Many sites at Department of Defense installations are contaminated with explosives as a result of the manufacture, handling, and storage of munitions. 2,4,6-Trinitrotoluene (TNT) and dinitrotoluenes (DNT) are major contaminants at these sites. The most common technologies for the treatment of heavily contaminated soils involve incineration or composting, both of which are expensive and do not result in complete degradation of the TNT molecule. Because there are no cost-effective strategies for elimination of DNT and TNT from water or moderately contaminated soil, most soils and groundwater have not been treated.

The objective of this project was to characterize bacterial strains with the ability to efficiently degrade mixtures of DNT isomers and to expand their degradative capability to TNT.

Bacteria with the ability to grow on DNT have the potential to remediate soil and water contaminated with mixtures of DNT isomers. Unfortunately, no bacteria have been isolated that can grow on and completely degrade TNT. The overall strategy for the development of a biocatalyst capable of degrading DNT and TNT was to introduce genes encoding enzymes for DNT and TNT degradation into an appropriate host strain. The organism would obtain carbon and energy from DNT and cometabolize and detoxify TNT. This approach required an understanding of the steps in the bacterial pathways for 2,4- and 2,6-DNT degradation, the identification of enzymes with the ability to utilize TNT and its metabolites, and detailed characterization of the conditions under which efficient degradation occurs.

In support of developing a biocatalyst capable of aerobic degradation of TNT while using DNT as the carbon and energy source, researchers (1) characterized the enzymes and identified intermediates in the bacterial degradation of both isomers of DNT; (2) characterized the substrate specificities of and elucidated the origin of nitroarene dioxygenases; (3) characterized the regulation of nitroarene dioxygenase genes and identified a potential regulatory gene for a constructed TNT degrading strain; (4) demonstrated that multiple operons for 2,4-DNT degradation can be integrated as a coordinately controlled genetic unit in a heterologous host; (5) cloned and sequenced genes encoding key enzymes in the 2,6-DNT degradation pathway, (6) demonstrated that bacteria degrading DNT can cometabolize TNT; (7) discovered enzymes in DNT degrading bacteria that catalyze all of the individual steps in the conversion of TNT to ring-fission products that contain no nitro groups; and (8) discovered nitroreductases specific for nitro groups at the 2-carbon position of TNT and characterized nitroreductase activities in potential host strains. Thus, all of the necessary elements have been identified for the construction of an efficient DNT/TNT degrader.

The development of a biocatalyst capable of degrading DNT and TNT would be of use in the cleanup of soil and groundwater at numerous sites, such as the Badger and Volunteer Army Ammunition Plants, where soil and groundwater are extensively contaminated with mixtures of TNT and DNT.