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).

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.

A hydride Meisenheimer complex of 2,4,6-trinitrotoluene (TNT) (TNTH-) was synthesized using tetramethylammonium octahydroborate. TNT and TNTH-(as the tetramethylammonium salt) were analyzed by direct exposure probe mass spectrometry using electron impact (EI), chemical ionization (CI) and negative-ion chemical ionization (NCI). For further identification, the ions of the mass spectra were investigated using tandem mass spectrometry with collision-induced dissociation (CID). Although the EI mass spectra of both compounds were similar, the CID mass spectra of the ions at m/z 227 (M' of TNT) of the two compounds showed large differences in daughter ion abundance. A major difference between the two compounds also appeared in their CI methane mass spectra. Two abundant ions, at m/z 183 and 198, appeared only in the CI mass spectrum of TNTH-. CID parent ion scans showed that the origin of these two ions was not the m/z 227 ion. We suggest that these ions are formed by chemical reactions of the complex on the surface of the heated probe tip, followed by ionization. These reactions, probably a reduction process forming m/z 198 and hydrolysis forming m/z 183, Occur preferentially in TNTH-, the already reduced form of TNT. Formation of the ions at m/z 183 and 198 was also observed in the liquid chromatography particle beam CI mass spectrum of TNTH-.

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Am Fraunhofer‐Zentrum für Chemisch‐Biotechnologische Prozesse CBP in Leuna wurde eine Pilotanlage zur Herstellung von Proteinen aus Raps eingeweiht. Nach dem Prinzip einer Bioraffinerie liefert sie durch Ethanolextraktion nicht nur hochwertiges Rapsöl in Vorraffinatqualität, sondern auch ein an hochwertigen Proteinen reiches Rapskernkonzentrat, sekundäre Pflanzenstoffe sowie Rapsschalen als weitere Produkte.