The sustainability of Department of Defense (DoD) training ranges is threatened by mobile explosive contaminants, such as RDX, which have migrated away from source areas and now contaminate surface and subsurface soils at relatively low but potentially toxic levels. In situ bioremediation is an attractive remedial action for these contaminated sites, but methods to assess the potential for in situ bioremediation of RDX have been generally lacking. Molecular biological tools that can detect the genes responsible for nitramine biodegradation would be valuable tools in demonstrating the capacity for microbial biodegradation of nitramines at contaminated sites. However, such molecular tools require a detailed understanding of the microorganisms, biochemical pathways, and genes used to degrade RDX in the environment. 

This project was focused on understanding the genetics, physiology, and biochemistry of RDX biodegradation in order to develop probes that could predict the potential for RDX biodegradation or monitor the progress of RDX bioremediation in the field. Specific objectives were to (1) elucidate the RDX degradation pathways in aerobic and anaerobic RDX-degrading bacteria; (2) design and develop molecular tools to identify genes responsible for RDX biodegradation; and (3) correlate the response of biomarker(s) to concentrations of RDX and/or rates of RDX degradation.

A combination of analytical chemistry, physiological, proteomic and genomic approaches were organized into five tasks. The first task determined biodegradation pathways, the second and third tasks identified RDX-related genes and proteins, and the final two tasks developed and evaluated markers of RDX biodegradation.

The bacterial strains used for these tasks were Gordonia sp. KTR9 and Shewanella oneidensis MR-1 as representative aerobic and anaerobic RDX-degrading bacteria. Each of these organisms uses a distinct mechanism to degrade RDX. Slightly different strategies were used for each strain based on the available genomic resources at the time. Two-dimensional (2-D) protein electrophoresis and mass spectrometry (MS), and genome annotation were initially used to characterize and identify RDX-related genes and proteins in strain KTR9. Later studies with KTR9 also included differential gene expression (transcriptomic) analysis using DNA microarrays developed from the annotated genome. Differential gene expression analysis also was conducted for strain MR-1 using commercially available microarrays and the publicly available annotated genome. These studies identified gene and protein targets that were considered as potential molecular biomarkers. A combination of genetic mutation/deletion studies, physiological, and proteomics data was used to characterize putative functions of gene targets with respect to RDX degradation and regulation of RDX genes. An existing gene target for aerobic RDX biodegradation, xplA which encodes for a cytochrome P450 enzyme, was used to develop and optimize a TaqMan quantitative polymerase chain reaction (qPCR) set of primers and hybridization probes. The ability of this TaqMan qPCR assay to detect the presence of RDX-degrading bacterial genes was validated with pure cultures and soil and groundwater samples.

Genome annotation and functional characterization of the plasmid pGKT2 in KTR9 revealed that xplA gene is both necessary and sufficient for RDX degradation. It was demonstrated that pGKT2 and its ability to degrade RDX could be disseminated via horizontal gene transfer (HGT) to other actinomycetes. Proteomic analysis of KTR9 grown on RDX as a nitrogen source identified multiple protein targets, including XplA, that were up-regulated. Physiological studies of KTR9 grown on RDX and competing inorganic nitrogen sources, in parallel with PCR analysis of xplAB, showed that ammonium sulfate (greater than 3 mM) and sodium nitrite (4 mM) have significant inhibitory effects on RDX degradation and xplAB expression. Transcriptome analysis of KTR9 grown on RDX suggested that the transcriptome response to RDX is likely a reaction to nitrogen limitation rather than a response to RDX. Of the gene targets identified as biomarker candidates, only xplA met criteria as a suitable biomarker. Validation of an xplA TaqMan quantitative polymerase chain reaction (qPCR) assay with field collected samples and microcosm incubations suggested that xplA may have value as a qualitative biomarker. The demonstration that xplA can be disseminated via HGT is significant, for bioaugmentation efforts utilizing xplA-containing bacteria may have the effect of seeding the indigenous microbial population with the ability to degrade RDX.

Shewanella oneidensis MR-1 was shown to efficiently degrade RDX anaerobically via two initial routes: (i) sequential N-NO₂ reduction to the corresponding nitroso (N-NO) derivatives; and (ii) mono-denitration followed by ring cleavage. Four RDX-defective mutants that had insertions in genes encoding for chorismate synthase, naphthoate synthase (MenB), and a cytochrome c, CymA were isolated. Characterization of RDX degradation in MenB and CymA mutants implied the participation of CymA in the nitrosation route, but could not definitively suggest a role for MenB. Network analysis of transcriptome data suggests that c-type cytochromes play a role in RDX transformation. However, CymA was not a suitable biomarker candidate due to its broad substrate specificity with other electron acceptors. While a functional gene was not identified in strain MR-1, a phylogenetic gene marker targeting Shewanella may have some benefit since a number of species from this genus transform RDX.

The xplA gene biomarker examined in this project is highly specific for the aerobic biodegradation pathway of RDX. The biomarker is found within a small group of bacteria belonging to the order Actinomycetales. These bacteria and the genetic marker have been found world-wide in aerobic surface soils and aerobic groundwater. This is significant to the application of the biomarker in prescribing an aerobic treatment scheme for shallow aerobic groundwater, surface waters and soils. Compared to anaerobic treatment schemes, aerobic stimulation of RDX biodegradation is expected to require a smaller mass of electron donor, be less expensive, and not generate undesirable end-products, such as methane, sulfides, and nitroso-RDX metabolites.

The fundamental information gained in this study suggests that xplA-mediated aerobic denitration of RDX may be subjected to inhibitory effects in response to nitrogen availability. This has implications for the role of xplA as a biomarker of RDX degradation, as it will also be necessary for bioremediation site managers to measure soil and groundwater concentrations for ammonium and nitrate. Ammonium concentrations in excess of 125 mg/L and nitrate concentrations in excess of 250 mg/L inhibited the rate of RDX degradation for strain KTR9. At these inorganic nitrogen concentrations, the extent of RDX degradation after 68 h was inhibited by approximately 60% and 15%, respectively. Concentrations between 100 and 1000 mg/L of ammonium and nitrate have been reported to inhibit RDX degradation for other aerobic RDX-degrading Rhodococcus species.

The developed qPCR molecular tools have the potential to be used by remediation specialists for site characterization, treatment recommendations, and for evaluation and optimization of the treatment process. The use of phylogenetic gene markers can provide information on the types and abundance of microorganisms associated with biodegradation of RDX. Catabolic gene markers can provide evidence for the potential for one of the specific pathways of RDX biodegradation at a site. These biomarkers could be used to complement traditional methods of determining the fate of RDX in the environment.