Military training activities have resulted in contamination of soil, sediment, and groundwater with nitroaromatic [2,4,6-trinitrotoluene (TNT) and dinitrotoluene (DNT)] and nitramine [hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX)] explosive compounds. Although a significant amount of information regarding the biological degradation of these contaminants has been published in recent years, little information is currently available relating how environmental conditions influence the composition and activity of the explosives-degrading microbial populations, how analysis of microbial communities can be used to predict the fate of explosives, and how site conditions can be manipulated to improve biodegradation activity.

The overall objective of this project was to gain a better understanding of the microbial ecology of explosives compound biodegradation in groundwater. Deciphering which organisms are involved with explosives degradation under various in-situ conditions could lead to better diagnostic and monitoring tools for bioremediation of energetics based on biomarkers, as well as better conceptual and predictive models.

Initially, the scope of this project included most of the major explosive compounds that have been detected in soil and groundwater at military installations. However, the scope was narrowed to RDX in the second half of the project because RDX is the compound of greatest concern in groundwater because of its mobility and recalcitrance. This research coupled chemical analyses to monitor RDX degradation and developed and applied the molecular techniques of polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), and stable isotope probing (SIP) to assess the microbial community. Individual members of the microbial community were identified based on recovered 16S rRNA gene sequences.

Through analysis of samples from laboratory enrichments, model aquifers, and actual bioremediation field demonstrations, the following conclusions can be drawn from the data generated during this project:

• RDX is amenable to biological degradation in groundwater when nutrients are added. Under some circumstances, the RDX can be used as the sole or supplemental nitrogen source, as well as a carbon source. In general, RDX was not readily degraded as the sole nitrogen source under the conditions tested.

• RDX was amenable to degradation in the presence of both complex (cheese whey, yeast extract) and defined (glucose, succinate, ± ammonium) nutrient sources.

• RDX degradation is more labile in groundwater under anoxic/anaerobic (low redox) conditions than under aerobic conditions. Aerobic degradation was not generally observed in groundwater, and aerobic RDX-degraders were not readily isolated or detected using molecular methods.

• RDX was amenable to degradation at typical groundwater temperatures of 15°C.

• Organisms detected in samples actively degrading RDX were generally not closely related to bacterial strains that have been previously described as being able to degrade RDX. The exception would be sequences identified as belonging to genera Clostridium and Pseudomonas, several strains of which have been shown to degrade RDX.

• Several nitrogen-fixing genera not previously associated with explosive compound degradation in general, or RDX degradation in particular, were detected in multiple samples. These genera included Azospira, Azospirillum, and Pleomorphomonas.

• The putative RDX-degrading genes (xenA, xenB, xplA, onr, hydA, nerA) were not detected in any samples with the exception of one of the Picatinny Arsenal model aquifer effluent samples. Given the wide range of samples screened (including many samples that were actively degrading RDX), these results seem to indicate that gene probing methods based on these specific genes are not likely relevant at this time.

• The application of SIP, based on molecular analysis of nucleic acids that become enriched in ¹³C and/or ¹⁵N as organisms degraded stable isotope labeled RDX, confirmed that bacterial genera other than those previously identified as RDX-degrading genera were present in samples exhibiting RDX degradation.

At the conclusion of this research, it appears that no single “biomarker” organism could be associated with RDX degradation in groundwater, at least under the anoxic/anaerobic conditions tested. However, the application of SIP to more directly probe which organisms are interacting with RDX (and/or RDX breakdown products) holds great promise to obtain more specific information and narrow the list of “organisms of interest.” SIP should also lead to insight into which bacterial genera may be best to study further in terms of developing bioremediation technologies for RDX in groundwater. (Project Completed – 2008)