Stable Isotope-Enabled Pathway Elucidation of 2,4-Dinitroanisole Metabolized by Rhizobium lichtii

Recently developed military explosives are safer to handle, but more environmentally mobile, than traditional explosives. The new insensitive munitions, including 2,4-dinitroanisole (DNAN), have been sparsely studied, making the use of bacterial and/or plant-based remediation approaches unfeasible without further information about the transformation of these compounds. To that end, Rhizobium lichtii isolated from willow tree tissues were studied and observed to completely degrade 5 mg L–1 DNAN within 24 h in liquid media with added carbon and nitrogen. The use of DNAN, [13C6]DNAN, and [15N2]DNAN enabled the identification of 11 previously unknown metabolites resulting from sulfation, acetylation, and/or methylation processes. Accurate mass analysis revealed DNAN metabolites with ion formulas of C7H7N2O6S, C7H7N2O7S, C9H9N2O5, C8H7N2O4, and C9H9N2O4. Further mass spectral analysis provided evidence of various combinations of amino and hydroxylamino DNAN metabolites. Nitro reduction was observed for the first time at the para position of DNAN to produce 4-amino-2-nitroanisole.