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The stability of soil bound metabolites of 2,4,6-trinitrotoluene (TNT) was investigated. Highly TNT-contaminated soil from a former production site was spiked with [ring-UL-14.C]TNT. An anaerobic short-term treatment (8 days) was carried out and compared with a previously described longterm anaerobic (51 day) treatment. In the short-term experiment the anaerobic treatment was stopped to examine the early-stage effects of the reduction process on the stability of TNT derivatives in soil at the time of the maximum accumulation of aminodinitrotoluenes, whereas the longterm anaerobic treatment was continued until the formation of triaminotoluene. In contrast to the soil from long-term anaerobic treatment, no significant reduction of aminodinitrotoluenes to diaminonitrotoluenes was observed after short-term anaerobic treatment, and the binding of reduced metabolites to the soil was incomplete. In both experiments the anaerobic phase was followed by an aerobic treatment, After the short-term anaerobic/aerobic treatment only 40% of the initial radioactivity was bound to the soil compared to 98% after the extended anaerobic incubation. A significant amount of this bound radioactivity could be released into organic solvents by silylation. The silylation extracts were analyzed by thin-layer chromatography and radiocounting. The released radioactivity from silylation after the long-term treatment was attributed to polar compounds cross-linked with humic material. For the silylated extracts after short-term anaerobic treatment conditions, a significant amount of released radioactivity (28%) found in fractions was attributed to TNT and aminodinitrotoluenes. This study indicates that with a shorter duration of the anaerobic phase in the overall treatment, physically entrapped (sequestered) metabolites remain in the soil. To eliminate physical entrapment, TNT derivatives should be completely reduced by a sufficiently extended anaerobic treatment.

DNT-degrading bacteria can completely degrade mixtures of DNT in liquid cultures without the production of aminonitrotoluenes. We determined whether specific DNT-degrading isolates could also degrade DNT from contaminated soil in the presence of indigenous microbial communities. When 2,4-DNT- and 2,6-DNT-degrading strains were added to a mixture of 2,4-DNT and 2,6-DNT in a soil slurry, disappearance of DNT was accompanied by 14.CO2 release and stoichiometric appearance of nitrite. When soil historically contaminated with mixed DNT isomers was used in slurries, the combination of DNT-degrading strains removed all of the DNT from the aqueous phase and over 99 per cent of the initial DNT. Traces of extractable DNT remained associated with the soil; however, the toxicity of the treated soil was low. After an extended acclimation period, the 2,4-DNT, and, much later, the 2,6-DNT, was degraded in the uninocutated control. The results show that aged DNT contamination can be removed effectivel y from soil. The addition of specific DNT-mineralizing bacteria dramatically enhances the mineralization of DNT in soil slurries. Native bacteria do not convert DNT to aminonitro-toluenes during the short incubation times required for mineralization of DNT.

Evidence is presented for the covalent binding of biologically reduced metabolites of 2,4,6-15.N3-trinitrotoluene (TNT) to different soil fractions (humic acids, fulvic acids, and humin) using liquid 15.N 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 2,4,6-15.N3trinitrotoluene and 14.C-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 per cent), whereas the radioactivity bound to humin increased to 71 per cent. 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 disappear ed 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.

3-Hydroxylaminophenol mutase from Ralstonia eutropha JMP134 is involved in the degradative pathway of 3-nitrophenol, in which it catalyzes the conversion of 3-hydroxylaminophenol to aminohydroquinone. To show that the reaction was really catalyzed by a single enzyme without the release of intermediates, the corresponding protein was purified to apparent homogeneity from an extract of cells grown on 3-nitrophenol as the nitrogen source and succinate as the carbon and energy source. 3-Hydroxylaminophenol mutase appears to be a relatively hydrophobic but soluble and colorless protein consisting of a single 62-kDa polypeptide. The pI was determined to be at pH 4.5. In a database search, the NH2-terminal amino acid sequence of the undigested protein and of two internal sequences of 3-hydroxylaminophenol mutase were found to be most similar to those of glutamine synthetases from different species. Hydroxylaminobenzene, 4-hydroxylaminotoluene, and 2-chloro-5-hydroxylaminophenol, but not 4-hyd roxylaminobenzoate, can also serve as substrates for the enzyme. The enzyme requires no oxygen or added cofactors for its reaction, which suggests an enzymatic mechanism analogous to the acid-catalyzed Bamberger rearrangement.

Ralstonia eutropha JMP134 utilizes 2-chloro-5-nitrophenol as a sole source of nitrogen, carbon, and energy. The initial steps for degradation of 2-chloro-5-nitrophenol are analogous to those of 3-nitrophenol degradation in R. eutropha JMP134. 2-Chloro-5-nitrophenol is initially reduced to 2-chloro-5-hydroxylaminophenol, which is subject to an enzymatic Bamberger rearrangement yielding 2-amino-5-chlorohydroquinone. The chlorine of 2-amino-5-chlorohydroquinone is removed by a reductive mechanism, and aminohydroquinone is formed. 2-Chloro-5-nitrophenol and 3-nitrophenol induce the expression of 3-nitrophenol nitroreductase, of 3-hydroxylaminophenol mutase, and of the dechlorinating activity. 3-Nitrophenol nitroreductase catalyzes chemoselective reduction of aromatic nitro groups to hydroxylamino groups in the presence of NADPH. 3-Nitrophenol nitroreductase is active with a variety of mono-, di-, and trinitroaromatic compounds, demonstrating a relaxed substrate specificity of the enzyme. N itrosobenzene serves as a substrate for the enzyme and is converted faster than nitrobenzene.

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.

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 m3 of contaminated soil (main contaminants were TNT, 2,4-dinitrotoluene, hexahydro-1,3,5-trinitro-1,3,5-triazine) and 10 m3 of water. The anaerobic stage was ca rried out by periodical feeding of sucrose. The sludge was subsequently dewatered and treated aerobically. Chemical analysis revealed an overall reduction of more than 99 per cent 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.

Untersuchungen des Fraunhofer-Instituts für Grenzflächen- und Bioverfahrenstechnik in Stuttgart haben gezeigt, daß sich der militärisch bedeutendste Sprengstoff TNT (Trinitrotoluol) und verwandte Explosivstoffe durch ein zweistufiges Verfahren mikrobiologisch entsorgen lassen. TNT-belastete Böden können auf diese Weise entgiftet werden. Die Firma Umweltschutz Nord hat das Verfahren auf dem Standort der Rüstungsaltlast Hessisch Lichtenau-Hirschhagen im technischen Maßstab erprobt. Die Pilotsanierung führte zu einer effizienten Abreinigung der Schadstoffe. Ökotoxikologische Untersuchungen des gereinigten Bodens ergaben keinerlei Hinweise auf toxische Effekte. Das erprobte Verfahren kann - abhängig vom jeweiligen Projekt - sowohl in mobilen Wannenreaktoren als auch in großvolumigen Behandlungstunneln einer Bodenreinigungsanlage eingesetzt werden.

Für Mono- und Dinitroaromaten, die als Zwischenprodukte der Synthese aromatischer Stickstoffverbindungen bedeutend sind, gibt es Mechanismen des vollständigen mikrobiellen Abbaus. Dagegen konnte für den Sprengstoff 2,4,6-Trinitrotoluol (TNT), die Haupkontaminante ehemaliger Rüstungstandorte, eine vollständige mikrobielle Mineralisation bislang nicht festgestellt werden. Unter anaeroben Bedingungen wird TNT in Gegenwart eines Auxiliarsubstrates zu 2,4,6-Triaminotoluol reduziert. Sowohl unter anaeroben als auch aeroben Bedingungen wird das reaktive 2,4,6-Triaminotoluol irreversibel im Boden gebunden. Basierend auf diesen Erkenntnissen wurde ein kombiniertes Anaerob-Aerob-Verfahren zur biologischen Sanierung von TNT-kontaminierten Böden entwickelt.

Chemikalien aus Altlasten der Sprengstoffproduktion sind zum einen besonders gefährdend, zum anderen - da sie normalerweise in der Natur nicht vorkommen - durch Mikroorganismen schwer oder gar nicht abbaubar. Hier können aber besondere verfahrenstechnische Maßnahmen erfolgreich sein.