Microbial and enzymatic biotransformation of 2,4,6-trinitrotoluene by aerobic bacteria

2,4,6-Trinitrotoluene (TNT), a toxic nitroaromatic explosive, accumulates in the environment, thus necessitating the remediation of contaminated areas and unused materials. While bioremediation has been utilized to detoxify TNT, the metabolic processes involved in the biotransformation of TNT have proven to be complex. This dissertation presents the characterization of three novel aerobic strains of bacteria that biotransform TNT in different ways. The strains were all isolated from TNT contaminated sites and include a Pseudomonas aeruginosa isolated by R. P. Naumova, and a Bacillus sp. and Staphylococcus sp. provided by Dr. Kirby C. Donnelly (Texas A&M University). They differ in their growth characteristics in the presence of TNT, ability to biotransform TNT, and partitioning of TNT in the biomass. In addition, a profile of the enzymatic activities of each strain indicates differences in the reduction of nitro groups, cofactor preferences, and the ability to eliminate nitrite from the ring. Three separate NADPH-dependent TNT reduction activities were purified from the Bacillus sp. These activities represent two different classes as determined by their substrate specificities, cofactor requirements, and kinetic profiles. One class is the first example of an enzyme exhibiting cooperativity toward TNT as a substrate. Furthermore, the enzymes initiate extensive transformation of TNT in the presence of reducing agents. This is the first reported instance of enzymatically facilitated conversion of TNT to 24DNT and 2NT. This transformation pathway has great potential for the complete mineralization of TNT.