TNT Metabolism Research Articles

Transformation of 2,4,6-Trinitrotoluene by Pseudomonas pseudoalcaligenes JS52

Pseudomonas pseudoalcaligenes JS52 grows on nitrobenzene via partial reduction of the nitro group and enzymatic rearrangement of the resultant hydroxylamine. Cells and cell extracts of nitrobenzene-grown JS52 catalyzed the transient formation of 4-hydroxylamino-2,6-dinitrotoluene (4HADNT), 4-amino-2,6-dinitrotoluene (4ADNT), and four previously unidentified metabolites from 2,4,6-trinitrotoluene (TNT). Two of the novel metabolites were identified by liquid chromatography/mass spectrometry and (sup1)H-nuclear magnetic resonance spectroscopy as 2,4-dihydroxylamino-6-nitrotoluene (DHANT) and 2-hydroxylamino-4-amino-6-nitrotoluene (2HA4ANT). A polar yellow metabolite also accumulated during transformation of TNT by cells and cell extracts. Under anaerobic conditions, extracts of strain JS52 did not catalyze the production of the yellow metabolite or release nitrite from TNT; moreover, DHANT and 2HA4ANT accumulated under anaerobic conditions, which indicated that their further metabolism was oxygen dependent. Small amounts of nitrite were released during transformation of TNT by strain JS52. Sustained transformation of TNT by cells required nitrobenzene, which indicated that TNT transformation does not provide energy. Transformation of TNT catalyzed by enzymes in cell extracts required NADPH. Transformation experiments with (sup14)C-TNT indicated that TNT was not mineralized; however, carbon derived from TNT became associated with cells. Nitrobenzene nitroreductase purified from strain JS52 transformed TNT to DHANT via 4HADNT, which indicated that the nitroreductase could catalyze the first two steps in the transformation of TNT. The unusual ability of the nitrobenzene nitroreductase to catalyze the stoichiometric reduction of aromatic nitro compounds to the corresponding hydroxylamine provides the basis for the novel pathway for metabolism of TNT.

Characterization of partial anaerobic metabolic pathway for 2,4,6-trinitrotoluene degradation by a sulfate-reducing bacterial consortium.

The anaerobic degradative pathway for metabolism of 2,4,6-trinitrotoluene (TNT) by a consortium of Desulfovibrio spp. isolated from a creek sediment was studied. This consortium has the metabolic capability to degrade TNT to fatty acids. The growth of the consortium and the metabolism of TNT were greatly enhanced in the presence of an additional carbon source like pyruvate. The optimal concentration of pyruvate for the maximum rate of TNT degradation was 15-20 mM. Various intermediates of TNT metabolism were identified. The first step in the pathway was reduction of TNT to 4-amino-2,6-dinitrotoluene and 2-amino-4,6-dinitrotoluene, which were further reduced to 2,4-diamino,6-nitrotoluene. The next intermediate to appear in the culture medium was nitrobenzoic acid, followed by cyclohexanone, 2-methyl pentanoic acid, butyric acid, and acetic acid. A study using radiolabeled TNT showed that no CO2 was produced from TNT during metabolism. The mass balance of the radiolabeled study showed that 49.6% of the TNT was converted to acetic acid, 28% was assimilated into biomass as trichloroacetic acid precipitable materials, and the rest was distributed as various TNT intermediates. Most Desulfovibrio spp. are incomplete oxidizers that are unable to carry out the terminal oxidation of organic substrates. The major end product of TNT metabolism was acetic acid. The bacteria grew on all the TNT intermediates tested as sole source of carbon, except on acetic acid, confirming that the Desulfovibrio spp. have the enzymes necessary for complete degradation of TNT to acetate.

Isolation and characterization of a sulfate-reducing bacterium that removed TNT (2,4,6-trinitrotoluene) under sulfate- and nitrate-reducing conditions

A sulfate-reducing bacterium isolated from a creek sediment and capable of metabolizing TNT (2,4,6-trinitrotoluene) using sulfate and nitrate as electron acceptors was tentatively characterized as Desulfovibrio desulfuricans strain A. The isolate was unable to use TNT as the sole source of carbon. TNT degradation was accomplished by a co-metabolic process using pyruvate as the main substrate. Two different metabolic steps were employed by this isolate under different electron-accepting conditions. Under sulfate-reducing conditions, TNT was reduced to 4-amino-2,6-dinitrotoluene, 2-amino-4,6-dinitrotoluene and 2,4-diamino-6-nitrotoluene. In contrast, under nitrate-reducing conditions, these amino compounds were not produced, instead, butyric acid was identified as the major metabolite of TNT metabolism. This organism also used a wide variety of other carbon sources, including ethanol, lactate, succinate, formate and malate. The isolate contained the electron-carrier desulfoviridin and used sulfate, nitrate, and thiosulfate as electron acceptors. The isolate had an optimal temperature of 25°C and an optimal pH of 6.8 and used ammonium chloride, nitrate and glutamate as nitrogen sources. The characteristic features of the sulfate-reducing bacterium closely resembled those of Desulfovibrio desulfuricans.

Metabolism and Detoxification of TNT by [formula omitted

Several lines of evidence suggest that TNT detoxification by Phanerochaete chrysosporium is through reduction. Rates of TNT reduction were directly correlated with mycelial mass and TNT concentration. Toxicity was inversely related to the amount of fungus. TNT toxicity was identical in both ligninolytic and nonhigninolytic cultures. Rapid disappearance of the reduced metabolites coincided with production of the manganese-dependent peroxidases and mineralization of TNT was not observed until the lignin peroxidases were detected.

Analysis of 2,4,6-trinitrotoluene and its transformation products in soils and plant tissues by high-performance liquid chromatography

Previous studies have failed to provide an acceptable mass balance for 2,4,6-trinitrotoluene (TNT) in soils and plants due to deficiencies in analytical methodology. A high-performance liquid chromatographic (HPLC) method for soil analysis is reported which allowed for a mass balance in excess of 88% during a 2-month study. A method for plant analysis was developed which involved fractionation of organic extracts on Florisil adsorbent, to remove interfering pigments, followed by HPLC analysis of TNT and the primary metabolites, 2-amino-4,6-dinitrololuene and 4-amino-2,6-dinitrotoluene. Chromatographic recovery of TNT from spiked tissues was 85 ± 6%. The methodology was utilized to investigate TNT uptake and metabolism in plants grown in TNT hydroponic solutions.

Oxidative pretreatment accelerates TNT metabolism in soils

The combined effect of ultraviolet (UV)-ozonation (O 3) of aqueous 14C-TNT solutions followed by direct addition of the solutions to aerobic soils was examined as a method of disposal. The effect of TNT concentration was studied on both UV-O 3 and soil metabolism. The amount of TNT degraded by either process decreased as the concentration increased. UV-O 3 of a 1 ppm solution of TNT using a laboratory 450 W lamp for 10, 20, and 30 minutes resulted in substantial fragmentation of the ring and an increase in polarity of the resultant products. Soil metabolism, as measured by metabolic CO 2 evolution, increased as the time of prior UV-O 3 increased. A large amount of the 14C associated with 14C-TNT recovered from soil was in the non-extractable fraction. When a Pseudomonas putida , adapted to metabolize ortho -nitrophenol or picric acid as a sole source of carbon and nitrogen, was substituted for the soil phase, about 25% of the added 14C appeared as 14CO 2. 1,3,5-Trinitrobenzene, 2,4,6-trinitrobenzaldehyde, 3,5-dinitrophenol, 3, 5-dinitrocatechol, 3,5-dinitrohydroquinone, and oxalic acid were identified as products of UV-O 3. Rapid destruction of TNT took place in a large 66 lamp unit, and the resultant distribution of 14C was similar to the results from the laboratory studies.