Soluble toxic compounds used in explosives are widely recognized as a significant barrier to attaining sustainable military training ranges. Movement of toxic munitions compounds, such as RDX, in groundwater and surface waters outside the boundaries of training ranges has the potential to threaten local water supplies and ecosystems. A sustainable solution to this problem must be inexpensive and self-maintaining. Microbial communities are known to use RDX as a source of nitrogen, but their natural activities in many soil and groundwater environments are not sufficient to remove the toxic compounds to levels required by regulation. Because rhizosphere microbial communities have enhanced populations and activity compared to surrounding soils and since specific microbial populations are associated with particular plant communities, manipulation of the plant community offers a way to enhance microbial degradation of RDX with a minimum of expense and maintenance.

The overall objectives of this project were to identify RDX-degrading bacteria in plant rhizospheres, discover the factors that control their abundance and diversity, and develop probes that can be used in the field to detect them. Carbon and nitrogen availability were anticipated to be important controlling variables in determining RDX persistence in soil, implying that soil bacteria do not effectively degrade energetic materials in situ unless they are associated with a carbon-rich environment that selects for populations active in either direct or cometabolic degradation of RDX. It was anticipated that activity of RDX-degrading bacteria would be favored by specific carbon sources in the root exudate spectrum, allowing the rhizosphere to be manipulated to enhance populations of RDX-degrading bacteria.

The following hypotheses were tested:

Hypothesis 1: Soil bacteria that can degrade RDX are carbon limited. Therefore, bacterial RDX degradation is enhanced by carbon compounds exuded by roots in the rhizosphere.

Hypothesis 2: The rhizosphere bacteria community is nitrogen limited. RDX serves as a nitrogen source for rhizosphere bacteria.

Hypothesis 3: The type of carbon compound in root exudate influences RDX degradation.

Root exudates of Arabidopsis mutant lines were characterized.  The rhizospheres of Arabidopsis and slender wheatgrass with R. rhodochrous and P. fluorescens were inoculated, and the survival of the inoculate strains was determined.   The RDX degrading strain, the xplA gene, was expressed in P. fluorescens. Degradation of RDX by defined cultures of sterile alfalfa and transformed P. fluorescens was assayed.

Wheatgrass grown in RDX degrading soil from training ranges was exposed to ¹⁴C-labeled CO₂ and soil samples were analyzed for the identity of rhizosphere bacteria grown on plant exudates using rRNA separation techniques.

The xplA gene was localized on extrachromosomal elements in known RDX degrading bacteria. Stable isotope probing with ¹⁵N-labeled RDX was used to identify bacteria in training range soils that assimilated nitrogen from RDX. A tandem real-time polymerase chain reaction terminal restriction fragment length polymorphism (qPCR-TRFLP) protocol was developed that improved SIP resolution and allowed the degree of label incorporation to be determined for individual members of the bacterial population. This method was applied to soils obtained from the Eglin Air Force Base training range.

High performance liquid chromatography (HPLC) profiles of hydroponic media of Arabidopsis mutants varied significantly between replicates, which complicated interpretation of differences between mutant lines. Treatment of the roots during extraction accounted for some of the variation, but not all. Culture conditions, especially the presence or absence of sucrose in the medium, or hydroponic, aeroponic, and vermiculite culture produced highly different root exudate profiles.

R. rhodochrous 11Y was inoculated into cultures of Arabidopsis, wheatgrass, and alfalfa, but it did not efficiently colonize the rhizospheres and RDX degradation in these rhizospheres was not enhanced. Two efficient root colonizer strains of Pseudomonas fluorescens were transformed with xplA under inducible control. When these transformed strains were introduced into soils, RDX removal increased compared to soils inoculated with P. fluorescens without xplA. Plants grown in the soil inoculated with xplA-transformed P. fluorescens contained less RDX. Transformed P. fluorescens persisted in alfalfa rhizospheres even as other bacteria colonized the soil, but the presence of RDX did not enhance persistence of the transformed species. RDX removal was 30% higher in soils inoculated with transformed P. fluorescens compared with controls. These transformed strains lacked xplB.

Efforts to label plant root exudates with ¹⁴C and to use single-stranded conformational polymorphism (SSCP) gel electrophoresis of RNA extracted from rhizosphere communities failed to detect labeled RNA. Group-specific capture probes for bacterial RNA and improved SSCP and RNA extraction methods were developed.

Pulse field gel analysis localized the RDX-degrading gene xplA to extrachromosomal elements in Rhodococcus and a distantly related Microbacterium. The R. rhodochrous 11Y and Microbacterium plasmid sequences in the vicinity of xplB and xplA were nearly identical and contained flanking IS elements, suggesting that xplA/B was transferred by horizontal gene transfer.

Because ¹⁵N SIP results in inadequate separation of labeled bands to clearly separate DNA variations due to C+G contents, a tandem qPCR-TRFLP protocol that improves resolution by quantifying labeling of the different taxonomic groups independent of their C+G content was developed.  Separation of ¹⁵N-labeled DNA extracted from low and high G+C bacterial isolates and from soil microcosms amended with known amounts of genomic DNA from bacterial isolates was verified.

Aerobic RDX degradation in surface soils extracted from a highly used target area of Eglin Air Force Base bombing range was measured. RDX-degradation activity was spatially heterogeneous and dependent upon the addition of exogenous carbon sources to the soils. Stable isotope probing (SIP) analysis of soils exposed to fully labeled (ring and nitro) ¹⁵N-RDX in microcosms revealed several bacteria species that were fully labeled with ¹⁵N-labeled DNA during and following RDX degradation, including xplA-bearing organisms. A Rhodococcus species was the most prominent genus in the RDX-degrading microcosms and was completely labeled with ¹⁵N-nitrogen from the RDX. Other highly labeled species identified in the gradient included Mesorhizobium sp., Variovorax sp., Rhizobium sp., and unspecified Proteobacteria. A Rhodococcus sp.(EG2B) and a Williamsia sp. capable of degrading RDX were isolated from these soils and each possessed the genetic element encompassing the xplB and xplA genes identified in the xplA-bearing strains R. rhodochrous 11Y and Microbacterium sp. MA1. The presence of these genes indicate that xplA/B can persist in military range soils and would be a candidate genetic biomarker indicating the potential for RDX degradation.

This work advances fundamental understanding of the distribution of xplA/B in soil microbial communities. The findings support the prevalence of Rhodococcus for RDX degradation in training range soils, while suggesting that RDX degradation may also occur as the result of Gram negative bacterial activity, resulting in assimilation of nitrogen derived from RDX.

An important observation was that RDX degradation potential and the occurrence of xplA was highly heterogeneous in samples taken from the target area at the Eglin Air Force Base training range.  More than half of the samples were unable to degrade RDX even with added carbon, and xplA was not detected in four of the soils. Munitions particulates deposited on soils that lack bacteria able to express XplA are likely to leach RDX into the subsurface.

The second observation of importance is that soil samples incubated without added carbon were unable to degrade RDX. Thus the presence of both xplA or other RDX biological degradation mechanism and carbon substrates were necessary for RDX degradation in Eglin Air Force Base training range soil samples. This observation is consistent with the role of xplA as a nitrogen releasing mechanism in bacteria isolates growing on RDX.

Results suggest that bioaugmentation (with xplA-bearing species) and biostimulation (with exogenous carbon sources) may be useful methods to increase RDX degradative potential in training range soils in target areas. Bioaugmentation and biostimulation could be accomplished using ground machinery, manned or remote controlled, by aerial application, or delivered on target ballistically.