The explosive 2,4,6-trinitrotoluene (TNT), found as a major contaminant at armament plants from the two world wars, is reduced by a variety of microorganisms when electron donors such as glucose are added. This study shows that the cometabolic reduction of TNT to 2,4,6-triaminotoluene by an undefined anaerobic consortium increased considerably with increasing TNT concentrations and decreased with decreasing concentrations and feeding rates of glucose. The interactions of TNT and its reduction products with montmorillonitic clay and humic acids were investigated in abiotic adsorption experiments and during the microbial reduction of TNT. The results indicate that reduction products of TNT particularly hydroxylaminodinitrotoluenes and 2,4,6-triaminotoluene bind irreversibly to soil components, which would prevent or prolong mineralization of the contaminants. Irreversible binding also hinders a further spread of the contaminants through soil or leaching into the groundwater.

A bacterial strain, Mycobacterium sp. strain HL 4-NT-1, enriched with 4-nitrotoluene as its sole source of nitrogen, was able to metabolize 2,4,6-trinitrotoluene under aerobic conditions. The dark red-brown metabolite, which accumulated in the culture fluid, was identified as a hydride-Meisenheimer complex by comparison with an authentic synthetic sample.In the course of large-scale manufacturing and handling of explosives, both soil and groundwater were contaminated extensively with polynitroaromatic compounds such as 2,4,6-trinitrotoluene (TNT) (5). Large amounts of TNT were synthesized during World War II. The high concentrations still found in the environment indicate the resistance of TNT to microbial degradation. Nitro groups, just like chloro substituents, reduce the electron density of the aromatic IT system, and thus hamper electrophilic attack by oxygenases. Initial reactions in the course of a degradative process of polynitroarenes thus must be reductive rather than oxidative. Several reports in the literature (1, 4, 15) deal with the reductive metabolism of TNT under anaerobic conditions. Little is known, however, about aerobic catabolism of TNT; most reports deal with cometabolic reactions by bacterial species leading to aminodinitrotoluenes (2, 6, 12, 14, 16). The hydroxylamino-or nitrosodinitrotoluenes, intermediates of such coreductions, usually couple with each other, generating biologically inert azoxy compounds (2,6,12,14,16).Recently, a novel reductive degradation mechanism by an aerobic organism has been described for the utilization of picric acid by Rhodococcus erythropolis HL PM-1 (9). An orange-red metabolite, accumulating transiently in the culture fluid, was characterized as the hydride-Meisenheimer complex formed by the nucleophilic addition of a hydride ion at C-3 of picric acid. Subsequently, this Meisenheimer complex was converted to 2,4-dinitrophenol and nitrite. From these data, though without any direct evidence, Duque et al. (3) proposed a corresponding mechanism for the metabolism of TNT yielding nitrite, dinitrotoluenes, nitrotoluenes, toluene, and major amounts of dead-end metabolites.We now present unequivocal evidence that the hydrideMeisenheimer complex of TNT (H --TNT complex) is the product of TNT bioconversion by Mycobacterium sp. strain HL 4-NT-1. 4-Nitrotoluene (4-NT)-grown cells of this organism produced sufficient amounts of the H --TNT complex to allow a spectroscopic characterization, in contrast to another newly isolated strain, CV TNT-8, which utilized TNT as a nitrogen source but accumulated only minor amounts of this rather unstable metabolite (iSa).

To analyze the fate of 2,4,6-trinitrotoluene (TNT) and its reduction products, a TNT-contaminated soil (350 mg TNT/ kg dry soil) was spiked with [ring-UL-14.C]TNT and treated in a laboratory slurry reactor. During an anaerobic/aerobic treatment, the total radioactivity measured in the supernatant and methanolic soil extracts decreased to 2 per cent. The decrease corresponded to an increase of strongly bound radioactivity to the soil. Throughout the whole treatment process, mineralization of TNT was not observed. During the reductive process, unidentified polar substances increased to a maximum amount of 23.2 per cent of the total radioactivity on the day after the start of the experiment. After the end of the anaerobic phase, still 9.7 per cent of the radioactivity was found in this fraction. Only during the aerobic phase did the polar substances disappear completely. The irreversible character of the binding of the reduced metabolites of TNT to the soil was indicated by the failure of desorption even under rigorous and longterm extraction conditions. A significant release of radioactivity could be measured only by using high concentrations of HCl (5.N) or EDTA (12.5 per cent and 5.9 per cent, respectively). However, in none of the extracts were TNT or any reduced metabolites detected by HPLC and 14.C-radiocounting. Size-exclusion chromatography of humic acids extracted from the treated soil indicated that the metabolites of TNT were evenly bound to the complete range of molecular size of the humic acids

Evidence is presented for the covalent binding of biologically reduced metabolites of 2,4,6-15N3-trinitrotoluene (TNT) to different soil fractions (humic acids, fulvic acids, and humin) using liquid 15N NMR spectroscopy. A silylation procedure was used to release soil organic matter from humin and whole soil for spectroscopic measurements. TNT-contaminated soil was spiked with 2,4,6-15N3-trinitrotoluene and 14C-ring labeled TNT, before treatment in a soil slurry reactor. During the anaerobic/aerobic incubation the amount of radioactivity detected in the fulvic and humic acid fractions did not change significantly (11−16%), whereas the radioactivity bound to humin increased to 71%. The 15N NMR spectra of the fulvic acid samples were dominated by a large peak that corresponded to aliphatic amines or ammonia. In the early stages of incubation, 15N NMR analysis of the humic acids indicated bound azoxy compounds. The signals arising from nitro and azoxy groups disappeared with further anaerobic treatment. At the end of incubation, the NMR shifts showed that nitrogen was covalently bound to humic acid as substituted amines and amides. The NMR spectra of the silylated humin suggest formation of azoxy compounds and imine linkages. Bound metabolites possessing nitro groups were also detected. Primary amines formed during the anaerobic incubation disappeared during the aerobic treatment. Simultaneously, the amount of amides and tertiary amines increased. Nitro and azoxy groups of bound molecules were still present in humin at the end of the incubation period. Formation of azoxy compounds from partially reduced TNT followed by binding and further reduction appears to be an important mechanism for the immobilization of metabolites of TNT to soil.

Because of its high electron deficiency, initial microbial transformations of 2,4,6-trinitrotoluene (TNT) are characterized by reductive rather than oxidation reactions. The reduction of the nitro groups seems to be the dominating mechanism, whereas hydrogenation of the aromatic ring, as described for picric acid, appears to be of minor importance. Thus, two bacterial strains enriched with TNT as a sole source of nitrogen under aerobic conditions, a gram-negative strain called TNT-8 and a gram-positive strain called TNT-32, carried out nitro-group reduction. In contrast, both a picric acid-utilizingRhodococcus erythropolis strain, HL PM-1, and a 4-nitrotoluene-utilizing Mycobacterium sp. strain, HL 4-NT-1, possessed reductive enzyme systems, which catalyze ring hydrogenation, i.e., the addition of a hydride ion to the aromatic ring of TNT. The hydride-Meisenheimer complex thus formed (H−-TNT) was further converted to a yellow metabolite, which by electrospray mass and nuclear magnetic resonance spectral analyses was established as the protonated dihydride-Meisenheimer complex of TNT (2H−-TNT). Formation of hydride complexes could not be identified with the TNT-enriched strains TNT-8 and TNT-32, or with Pseudomonas sp. clone A (2NT−), for which such a mechanism has been proposed. Correspondingly, reductive denitration of TNT did not occur.

Anaerobic treatment of originally contaminated soil from a former ammunition plant was carried out in a laboratory slurry reactor. While fermenting glucose to ethanol, acetate, and propionate, the anaerobic bacteria completely reduced the nitro groups of 2,4,6-trinitrotoluene (TNT) and aminodinitrotoluenes, which led to a complete and irreversible binding of the reduced products to the soil. 2,4-Dinitrotoluene and hexahydro-1,3,5-trinitro-1,3,5-triazine were also reduced in the soil slurry and were no longer detectable after the anaerobic treatment. To mineralize the fermentation products, a subsequent aerobic treatment was necessary to complete the bioremediation process. This bioremediation process was tested in a technical scale at Hessisch Lichtenau-Hirschhagen, Germany. A sludge reactor (Terranox system) was filled with 18 m 3 of contaminated soil (main contaminants were TNT, 2,4dinitrotoluene, hexahydro-1,3,5-trinitro-1,3,5-triazine) and 10 m 3 of water. The anaerobic stage was carried out by periodical feeding of sucrose. The sludge was subsequently dewatered and treated aerobically. Chemical analysis revealed an overall reduction of more than 99% of the contaminants. Ecotoxicological tests performed with various aquatic systems (luminescent bacteria, daphnids, algae) and terrestrial systems (respiring bacteria, nitrifying bacteria, cress plants, earth worms) showed that residual toxicity could not be detected after the anaerobic/aerobic treatment.

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Rhodococcus erythropolis HL 24-2, which was originally isolated as a 2,4-dinitrophenol-degrading bacterium, could also utilize picric acid as a nitrogen source after spontaneous mutation. During growth, the mutant HL PM-1 transiently accumulated an orange-red metabolite, which was identified as a hydride-Meisenheimer complex of picric acid. This complex was formed as the initial metabolite and further converted with concomitant liberation of nitrite. 2,4,6-Trinitrocyclohexanone was identified as a dead-end metabolite of the degradation of picric acid, indicating the addition of two hydride ions to picric acid.