Biodegradation of RDX and HMX under Aerobic Conditions

Several groups have studied aerobic metabolism of RDX and HMX and have demonstrated the potential for these energetic chemicals to be mineralized (Binks et al. 1995; Jones et al. 1995a, b; Greer et al. 1997; Coleman et al. 1998; Tekoah and Abeliovich 1999; Sheremata and Hawari 2000). Binks et al. (1995) showed that Stenotrophomonas maltophilia can degrade RDX, but not HMX when the cyclic nitramine is used as a nitrogen source. Harkins et al. (1999) reported the formation of five nitroso derivatives (two di-isomers, and mono-, tri- and tetra-nitroso derivatives) when HMX was treated in water under aerobic conditions using livestock manure and indigenous microorganisms from a contaminated site. Several aerobic bacteria have been isolated based on their ability to use RDX as a sole nitrogen source incorporating three of the six RDX nitrogen atoms into biomass. Aerobic RDX degraders, such as Stenotrophomonas strain PB1 (Binks et al. 1995), Rhodococcus sp. strain A (Jones et al. 1995b), Rhodococcus sp. strain DN22 (Coleman et al. 1998), and Rhodococcus rhodochrous strain 11Y (Seth-Smith et al. 2002) grow on RDX using as sole nitrogen source. Van Aken et al. (2004) have also reported that aerobic Methylobacterium sp. strain BJ001 was able to metabolize RDX and HMX under aerobic conditions.

Products from biodegradation of cyclic nitramine explosives under aerobic conditions are poorly understood, particularly ring cleavage products (Binks et al. 1995; Hawari 2000). Jones et al. (1995b) 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. Binks et al. (1995) reported the formation of two products from the degradation of RDX with Stenotrophomonas maltophilia. One product was identified as methylene-N-(hydroxymethyl)-hydroxylamine-N-(hydroxymethyl) nitramine and the second as the chloride salt of methylene-N-nitroamino-N-acetoxyammonium chloride. Coleman et al. (1998) reported the isolation and characterization of another Rhodococcus sp. strain DN22, which efficiently degrades RDX with the production of NO2-. No other products have been identified during RDX biodegradation with DN22. Several authors (Binks et al. 1995; Jones et al. 1995b; Coleman et al. 1998; Coleman and Duxbury 1999; Tekoah and Abeliovich 1999) did not observe any of the nitroso derivatives under aerobic conditions. Coleman and Duxbury (1999) suggested the involvement of cytochrome P-450 in the degradation of RDX by Rhodococcus sp. Strain DN22. No products were identified other than nitrite. Tekoah and Abeliovich (1999) also reported the involvement of cytochrome P-450 in the degradation of RDX by Rhodococcus sp. strains YH11. However, the study did not identify any metabolites other than nitrite. Harkins (1998) employed mixed cultures from horse manure to degrade RDX and HMX separately using dextrose and alfalfa as supplementary carbon sources. Gram-negative bacteria (Alcaligenes sp., Hydrogen-ophaga flava and Xanthomonas oryzae) and several facultative bacteria (Escherichia coli, Kingella kingae and Capnocytophaga canimorus) were tested. The nitramines RDX and HMX disappeared in 9 days and produced the

2e"

2e"

2e"

1NO-HMX

2NO-HMX

3NO-HMX

4NO-HMX

Path A: Reduction of HMX to nitroso derivatives before ring cleavage (McCormick et al, 1981)

MDNA + BHNA

N2O + HCHO

Path B: Direct enzymatic cleavage of HMX (Hawari et al, 2001)

1e"

NO2-

NO2-

Path C: Denitration of HMX via xanthine oxidase enzyme (Bhushan et al, 2003a)

Fig. 9.3 Proposed biodégradation pathways for HMX. 1NO-HMX octahydro-1-nitroso-3,5,7-trinitro-1,3,5,7-tetrazocine, 2NO-HMX octahydro-1,3-dinitroso-5,7-dinitro-1,3,5,7-tetrazo-cine or octahydro-1,5-dinitroso-3,7-dinitro-1,3,5,7-tetrazocine, 3NO-HMX octahydro-1,3, 5-trinitroso-7-nitro-1,3,5,7-tetrazocine, 4NO-HMX octahydro-1,3,5,7-tetranitroso-1,3,5,7-tetrazo-cine, MDNA methylenedinitramine, BHNA bis-(hydroxymethyl)nitramine, NDAB 4-nitro-2,4-diazabutanal

> HCOOH

Path C: Denitration of HMX via xanthine oxidase enzyme (Bhushan et al, 2003a)

Fig. 9.3 Proposed biodégradation pathways for HMX. 1NO-HMX octahydro-1-nitroso-3,5,7-trinitro-1,3,5,7-tetrazocine, 2NO-HMX octahydro-1,3-dinitroso-5,7-dinitro-1,3,5,7-tetrazo-cine or octahydro-1,5-dinitroso-3,7-dinitro-1,3,5,7-tetrazocine, 3NO-HMX octahydro-1,3, 5-trinitroso-7-nitro-1,3,5,7-tetrazocine, 4NO-HMX octahydro-1,3,5,7-tetranitroso-1,3,5,7-tetrazo-cine, MDNA methylenedinitramine, BHNA bis-(hydroxymethyl)nitramine, NDAB 4-nitro-2,4-diazabutanal corresponding nitroso derivatives. Harkins (1998) concluded that the nitramine explosives were neither used as a carbon source nor as a nitrogen source. Fournier et al. (2002) detected nitrite, nitrous oxide, ammonia, formaldehyde and CO2 in addition to a dead-end metabolite (C2H5N3O3), possibly a structural isomer of formyl-methylamino-nitroamine. The initial denitration of the RDX molecule by Rhodococci requires the involvement of a cytochrome P-450 (Coleman et al. 2002; Seth-Smith et al. 2002; Bhushan et al. 2003b). Denitration is followed by ring cleavage and the production of nitrous oxide (N2O), ammonia (NH3), formaldehyde (HCHO), and a dead-end product identified as 4-nitro-2,4-diazabutanal (NDAB) (Fournier et al. 2004a). Hawari et al. (2000b) assumed that following any initial enzymatic attack on RDX leading to the cleavage of any of the bonds in RDX (inner NOC or external NONO2 and COH bond), the resulting intermediate becomes very unstable. Once an external bond in RDX is cleaved, some of the inner CON bonds would spontaneously decompose to produce N2, N2O, HCHO, and HCOOH. The fate of the resulting intermediates will be determined by competitive microbial and chemical processes. Most of the products are thermally unstable and undergo fast hydrolytic cleavage in water.

Some potential pathways have been postulated for the biodegradation of cyclic nitramines under aerobic conditions based on the information of the product distribution and the enzymes involved. Reduction of the nitro groups in RDX by nitroreductases can take place either via two electron transfer process to produce nitroso derivatives or via a one electron transfer process to produce the free anion radical of RDX (or HMX). It has also been reported that RDX nitro anion radical reverts back to the nitro compound in the presence of oxygen (McCalla et al. 1970; Peterson et al. 1979). The denitration of the anion radical to give the corresponding RDX (or HMX) radical is also proposed. This later transient intermediate would then undergo rapid ring cleavage to produce the end product. The absence of nitroso RDX derivatives as degradation products in several studies (Binks et al. 1995; Coleman et al. 1998) supports the occurrence of a denitration mechanism. Furthermore, the superoxide anion radical, O2-, generated from the reaction of oxygen with RDX anion radical is extremely reactive and its presence may contribute to the degradation of the cyclic nitramine. Several studies identified nitrite as the metabolite during biodegradation of RDX under aerobic conditions (Binks et al. 1995; Coleman et al. 1998). However, as the nitrite did not accumulate, it was suspected that the microorganisms used this ion as their nitrogen source. Jones et al. (1995a) showed that the disappearance of RDX initially produced nitrite that subsequently disappeared with the detection of traces of ammonium ions. Coleman et al. (1998) isolated and identified Rhodococcus sp. Strain D22 from an RDX-contaminated site that used nitrite liberated from RDX as its sole nitrogen source. The liberated nitrite was transformed to ammonium enzymatically prior to its use by the microorganism. In contrast, the presence of ammonium (NH4+) has been shown to inhibit RDX mineralization (Yang et al. 1983; Binks et al. 1995; Coleman et al. 1998). Ammonium competes with RDX as a nitrogen source, and thus RDX degradation is repressed. Fournier et al. (2002) have proposed a pathway for aerobic RDX degradation by analogy to the abiotic alkaline hydrolysis of RDX, in which initial denitration yields unstable cyclohexenyl derivatives (Hoffsommer et al. 1977).

Denitration of RDX by aerobic bacteria involves two one-electron transfer steps and releases two nitro groups before ring cleavage (Fig. 9.2, Path G). This results in the formation of one molecule of 4-nitro-2,4-diazabutanal (NDAB) along with nitrous oxide, ammonium, formaldehyde, and carbon dioxide (Fournier et al. 2002; Bhushan et al. 2003b). RDX appears to be mineralized according to this mechanism and the nitrite generated is utilized as a nitrogen source for growth by the aerobic bacteria Rhodococcus rhodochrous strain 11Y (Seth-Smith et al. 2002), Rhodococcus sp. strain DN22 (Coleman et al. 1998; Fournier et al. 2002), Williamsia sp. strain KTR4 (Thompson et al. 2005), and Gordonia sp. strain KTR9 (Thompson et al. 2005). The NDAB produced is apparently not further metabolized by strains 11Y, DN22, KTR4, or KTR9 and hence gets accumulated (Fournier et al. 2002, 2004a; Thompson et al. 2005). However, it has recently been shown that NDAB may not be an environmentally recalcitrant end-product, as it can be biodegraded by a methylotrophic bacterium producing nitrous oxide and carbon dioxide (Fournier et al. 2004a, 2005).

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