Anaerobic bacteria have been used to produce 2,4-dihydroxylamino-nitrotoluene (2,4DHANT), a reductive metabolite of 2,4,6-trinitrotoluene (TNT). Here, an aerobic TNT biodegrader Pseudomonas sp. strain TM15 produced 2,4DHANT as evidenced by the molecular ion with m/z of 199 identified from LC-TOFMS analyses. TNT biodegradation with a high cell concentration (10(9) cells/ml) led to a significant accumulation of 2,4DHANT in the culture medium, as well as hydroxylamino-dinitrotoluenes (HADNTs), although these products were not accumulated when a low cell concentration was used; also, the accumulation of diamino-nitrotoluene and of an unidentified metabolite were observed in the culture medium with the high cell concentration (10(10) cells/ml). 2,4DHANT overproduction was a function of the aeration speed since cultures with low aeration speeds (30 rpm) had a 19-fold higher DHANT productivity than those aerated with high speeds (180 rpm); this indicates that molecular oxygen was related to the formation of 2,4DHANT. The quantification of dissolved oxygen (DO) in the media demonstrated that the productivity of 2,4DHANT was increased at low DO values. Moreover, supplying oxygen to the culture media produced a remarkable decrease of 2,4DHANT accumulation; these results clearly indicate that high 2,4DHANT production was a consequence of the oxygen deficit in the culture medium. This finding is useful for understanding the TNT biodegradation (bioremediation technology) in an anoxic environment.

Two kinds of hydroxylamino-dinitrotoluenes (HADNTs), 2-hydroxylamino-4,6-dinitrotoluene (2HADNT) and 4-hydroxylamino-2,6-dinitrotoluene (4HADNT), are known to be major metabolites produced from 2,4,6-trinitrotoluene (TNT) by bacteria. These chemicals could not be identified as TNT metabolites produced by Pseudomonas sp. strain TM15 because the mass spectra of these chemicals could not be obtained by liquid chromatography-mass spectrometry (MS) or gas chromatography-MS, which are the classic methods for identifying the metabolites of xenobiotics. However, these problems are overcome by isolating 2HADNT and 4HADNT from TNT metabolites with one-step thin-layer chromatography using dichloromethane as the developing solvent, and individually extracting them into acetonitrile by collecting spots of 2HADNT and 4HADNT. The purity of each HADNT was approximately 98%, based on the results of high-performance liquid chromatographic analyses. 2HADNT and 4HADNT are identified by obtaining their mass spectra with laser time-of-flight MS. 2HADNT and 4HADNT dissolve in distilled water and are spontaneously broken down with time. Also, heat treatment (increasing temperatures) and dissolved oxygen accelerate the destruction of HADNTs. This technique may be applicable for the identification and exact quantitative analysis of unstable and fragile compounds such as HADNTs.

Two kinds of hydroxylamino-dinitrotoluenes, 2-hydroxylamino-4, 6-dinitrotoluene (2HADNT) and 4-hydroxylamino-2, 6-dinitrotoluene (4HADNT), are known to be major metabolites produced from 2,4,6-trinitrotoluene (TNT) by bacteria. The amounts of 2HADNT and 4HADNT in distilled water are found to spontaneously decrease with time, albeit abiotic conditions, and many white precipitates are educed in the processes of this phenomenon; however, how these compounds are converted is unclear. We evinced the mystery of this phenomenon by using thin-layer chromatography (TLC) and laser time-of-flight mass spectrometry (TOFMS). TLC analyses in the spontaneous conversion processes of 2HADNT, 4HADNT, and 2HADNT plus 4HADNT demonstrats that three novel spots emerge on the TLC plate, respectively. These products are individually extracted into acetonitrile by collecting each spot. The purity of these extracts, which have retention times of 14.0, 17.7, and 15.4 min, is approximately 98%, judging from the results of high-performance liquid chromatographic analyses. The spontaneous conversion products of 2HADNT, 4HADNT, and 2HADNT plus 4HADNT are identified as 4,4',6,6'-tetranitro-2,2'-azoxytoluene (2,2'AZT), 2,2',6,6'-tetranitro-4,4'-azoxytoluene (4,4'AZT), and 4,2',6, 6'-tetranitro-2,4'-azoxytoluene (2,4'AZT) by obtaining their mass spectra with laser TOFMS. It is confirmed that most of the spontaneous conversion products are 2,2'AZT, 4,4'AZT, or 2,4'AZT, judging from the results of mass balance in the spontaneous conversion processes of 2HADNT, 4HADNT, and 2HADNT plus 4HADNT.

No abstract