Mechanistic studies on enzymatic nitroarene reduction and implications for the fate of nitroarene mixtures in redox-stratified biofilm

At military bases and munitions factories, 2,A,6-trinitrotoluene (TNT), 2,4-dinitrotoluene (24DNT), and 2,6-dinitrotoluene (26DNT) are persistent soil and groundwater contaminants. Although the reduction of nitro groups in these compounds has been extensively investigated, few researchers have studied the link between the reduction rates and the electrochemical properties of these compounds. In this work, the standard one-electron redox potentials at pH 7 (E1°′) for TNT and related nitroorenes were measured for the first time by pulse rodiolysis. The reduction kinetics were investigated using a bacterial nitroreduclase. Contrary to most whole cell studies wherein amino group formation has been observed, nitro group reduction halted at the level of the hydroxylamino group. A linear free energy relationship was observed between the enzymatic reduction rates and the E1°′ values. Enzymatic TNT reduction did not obey simple Michoelis-Menten kinetics, and clear evidence of enzyme inactivation during TNT transformation was obtained. ^ Batch experiments were conducted to test whether 2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene, and 2,4-diamino-6-nitrotoluene, potential TNT degradation products, were aerobically transformed by a mixed culture that mineralizes DNTs. None of the compounds were degraded to any extent when provided as sole carbon and nitrogen sources or when DNTs were provided as primary substrate. 26DNT degradation was inhibited in the presence of 4-amino-2,6-dinitrotoluene. ^ As a first step in testing the feasibility of using a redox-stratified biofilm for simultaneous DNT and TNT mineralization, a comprehensive biofilm model was developed. A novel model form was derived to depict nitroarene reduction as a function of the compound E1°′ and the intracellular NADH concentration, an indicator for solution redox potential. Using the comprehensive biofilm model, several reactor types (fluidized bed, hollow fiber membrane, and extractive membrane) were investigated. Model results suggested that membrane biofilm reactors are most effective because they promote thicker biofilm growth and redox stratification. A sensitivity analysis demonstrated that additional investigation of aminodinitrotoluene mineralization, oxygen limitation, and ozoxy dimer production ore necessary to better predict this technology. This model will guide application of this treatment process, as more mechanistic detail becomes available. However, these model results are preliminary due to several assumptions that require more stringent validation.