Residual munition constituents (MCs) generated from live fire training exercises persist in soil and can migrate to groundwater and surface waters. Some legacy MCs (e.g., RDX) and newer insensitive explosives (insensitive high explosives [IHEs], e.g. 3-nitro-1,2,4-triazole-5-one [NTO]) are mobile and exhibit significant migration risks. Microbial biodegradation is an important route affecting the fate and attenuation of explosives. Recently, cultivation independent genomics techniques have inferred that the indigenous explosive-degrading community extends to taxa that were previously unknown to have roles in RDX and NTO biotransformation. In addition, known RDX-degradative genes are often not detected in groundwater or soil, which suggests that bacteria with these genes are rare at these sites or the conditions required to stimulate these bacteria are still poorly known. Therefore, bioaugmentation (i.e., inoculation with explosive-degrading bacteria) may be necessary to promote in situ biodegradation of MCs. These recently identified taxa (known only by their 16S rRNA sequences) represent an unexplored resource that could reveal novel and highly-efficient enzymes for explosives biotransformation in soils.

The objectives of this project are to: (1) determine the ecological role of the inferred taxa in RDX and NTO biotransformation in range soils; and (2) quantify contaminant flux reduction achieved through bioaugmentation of range soils with an explosive-degrading bacterial consortium. The central hypothesis is that range soils contain novel explosives-degrading microbial species, which can be isolated and used to develop bioaugmentation consortia as a sustainable range technology to accelerate explosives degradation, thus minimizing migration to ground and surface waters. 

Project objectives will be accomplished by performing the following four tasks:

• Isolate novel RDX- and NTO-degrading bacterial strains that were identified in metagenomics studies and which are known only by a 16S rRNA sequence. Based on the assumption that the natural environment provides all of the nutrients needed for growth, novel diffusion chamber arrays will be used for the in situ cultivation of bacteria that are recalcitrant to conventional isolation approaches;
• Optimize media for the cultivation and characterization of these new explosive-degrading isolates in order to develop an explosive-degrading consortium;
• Optimize techniques to deliver the soil bioaugmentation consortium to range soils in a manner that enhances consortium survival and activity over time; and
• Assess bioaugmentation treatment performance using constructed soil columns.

In situ soil array cultivation and isolation of RDX-degrading bacteria

This project will characterize an RDX- and NTO-degradative bioaugmentation consortium, as well as develop the means to deliver the consortium to range soils that considers safety and accessibility issues. The bioaugmentation technology can be used in a broad variety of military range soils and is expected to be very effective for the localized mitigation of MC at firing points, burn-pan sites, and various mortar and rocket target sites. Soil-to-groundwater migration of explosive compounds (e.g., RDX) has resulted in groundwater plumes, which continue to cost the DoD hundreds of millions of dollars to contain or cleanup. Development of a bioaugmentation consortium and delivery scheme, which can prevent ground and surface water contamination, has tremendous potential environmental and cost savings benefits. (Anticipated Project Completion - 2023)