Biodegradation of the hexahydro-1,3,5-trinitro-1,3,5-triazine ring cleavage product 4-nitro-2,4-diazabutanal by Phanerochaete chrysosporium

Abstract

Initial denitration of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by Rhodococcus sp. strain DN22 produces CO2 and the dead-end product 4-nitro-2,4-diazabutanal (NDAB), OHCNHCH2NHNO2, 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 micromol kg(-1)), HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine; 3,057 micromol kg(-1)), MNX (hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine; 155 micromol kg(-1)), and traces of NDAB (3.8 micromol 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 micromol 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 (N2O). In cultures with the fungus alone NDAB degraded to give a stoichiometric amount of N2O. To determine C stoichiometry, we first generated [14C]NDAB in situ by incubating [14C]RDX with strain DN22, followed by incubation with the fungus. The production of 14CO2 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.

FIG. 1.

Structures of RDX, MNX, HMX, and NDAB.

FIG. 2.

Distribution of cyclic nitramines and products in soil collected from an ammunition plant containing RDX (342 μmol kg⁻¹), HMX (3,057 μmol kg⁻¹), MNX (155 μmol kg⁻¹), and traces of NDAB (3.8 μmol kg⁻¹) with no added reagents (A), after 5 days of incubation in m-succinate medium (20%, wt/vol) (B), and after 5 days of incubation in m-succinate medium with the RDX-induced DN22 strain (20%, wt/vol) (C). DN22 was induced with RDX, harvested at mid-log phase, and concentrated to an optical density at 530 nm of approximately 4.0. The bacterial suspension (0.6 ml) was used to inoculate the slurries. Incubation was performed at 30°C with agitation at 180 rpm.

FIG. 3.

Formation of NDAB in aerobic soil slurries (20%, wt/vol) prepared with an ammunition plant soil contaminated with RDX (342 μmol kg⁻¹), HMX (3,057 μmol kg⁻¹), MNX (155 μmol kg⁻¹), and traces of NDAB (3.8 μmol kg⁻¹) in m-succinate medium inoculated with either RDX-induced Rhodococcus sp. strain DN22 (▪), Rhodococcus sp. strain A (○), or R. rhodochrous sp. strain 11Y (▵). The strains were induced with RDX, harvested at mid-log phase and concentrated to an optical density at 530 nm of approximately 4.0. The bacterial suspensions (0.6 ml) were used to inoculate the slurries. Incubation was performed at 30°C with agitation at 180 rpm. Values represent the averages and standard deviations of triplicate experiments.

FIG. 4.

Degradation of NDAB added to uninoculated MC1 medium (□), to ligninolytic P. chrysosporium in MC1 (▪), and to P. chrysosporium grown in 20% (wt/vol) slurries prepared with VT (○), with SSL (×), and with an ammunition plant soil contaminated with RDX, MNX, HMX, and traces of NDAB (•). Values represent the averages and standard deviations of triplicate experiments.

FIG. 5.

Sequential biodegradation of [¹⁴C]RDX (40 ppm). Phase 1 was incubation with Rhodococcus sp. strain DN22 in 10 ml of m-succinate medium (for in situ production of [¹⁴C]NDAB); phase 2 was the period after the addition of MC1 medium (10 ml) to 10 ml of DN22 culture (•) and after the addition of P. chrysosporium culture (10 ml) to 10 ml of DN22 culture (□). Values represent the averages and standard deviations of triplicate experiments.