Selecting the best electron donor and operational temperature for the rapid biotransformation of the insensitive munitions compound 2,4-dinitroanisole (DNAN) by anaerobic sludge.

2,4-Dinitroanisole (DNAN) is a toxic compound increasingly used by the military that can be released into the environment on the soil of training fields and in the wastewater of manufacturing plants. DNAN's nitro groups are anaerobically reduced to amino groups by microorganisms when electron donors are available. Using anaerobic sludge as the inoculum, we tested different electron donors for DNAN bioreduction at 20 and 30 °C: acetate, ethanol, pyruvate, hydrogen, and hydrogen + pyruvate. Biotic controls without external electron donors and abiotic controls with heat-killed sludge were also assayed. No DNAN conversion was observed in the abiotic controls. In all biotic treatments, DNAN was reduced to 2-methoxy-5-nitroaniline (MENA), which was further reduced to 2,4-diaminoanisole (DAAN). Ethanol or acetate did not increase DNAN reduction rate compared to the endogenous control. The electron donors that caused the fastest DNAN reductions were (rates at 30 °C): H2 and pyruvate combined (311.28 ± 10.02 µM·d-1·gSSV-1), followed by H2 only (207.19 ± 5.95 µM·d-1·gSSV-1), and pyruvate only (36.35 ± 2.95 µM·d-1·gSSV-1). Raising the temperature to 30 °C improved DNAN reduction rates when pyruvate, H2, or H2 + pyruvate were used as electrons donors. Our results can be applied to optimize the anaerobic treatment of DNAN-containing wastewater.

The key role of oxygen in the bioremoval of 2,4-diaminoanisole (DAAN), the biotransformation product of the insensitive munitions compound 2,4-dinitroanisole (DNAN), over other electron acceptors.

Insensitive munitions compounds, such as 2,4-dinitroanisole (DNAN), are replacing conventional explosives. DNAN is anaerobically reduced to 2,4-diaminoanisole (DAAN), a toxic aromatic amine. However, the removal of DAAN under different redox conditions is yet to be elucidated. Herein, we analyzed DAAN consumption in biotic and abiotic microcosms when exposed to different redox conditions (without added electron acceptor, without added electron acceptor but with pyruvate as a co-substrate, with sulfate, with nitrate, and with oxygen), using an anaerobic sludge as inoculum. We observed that DAAN autoxidation, an abiotic reaction, was significant in microaerobic environments. DAAN also reacted abiotically with heat-killed sludge up to a saturation limit of 67.4 µmol DAAN (g VSS heat-killed sludge)-1. Oxygen caused the fastest removal of DAAN in live sludge among the conditions tested. Treatments without added electron acceptors (with or without pyruvate) presented similar DAAN removal performances, although slower than the treatment with oxygen. Sulfate did not exhibit any effect on DAAN removal compared to the treatment without added electron acceptors. Nitrate, however, inhibited the process. An enrichment culture from the microcosms exposed to oxygen could be developed using DAAN as the sole substrate in microaerobic conditions. The enrichment profoundly changed the microbial community. Unclassified microorganisms accounted for 85% of the relative abundance in the enrichment culture, suggesting that DAAN microaerobic removal might have involved organisms that were not yet described. Our results suggest that DAAN microaerobic treatment can be coupled to DNAN anaerobic reduction in sludge, improving the treatment of DNAN-containing wastewaters.