Biodegradation of the cyclic nitramine explosives RDX , HMX , and CL - 20 Fiona

Cyclic nitramine explosives are synthesized globally mainly as military munitions, and their use has resulted in environmental contamination. Several biodegradation pathways have been proposed, and these are based mainly on end-product characterization because many of the metabolic intennediates are hypothetical and unstable in water. Biodegradation mechanisms for cyclic nitramines include (a) f(mnation of a nitramine free radical and loss of nitro functional groups, (b) reduction of nitro functional groups, (c) direct enzymatic cleavage, (d) a-hydroxylation, or (e) hydride ion transfer. Pathway intem1ediates spontaneously decompose in water producing nitrite, nitrous oxide, formaldehyde, or formic acid as common endproducts. In vitro enzyme and functional gene expression studies have implicated a limited number of enzymes/genes involved in cyclic nitramine catabolism. Advances in molecular biology methods such as high-throughput DNA sequencing, microarray analysis. and nucleic acid sample preparation are providing access to biochemical and genetic information on cultivable and uncultivable microorganisms. This infom1ation can provide the knowledge base for rationa I engineering of bioremediation strategies, biosensor development, environmental monitoring, and green biosynthesis of explosives. This paper reviews recent developments on the biodegradation of cyclic nitramines and the potential of genomics to identify novel functional genes of explosive metabolism. F. H. Crocker (v·1) · K. J. lndest ·H. L. Fredrickson U.S. Army Engineer Research and Development Center. Environmental Laboratory, 3909 Halls Ferry Road, Vicksburg, MS 39180, USA e-mail: Fiona.H.Crocker(£1,erdc.usace.army.mil ~Springer Introduction Surveys of the extent and distribution of explosive contamination on military ranges in the United States (U.S.) and Canada have shown that explosive residues can be widely and heterogeneously dispersed in soil. Generally, the concentrations of explosives are relatively low (I 00,000 iJ.g/kg) and large chunks of explosive fonnulations can be found (Hewitt 2002; Jenkins et a!. 1998, 200 I; Pennington et a!. 200 I, 2005; Walsh et al. 2001 ). The extent of contamination is not well known outside of North America because only a limited number of site characterizations have been completed at training or manufacturing facilities in Germany, Australia, and the United Kingdom (Spain 2000). A recent survey of the A.lvdalen Shooting Range in Sweden found generally low levels of explosive and propellant residues in soil samples, with a few samples exhibiting concentrations as high as 4,200 f.l.g/kg for octahydro1 ,3.5,7-tetranitro-1,3,5,7-tetrazocine (HMX), 59,000 f.l.g/kg for 2,4,6-trinitrotoluene (TNT), and 6,500 f.l.g/kg for hexahydro-1.3,5-trinitro-1 ,3,5-triazine (RDX) (Wingfors et a!. 2006). Explosive residues have the potential to move into surface water and groundwater (Clausen et al. 2004) and to impact various ecological and human receptors. The U.S. Environmental Protection Agency (2004) has listed the cyclic nitramine RDX as a priority pollutant and HMX as a contaminant of concern. Many microorganisms have been shown to transfom1 RDX and HMX, including aerobic bacteria. aerobic fungi, and anaerobic bacteria (Table 1 ). Thus, biological remediation is favored as a cost-effective ex situ or in situ treatment method for the removal of these compounds from contaminated environments. 11111111111111111111111111111111111 14680 Appl Microbiol Riotechnol (2006) 73:274-290 275 Table I Cyclic nitramine-degrading bacteria and possible biochemical mechanisms for degradation Strain RDX'' IIMXh CL-20" Mcchanismd Reference Serralill n1arcesccns +" NT NT A Young et al. !9l)7 Entcmhactcr cloacae NT NT A Kitts ct al. 2000 Clostridium sp. I[;\ W-G3 NT NT A, F Zhao et al. 2003b Clostridium a<·etohutl·licum NT NT c Zhang and Hughes 21l03 Dcsulfovihrio sp. ITAW-ES2 NT NT A. F Zhao et al. ?OOJb ;lcctohar"/crium malicum + NT NT E. F Adrian and Arnett 2004 ;I cctohacterium pal/1(/osum NT NT ND Sherburne et al. :2005 Shnmnc/la scdiminis IIAW-EBJ NT A, F Zhao ct al. 2004c. 21l05 Shnmnc/la lwlifilxelllis I-lA W-EB4 NT A. F Zhao et al. 2004c, :2001> Shewancl/,1 sp. HAW EBI NT A, F Zhao ct al. 2004c Shemmdla sp. I lAW EB2 NT A. F Zhao ct al. 2004c Shemtndla sp. I!AW-EB'\ NT A. F Zhao et al. 2004c Srenolrophomonas maltophila PHI NT NT ND Rinks ct al. I'N.'i Rhodococcus sp. DN22 NT NT G Coleman ct al. 19% Rhodococcus rhodochrous sp. IIY NT NT c _T Seth-Sm1th et al. 2002 ff/"1/iamsia sp. KTR4 NT Ci.ND Thompson et al. 2005 <Iordonia sp. KTR9 NT G. ND Thompson ct al. 20()5 Metln•lohacrcriflm sp. JSI7H NT NT A Fournier et al. 2005 liJorJ!.andla morganii B2 NT A, II Kitts et al. 1994 Citmhacterfieundii NS2 NT A.ND Kitts ct al. 11)94 Providcncia r<'llgcri B I NT A. ND Kitts et al. 1994 Klchsidla pncw1wniac SCZI NT A. E, F, J Zhao ct al. 2002, 2004b Clostridium hiflc:rmentans HAW-I + + NT A, F, J Zhao et al. 2003a,b. 2o04h Clostridium sp. HAW-G4 NT A, F, H, J Zhao et al. 2003h. 2004b Clostridium sp. IIAW-E3 NT A, F, II, J Zhao et al. 200:\b, 20(l4b Clostridium sp. HAW-HCI + NT A. F. H. J Zhao et al. 200~b. 2004b Clostridium sp. IIAW-EB 17 NT A. F. II Zhao et al. :~004c Desulfm·ihrio sp. I !A W-EB I X NT A. F. II Zhao ct al. 2004c Fusobacteria isolate !IAW-EB21 NT A, F, H. J Zhao et al. 2004h.c Methrlohactcriwn sp. BJOO I NT A. II Van Akcn ct al. 2004 J\1cth<,1ohac/crium exlriiYfUl!IIS NT A. ND Van Akcn et al. ::'004 lvfctln1ohacteritllfl organophilum NT A. ND Van Aken et al. 2004 AicthvlrJhacterill/11 rh<xlcsiarlll//1 NT A. ND Van Akcn ct al. 2004 Gordoffitl sp. KTC 13 NT -f ND Crocker et al.. unpublished Pscudomon"s sp. FA I NT NT K Rhushan et al. 20fn Agmhaclcrium sp. JS 71 ND Trott ct al. 2003 /rpcr lactcus NT NT + ND Foumier ct al. 20[)(, Clostridium sp. EDB2 F. J, K. L, M Bhushan ct al. 2il04c. 2005a Phai/Crll<'haetc' chrl'S<IS]JIII"ium ND. H. J, K Shercmata and Hawari 2000; Fournier ct al. 2004b, 2006 "RDX hcxahvdro-1.3.5-trinitro1.3,5-triazine h I I MX octahydro-1 ,3.5. 7 -tctranitro-1.3.5, 7 -tctruzocine '"CL-:20 2,4.6,X, I 0, 12-hcxanitro-2.4.6,8, I 0, 12-hexaazaisowurtzitanc d Mechanisms are described using the same letter codes in Figs. 2 • . \. and 4. which are used to desc1ibe the different pathways of biodegradation for RDX, HMX, and CL-20 ,. + • Positive fi.)r degradation of the explosive; . does not degrade the explosive; NT, not tested; ND, not detennined due to lack of infonnation on metabolites The biochemical details of the metabolic pathways involved in the degradation of the cyclic nitramines RDX and H MX have been reviewed (Hawari et aL 2000a). Since then. research into the biological fate of a new cyclic nitramine explosive 2,4,6,8, 1 0, 12-hexanitro-2,4,6.8, I 0,12hexaazaisowmizitane (CL-20) has been initiated. Unlike RDX and HMX. CL-20 is composed of a rigid and highly strained cage limned from two five-member rings and one six-member ring (Fig. l ). It also differs from RDX and HMX in that it has three carbon--<:arbon (CC) bonds, and the repeating structural unit is CH--N~N02 instead of CH2~ N~N02 . As with RDX and HMX, biological transformation of CL-20 has been repotied in soils (Crocker et al. 2005; Strigul et aL 2006; Trott et aL 2003) and by pure cultures of bacteria and fungi (Table I). However, CL-20 was more susceptible to abiotic transfom1ation than RDX and HMX