Biodegradation of the Hexahydro-1,3,5-Trinitro-1,3,5-Triazine Ring Cleavage Product 4-Nitro-2,4-Diazabutanal by Phanerochaete chrysosporium

Abstract

ABSTRACT Initial denitration of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by Rhodococcus sp. strain DN22 produces CO 2 and the dead-end product 4-nitro-2,4-diazabutanal (NDAB), OHCNHCH 2 NHNO 2 , in high yield. Here we describe experiments to determine the biodegradability of NDAB in liquid culture and soils containing Phanerochaete chrysosporium . A soil sample taken from an ammunition plant contained RDX (342 μmol kg −1 ), HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine; 3,057 μmol kg −1 ), MNX (hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine; 155 μmol kg −1 ), and traces of NDAB (3.8 μmol kg −1 ). The detection of the last in real soil provided the first experimental evidence for the occurrence of natural attenuation that involved ring cleavage of RDX. When we incubated the soil with strain DN22, both RDX and MNX (but not HMX) degraded and produced NDAB (388 ± 22 μmol kg −1 ) in 5 days. Subsequent incubation of the soil with the fungus led to the removal of NDAB, with the liberation of nitrous oxide (N 2 O). In cultures with the fungus alone NDAB degraded to give a stoichiometric amount of N 2 O. To determine C stoichiometry, we first generated [ 14 C]NDAB in situ by incubating [ 14 C]RDX with strain DN22, followed by incubation with the fungus. The production of 14 CO 2 increased from 30 (DN22 only) to 76% (fungus). Experiments with pure enzymes revealed that manganese-dependent peroxidase rather than lignin peroxidase was responsible for NDAB degradation. The detection of NDAB in contaminated soil and its effective mineralization by the fungus P. chrysosporium may constitute the basis for the development of bioremediation technologies.