Denitration of 2,4,6-trinitrotoluene by Pseudomonas savastanoi

Past disposal of wastewaters containing 2,4,6-trinitrotoluene (TNT) at the former Nebraska Ordnance Plant has resulted in numerous acres of TNT-contaminated soil. Examining the microbial population of these soils revealed several TNT-tolerant Pseudomonas spp. We selected one species, P. savastanoi, to determine its ability to transform TNT. Pure culture experiments were performed in pseudomonas minimal medium containing 0.31 mM TNT (70 mg TNT . L(-1)) under varied nutrient and cell density regimes. Experiments with TNT as a sole C or N source showed that P. savastanoi has the ability to denitrate TNT, as evidenced by production of 2,4-dinitrotoluene (2,4-DNT) and NO2- with time. TNT denitration and formation of 2,4-DNT were enhanced by removing NH4+ and adding NO2- to the growth medium. In all experiments, 2-amino-4,6-dinitrotoluene (2-ADNT) and 4-amino-2,6-dinitrotoluene (4-ADNT) appeared as incidental reduction products. Glucose addition to the medium enhanced 2-ADNT and 4-ADNT production and decreased denitration of TNT. Mid-log phase cells rapidly transformed [ring-14C(U)]TNT but were unable to mineralize significant quantities of TNT, as evidenced by conversion of less than 1% of the label to 14CO2. These results indicate that P. savastanoi is a TNT-tolerant pseudomonad that can promote TNT degradation through reductive denitration and nitro moiety reduction.