Explosives from manufacture and military use now contaminate large areas of land and groundwater across the United States and Europe. These energetic compounds are both toxic and recalcitrant to degradation and removing them is a difficult and massive logistical task. While phytoremediation is a technique that potentially offers an environmentally friendly, low-cost alternative to current remediation techniques, the use of this method is hindered by the phytotoxicity of 2,4,6-trinitrotoluene (TNT) and the low inherent metabolic abilities of plants towards these xenobiotic compounds. The overall objectives of this project were to engineer model plant species to express bacterial genes that detoxify or degrade the explosive compounds TNT and royal demolition explosive (RDX) and to produce robust transgenic plants with desirable structural and biological characteristics that can be used to contain and degrade energetic materials.

Plants growing in 0.1 mM TNT. In the laboratory, non-transgenic plants extracted very little TNT and the toxic effects were clearly visible (left). Transgenic plants with bacterial NR displayed minimal signs of phytotoxicity (right) and removed all of the TNT

Bacterial enzymes capable of detoxifying these energetic compounds have been identified. These genes have been introduced into the model plant species, tobacco and Arabidopsis where, in combination, they confer tolerance to TNT and the ability to detoxify TNT and degrade RDX. Past studies have shown that expression of the bacterial gene on encoding pentaerythritol tretranitrate reductase (PETNR) in tobacco resulted in plants with a greatly increased ability to detoxify and degrade nitroglycerin. PETNR also displays activity towards TNT, and plants expressing PETNR tolerate and detoxify low levels of TNT. Tobacco plants were constructed that express the aromatic nitroreductase (NR) from the bacterium Enterobacter cloacae. These transgenic plants showed a remarkable increase in their ability to tolerate and remove TNT, even at the saturating levels that would be found in heavily contaminated environments such as training ranges. A Rhodococcus sp. was isolated that can utilize RDX as a sole source of nitrogen, completely degrading RDX. The gene that encodes the enzyme (RDX P450) that catalyzes the dissimilation of RDX was cloned and sequenced, allowing for the engineering of plants that are capable of degrading RDX. Tobacco is clearly not a particularly robust plant for phytoremediation; therefore, trees and other plants found on firing ranges were engineered to contain PETNR, RDX P450, and TNT-detoxifying NR.

The results from this project demonstrate that use of transgenic plants for the phytoremediation of RDX and TNT at contaminated military training ranges is viable, in principle. The NR-expressing plants have increased tolerance to, and are able to detoxify, the co-contaminating explosive TNT, and researchers have produced transgenic plants containing XplA and NR that are able to tackle both TNT and RDX together. These plants increase the functional and genetic diversity of the rhizosphere bacterial community in acutely contaminated soil. As Arabidopsis, the model plant system studied, is a relatively small growing annual, a further magnified effect is predicted in the more robust and larger tree and perennial grass species transformed with these transgenes. These transformed grasses and tree species could be planted along range borders to remove RDX from soil leachate as it leaves the training range.

Transgenic plants developed in this project are able to remove explosives from soil and groundwater. The knowledge gained using these model plant species has been critical to the production and characterization of more robust tree species for both laboratory and field testing. Use of these plants on Department of Defense training ranges could provide a nonintrusive, inexpensive, self-sustaining, easily confined, and environmentally friendly method for preventing contamination of groundwater from munitions compounds used on training ranges. (Project Completed – 2009)