Mineralization of the cyclic nitramine explosive hexahydro-1,3,5-trinitro-1,3,5-triazine by Gordonia and Williamsia spp

Hexahydro-1,3,5-trinitro-1,3,5-triazine(RDX) is a cyclic nitroamine that is one of the most powerful and commonly used military explosives. RDX has contaminated soils and groundwater at army ammunition plants and other military sites through its manufacture, use on testing and firing ranges, and disposal (22). RDX is a toxic compound that affects the central nervous system of laboratory animals, causing convulsions (10, 37), and it has been proposed as a possible human carcinogen (38). Information on the environmental factors that determine the transport and fate of RDX in soils is needed to accurately assess the risk posed by RDX and to ensure the sustainability of live-fire training exercises. Anaerobic biodegradation of RDX has been extensively studied (1, 6, 21, 23, 25, 26, 29, 42-45). McCormick et al. (29) proposed a pathway where RDX undergoes sequential reduction of the nitro groups to form hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine; hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine; and hexahydro-1,3,5-trinitroso-1,3,5-triazine. According to the proposed pathway, this transformation then produces formaldehyde, methanol, hydrazine, and dimethyl hydrazine. Hawari et al. (23) proposed another pathway where ring cleavage occurs before sequential reduction, producing the metabolites methylenedinitramine and bis(hydroxymethyl)nitramine. Although most of the initial strains isolated could only degrade RDX anaerobically (25, 32, 41, 42), several pure strains have subsequently been isolated that aerobically degrade RDX as a nitrogen source. Binks et al. (8) isolated Stenotrophomonas maltophilia PB1 and tentatively identified an RDX metabolite as methylene-N-(hydroxymethyl)-hydroxylamine-N′-(hydroxymethyl)nitroamine. Rhodococcus sp. strain DN22 was isolated by Coleman et al. (12) and was found to produce nitrite as a metabolite. Fournier et al. (19) found that DN22 also produces the metabolites ammonia, nitrous oxide, formaldehyde, carbon dioxide, and a dead-end product with a molecular weight of 119 that was later identified as 4-nitro-2,4-diazabutanal by Bhushan et al. (7). Fournier et al. (19) also proposed a pathway for aerobic RDX degradation in which denitration occurs, followed by spontaneous ring cleavage. Further experiments determined that the enzyme responsible for the degradation of RDX by DN22 is a cytochrome P450 enzyme (7, 13). Rhodococcus rhodochrous strain 11Y, which was isolated by Seth-Smith et al. (33), was found to produce the metabolites nitrite, formate, and formaldehyde. Seth-Smith et al. (33) also identified the gene responsible for RDX degradation as a cytochrome P450-like gene, xplA, and reported that the mechanism of action was initial denitration, followed by spontaneous ring cleavage and mineralization. In this study, we report on the isolation of two bacteria that, unlike all previously described bacteria, degrade RDX aerobically as a sole carbon and nitrogen source. These bacteria are able to mineralize RDX when used as a source of carbon, nitrogen, or carbon and nitrogen. Their unique metabolisms, ease of culture, and rapid growth make these strains excellent models for the basic genomic and proteomic studies required for the development of RDX bioremediation systems and potentially green RDX biosynthetic systems.