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

Previously we demonstrated that Rhodococcus sp. strain DN22 can degrade RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) aerobically via initial denitration. The present study describes the role of oxygen and water in the key denitration step leading to RDX decomposition using O-18(2) and (H2O)-O-18 labeling experiments. We also investigated degradation of MNX (hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine)with DN22 under similar conditions. DN22 degraded RDX and MNX giving NO2-, NO3-, NDAB (4-nitro-diazabutanal), NH3, N2O, and HCHO with NO2-/NO3- molar ratio reaching 17 and ca. 2, respectively. In the presence of O-18(2), DN22 degraded RDX and produced NO2- with m/z at 46 Da that subsequently oxidized to NO3- containing one O-18 atom, but in the presence of (H2O)-O-18 we detected NO3- without O-18. A control containing NO2-, DN22, and O-18(2) gave NO3- with one O-18, confirming biotic oxidation of NO2- to NO3-. Treatment of MNX with DN22 and O-18(2) produced NO3- with two mass ions, one (66 Da) incorporating two O-18 atoms and another (64 Da) incorporating only one O-18 atom and we attributed their formation to bio-oxidation of the initially formed NO and NO2-, respectively. In the presence of (H2O)-O-18 we detected NO2- with two different masses, one representing NO2- (46 Da) and another representing NO2- (48 Da) with the inclusion of one O-18 atom suggesting auto-oxidation of NO to NO2-. Results indicated that denitration of either RDX or MNX and denitrosation of MNX by DN22 did not involve direct participation of either oxygen or water, but both played major roles in subsequent secondary chemical and biochemical reactions of NO and NO2-.