Enhanced RDX mineralization using mixed abiotic-biotic reactions mediated by extracellular electron-shuttling compounds

Bioremediation is a common approach for the cyclic nitramine contaminant hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). While aerobic biodegradation has been reported, anaerobic biodegradation is more prevalent. While several different strategies are very promising, few have shown that mineralization (to CO₂ or CH₄) can be accelerated; it is usually just the initial steps in RDX transformation that are enhanced (such as the formation of the nitroso intermediates). We have been investigating RDX transformation in contaminated aquifer material mediated by Fe(III)-reducing microorganisms and stimulated by electron shuttles. The mixed biological-abiotic reactions promote RDX transformation through several intermediates. However, in the presence of electron shuttling compounds RDX mineralization was much greater; the extent of mineralization with shuttles was ∼50% more than with electron donor alone (which is the typical strategy). In addition, a unique Fe(III)-reducing microbial community developed during biodegradation, which was not dominated by members of the delta proteobacteria. RDX-contaminated aquifer material was obtained from the Picatinny Arsenal in New Jersey. Incubations were constructed to mimic in situ conditions; therefore the only amendments were either electron donor (10mM acetate) or electron donor plus electron shuttles. The shuttles used were 100μM anthraquinone-2,6-disulfonate (AQDS) or 0.1g/L purified humic substances. RDX reduction was similar in all acetate amended systems, and the shuttles increased the rate only slightly. However, nitroso intermediates accumulated in the acetate-alone system, whereas the shuttle-amended bottles did not accumulate the nitroso compounds. 4-nitro-diazabutanol (NDAB) was a critical intermediate in the electron-shuttle amended systems, and it did not accumulate in any other incubations. This was interesting because recent reports suggest that NDAB is an intermediate only during aerobic RDX biodegradation, while these results show that it is significant during anaerobic transformation as well. Most significantly, 50-60% of U-[¹⁴C]-RDX was mineralized to ¹⁴CO₂ (no ¹⁴CH₄) in electron shuttle-amended incubations. Only 12% of the RDX was mineralized in corresponding acetate-alone incubations. These data demonstrate that the rate and extent of RDX mineralization can be improved under in situ conditions. All activity was coupled to Fe(III) reduction. Nitrate was depleted at least 10 days before the onset of RDX degradation and sulfate was not reduced during the timeframe of the experiments. Given the conditions (acetate plus electron shuttles) we expected the microbial community to become dominated by delta proteobacteria, with Geobacteraceae in particular. While Geobacteraceae were enriched to about 10-13%, they were not the dominant Fe(III) reducers present. The microbial community enriched during most active RDX transformation were primarily made up of organisms within the gamma and beta proteobacteria; most closely related to the genera Pseudomonas, Herbaspirillum, Aquaspirillum, and Rhodoferax. RDX must exert unique selective pressure on the microbial community to enrich the organisms that became dominant over time.