Chromatographic, spectroscopic and microscopic analyses reveal the impact of iron oxides and electron shuttles on the degradation pathway of 2,4,6- trinitrotoluene (TNT) by a fermenting bacterium

Abstract

Contamination of surface and subsurface environments with explosives such as 2,4,6-trinitrotoluene (TNT) is a worldwide problem. The fate and analysis of TNT were investigated in numerous artificially contaminated model systems. We developed a unique high performance liquid chromatography gradient elution method for the analysis of commonly observed TNT metabolites and EPA explosives. Column temperature was identified as the key parameter for optimal separation. Iron (hydr)oxides play an important role in the reduction, sorption and fate of TNT in soil and sediment. Consequently, characterization of the nature and properties of natural and synthetic Fe (hydr)oxides is important for determining reaction mechanisms and surface-associated chemical processes. This work thus summarizes the potential applicability of imaging and spectroscopic techniques for eliciting chemical and physical properties of iron (hydr)oxides. TNT is persistent in soils due to its low redox potential and sorption. Batch and column studies revealed some of the first results on TNT desorption behavior in two well-defined model soil systems. Biosurfactants were found to be the most promising technique for enhanced TNT desorption. Batch studies with a Cellulomonas sp. in the presence of ferrihydrite and the electron shuttle anthraquinone-2,6-disulfonate (AQDS) were conducted to reveal biotic and abiotic mechanisms contributing to the degradation of TNT. Strain ES6 was found to reduce TNT and ferrihydrite with enhanced reduction in the presence of AQDS. Ferrihydrite stimulated the formation of more reduced TNT metabolites such as 2,4-diamino-6-nitrotoluene. Interestingly, a completely different degradation pathway was observed in AQDS-amended iron-free cell suspensions, showing a rapid transformation of TNT to 2,4-dihydroxylamino-6-nitrotoluene, which transformed into unidentified polar products. The influence of iron phases (i.e. hematite, magnetite, and ferrihydrite) and secondary Fe mineral formation on the degradation of TNT was also evaluated. The initial reduction of TNT was fastest in the presence of hematite; however, the further reduction of hydroxylamino-dinitrotoluenes was fastest, in the presence of magnetite and ferrihydrite (no AQDS). The impact of AQDS was predominant in the presence of hematite resulting in the formation of 2,4,6-triaminotoluene. Ferrihydrite underwent reductive dissolution with the formation of secondary hematite. The enhanced TNT reduction in ferfihydrite-amended systems was therefore most likely due to redox-active Fe(II) rather than secondary Fe phases.