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...

Recently we demonstrated that Rhodococcus sp. strain DN22 degraded hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) (1) aerobically via initial denitration followed by ring cleavage. Using UL 14C-[RDX] and ring labeled 15N-[RDX] approximately 30% of the energetic chemical mineralized (one C atom) and 64% converted to a dead end product that was tentatively identified as 4-nitro-2,4-diaza-butanal (OHCHNCH2NHNO2). To have further insight into the role of initial denitration on RDX decomposition, we photolyzed the energetic chemical at 350 nm and pH 5.5 and monitored the reaction using a combination of analytical techniques. GC/ MS-PCI showed a product with a [M+H] at 176 Da matching a molecular formula of C3H5N5O4 that was tentatively identified as the initially denitrated RDX product pentahydro-3,5-dinitro-1,3,5-triazacyclohex-1-ene (II). LC/MS (ES-) showed that the removal of RDX was accompanied by the formation of two other key products, each showing the same [M-H] at 192 Da matching a molecular formula of C3H7N5O5. The two products were tentatively identified as the carbinol (III) of the enamine (II) and its ring cleavage product O2NNHCH2NNO2CH2NHCHO (IV). Interestingly, the removal of III and IV was accompanied by the formation and accumulation of OHCHNCH2NHNO2 that we detected with strain DN22. At the end of the experiment, which lasted 16 h, we detected the following products HCHO, HCOOH, NH2CHO, N2O, NO2-, and NO3-. Most were also detected during RDX incubation with strain DN22. Finally, we were unable to detect any of RDX nitroso products during both photolysis and incubation with the aerobic bacteria, emphasizing that initial denitration in both cases was responsible for ring cleavage and subsequent decomposition in water.