other biological systems (9, 19); hence, there is an urgent need for safe removal of this compound from the environment. In several reports workers have described biodegradation of cyclic nitramine explosives, such as RDX, under both aerobic (2, 3, 5, 18) and anaerobic (8,10,12,13,15,20) conditions, but little information is available on the initial enzymatic processes involved in RDX degradation. Such information is necessary for understanding the pathway by which RDX can serve as a nitrogen source for aerobic bacteria.Unlike 2,4,6-trinitrotoluene, the cyclic nitramine compounds lack the electronic stability of aromatic compounds. Therefore, a successful microbial or chemical attack on one of the ONO 2 or OCH 2 O groups of RDX is sufficient for ring cleavage and subsequent spontaneous decomposition. Binks et al. (2) provided strong evidence that RDX ring cleavage occurs during aerobic degradation of RDX by Stenotrophomonas maltophilia PB1. Previously, we proved that initial denitration of RDX by Klebsiella sp. strain SCZ-1, an isolate from anaerobic sludge (21), and Rhodococcus sp. strain DN22, a soil isolate (5), can lead to ring cleavage and spontaneous decomposition to HCHO, CO 2 , N 2 O, and NH 3 . However, we did not determine the enzymes responsible for initiating the biotransformation of RDX. During RDX degradation with Rhodococcus sp. strain DN22, we detected a dead end product with a molecul...