Explosives have contaminated many military sites in North America and Europe. The explosive RDX is a particularly problematic groundwater contaminant because of its relatively high solubility, recalcitrance, toxicity, and frequency of use. The most cost-effective remediation approach for RDX is likely to be bioremediation; however, because little is known about the microorganisms involved, it is not yet possible to predict the effectiveness and reliability of this cleanup strategy.

This project was conducted in two phases. The objective of Phase I was to identify the microorganisms responsible for the biodegradation of RDX in complex, mixed culture samples through the application of stable isotope probing. This effort was completed in 2010. The objective of Phase II was to identify the microorganisms and genes responsible for the biodegradation of RDX in mixed culture samples through the application of stable isotope probing (SIP), quantitative PCR (qPCR) and high throughput sequencing (Illumina MiSeq). This research specifically targeted RDX biodegradation in mixed community samples, as these microbial communities are more representative of those at contaminated sites.

Phase I efforts involved the screening of ten soils for RDX degradation and the development and application of DNA stable isotope probing to identify RDX degraders. This approach had two distinct advantages: i) RDX degradation could be investigated in mixed cultures and environmental samples (more typical of contaminated site conditions) and ii) only active organisms were targeted.

Phase II efforts involved four major tasks, the first of which was a literature review that identified the dominant microorganisms linked to RDX biodegradation in pure and mixed culture samples. Researchers then examined the RDX degrading communities in four different soil slurries. High throughput sequencing was used to determine which phylotypes experienced an increase in relative abundance following RDX degradation. For this, total genomic DNA was sequenced from the 1) initial soils, 2) soil slurry microcosms following RDX degradation, and 3) control soil slurry microcosms without RDX addition.

Researchers then examined the microorganisms involved in RDX biodegradation from surface soils collected at a detonation area at a Naval Base. Microbial communities were compared between the initial sample, samples following RDX degradation, and controls not amended with RDX to determine which phylotypes increased in abundance following RDX degradation. The effect of glucose on these communities also was examined as well as performing stable isotope probing (SIP) using labeled (¹³C₃, ¹⁵N₃ – ring) RDX.

Lastly, researchers investigated the microorganisms and functional genes (xenA, xenB and xplA) linked to RDX biodegradation in microcosms composed of sediment or groundwater from two Navy sites using qPCR and high throughput sequencing. Experiments were completed using sediment samples from three different depths (5 to 30 feet) collected at two wells located on one U.S. Navy site. In addition, the groundwater upstream and downstream of an emulsified oil biobarrier was examined from another U.S. Navy site. Further, for the groundwater experiments, the effect of glucose addition was explored. 

In Phase I, RDX degradation activity was noted in six of ten soils and degradation occurred only under anaerobic conditions. The stable isotope probing experiments involved exposing soil microcosms to labeled RDX, DNA extraction, DNA ultracentrifugation (to separate the labeled nucleic acid from the unlabeled background nucleic acid), terminal restriction fragment length polymorphism (TRFLP) and 16S rRNA gene sequencing to identify the organisms responsible for label uptake from RDX. Partial 16S rRNA gene sequencing indicated the organisms responsible belonged to either the Sphingobacteria or the Acidobacteria. These bacteria have had no previous links to RDX degradation, indicating the discovery of novel RDX degraders. In summary, the proof-of-concept that stable isotope probing could be used to identify in situ RDX degraders in complex, mixed culture samples was proven successful. The results of this study can be found in the Phase I Final Report.

In Phase II, the literature review indicated that from the phyla with known RDX degrading isolates, Firmicutes and Proteobacteria (particularly Gammaproteobacteria) were the most dominant microorganisms in many contaminated site derived samples. There was found to be an increase in Brevundimonas and/or unclassified Bacillaceae 1 in the four soils studied during RDX degradation. Although isolates of the family Bacillaceae 1 have previously been linked to RDX degradation, isolates of the genus Brevundimonas have not been previously associated with RDX degradation. Overall, the data suggested these two phylotypes have key roles in RDX degradation in these soil communities. The focus on surface soils from a denotation area revealed that several phylotypes were more enriched during RDX degradation. This trend was strong for unclassified Pseudomonadeae, Comamonas and Acinetobacter. Rhodococcus, a known RDX degrader, also increased in abundance following RDX degradation. SIP indicated that unclassified Pseudomonadaceae was the most abundant phylotype in the heavy fractions. In the glucose amended heavy fractions, Comamonas and Anaeromxyobacter also were present. For the sediment experiments, the most enriched phylotypes during RDX degradation varied over time, by depth and well locations. However, several trends were noted, including the enrichment of Pseudomonas, Rhodococcus, Arthrobacter, and Sporolactobacillus in the sediment microcosms. For the groundwater experiments, Pseudomonas, unclassified Rhodocyclaceae, Sphingomonas, and Rhodococcus also were highly abundant during RDX degradation. Both xplA and xenA increased during RDX degradation for many treatments. In a limited number of microcosms, xenB gene copies increased. Phylotype data were correlated with functional gene data to highlight potentially important biomarkers for RDX biodegradation at these two Navy sites. The results of this study can be found in the Phase II Final Report.

Most data on RDX biodegradation has originated from pure cultures or enrichments, with little information of how these organisms act in environmental samples or in situ. This lack of knowledge makes it difficult to determine if natural attenuation is an appropriate remediation approach for RDX. The innovative molecular methods applied in this project could provide this much-needed information. Several key microorganisms were identified as being associated with RDX removal in these mixed communities. The phylogenetic and functional biomarkers identified can be used to determine the biological potential for RDX degradation across DoD sites in order to facilitate the establishment of more cost-effective and efficient RDX remedial plans, expediting the cleanup of RDX-contaminated DoD sites.