Environ. Sci. Technol. 2009, 43, 2773–2776
Microorganisms and Explosives: Mechanisms of Nitrogen Release from TNT for Use as an N-Source for Growth ROLF-MICHAEL WITTICH,* JUAN LUIS RAMOS, AND PIETER VAN DILLEWIJN Departamento de Proteccio´n Ambiental, Estacio´n Experimental del Zaidı´n, Consejo Superior de Investigaciones Cientı´ficas, Calle Profesor Albareda 1, E-18008 Granada, Spain
Received November 27, 2008. Revised manuscript received February 16, 2009. Accepted February 23, 2009.
Unstable reduced derivatives of 2,4,6-trinitrotoluene (TNT) produced by microorganisms have been found to release nitrite by rearomatization and/or condensation. Here, we present further information regarding the novel mechanism of the condensation of reactive hydroxylaminodinitrotoluene and the Meisenheimer dihydride complex of TNT to produce two secondary diarylamine isomers. Using uniformly 15N-labeled (15N3) TNT, we show that the nitrite is being released by the condensation reaction and, also under environmental conditions, will originate from the microbiologically generated dihydride complex.
Introduction Biodegradation of explosives, and especially of 2,4,6-trinitrotoluene (TNT), has attracted much attention in the last two decades as this compound constitutes a health and environmental hazard. Although generally recalcitrant, TNT can be degraded by several microorganisms. The utilization of TNT and other nitroaromatics by microorganisms as a potential nitrogen source is a well-established fact (1-3). However, with the exception of a very few reports (4), no proof for the complete mineralization of TNT has been found as experiments performed with the 14C-labeled compound showed only negligible detection of 14CO2 and/or incorporation into biomass under either aerobic or anaerobic conditions. On the other hand, bioremediation technologies require not only the mineralization but, often much more importantly, the detoxification and inactivation of the chemical compounds of concern, including their potentially toxic metabolites. Recent investigations on the mechanism of the elimination of nitro groups from TNT by the marine yeast, Yarrowia lipolytica (5), reported nitrite release from an intermediary Meisenheimer dihydride complex of TNT without the identification of the final products, a finding which contrasts with an earlier proposal (6). In the latter, the authors proposed biological release of nitrite to occur from the monohydride complex. This compound, obtained upon bacterial reduction of the aromatic ring system low in electron density, is known to rearomatize to a minute extend to its parent TNT structure * Corresponding author phone: (+34) 958 181 600, ext. 138; fax: (+34) 958 135740; e-mail:
[email protected]. 10.1021/es803372n CCC: $40.75
Published on Web 03/23/2009
2009 American Chemical Society
(7) but generally, in the presence of reduction equivalents, is spontaneously further reduced to the dihydride complex (8). Although the dihydride complex is quite stable under experimental conditions, it slowly rearomatizes with the concomitant release of nitrite (9). Nitrite release has also been reported to occur by a reductive denitration mechanism in the presence of a Pseudomonas strain down to toluene (4). On the other hand, the (bio-) chemical mechanism of nitrite release initiated by enzymes of Pseudomonas putida strain JLR11 (10, 11) and, probably, the majority of TNT-utilizing bacteria, was elucidated only recently (12). Other oxidative and fortuitous dioxygenase reactions may release a nitro group during enzymatic co-oxidations of TNT or upon its initial reduction to an aminodinitrotoluene, thereby leading to the accumulation of reactive and toxic nitrocatechols and other intermediates accumulating as dead-end products (13-15). Hydroxylamine and amine derivatives of TNT obtained from biological reductions of the nitro groups (16, 17) have been suggested to furnish ammonium upon Bamberger rearrangements (9-11, 18–20) but this mechanism or any further breakdown products have yet to be unambiguously demonstrated. The only mechanism for nitrogen release from TNT has been shown for the condensation of hydroxylamines and Meisenheimer dihydride complexes to secondary diarylamines (12). The abovementioned reactions have been compiled in Figure 1 as an overview of hitherto proposed reactions for the aerobic release of nitrogen from TNT which could serve as possible N-sources for microorganisms. Here we sought to determine the reaction mechanism of this condensation reaction and the source of the nitrite released.
Materials and Methods Chemicals. TNT was obtained from Unio´n Espan ˜ ola de Explosivos (Madrid, Spain) and was >99% pure upon GC and HPLC analysis. Uniformly 15N-labeled 2,4,6-trinitrotoluene (15N3-TNT) was synthesized according to methods compiled by Kro¨ger and Fels (21). The starting material was analytical grade toluene (Fluka) and 98 atom % of enriched 15N nitric acid in an aqueous solution of 40% (Sigma-Aldrich, Steinheim, Germany). The reaction was performed in combination with oleum (H2SO4 with 20% SO3, Riedel-de Hae¨n, Seelze, Germany), giving a yield of 8% 2,4-dinitrotoluene and 92% TNT. The molecular masses of the obtained products were confirmed by GC-MS. Three-fold crystallization from boiling methanol yielded a highly pure fraction the molecular mass of which was confirmed by GC-MS and HPLC-MS. The Meisenheimer dihydride complex of 15N3-TNT was generated through stepwise reduction of a 0.5 mM aqueous solution using KBH4 (Fluka, Buchs, Switzerland) and the progress of the reaction was monitored by HPLC. Authentic 14N3 4-hydroxylaminodinitrotoluene was from Accustandard (New Haven, CT). All other reagents and solvents were of analytical grade. Analytical Methods. GC-MS was performed on an Agilent (Waldbronn, Germany) 6890 GC coupled to a 5972A mass detector. Standard HPLC analyses were performed on an Agilent model 1050 as described previously (12). HPLC-MS data were generated on an HPLC system (Waters, Milford, MA) consisting of a 2695 XE Alliance separation module coupled to a 2996 photodiode array detector and a Quattro micro API-ESI tandem quadrupole mass spectrometer. Instrument control, data collection, analysis, and management were by the Empower 2/MassLynx 4.0 software packages. Separation was performed on a narrow-bore, 4 µm C-8 Nova-Pak column (Waters) of 2.1 by 150 mm with VOL. 43, NO. 8, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Hitherto proposed reactions for the aerobic release of nitrite from biotransformed derivatives of TNT. (1) Nitroarene reductase; (2) nitro reductase; (3) putative mutase; (4) ammonium lyase; (5) nitrobenzene dioxygenase; (6) dinitrotoluene dioxygenase; (7) hypothetic reductive denitrase; and (8) chemical rearomatization of the Meisenheimer dihydride complex. 50% (v: v) acetonitrile: water as the eluent, at a flow rate of 0.25 mL min-1. Detection of compounds was performed in the negative ionization method. Searches using the SIR method for scanning further potential products for molecular masses of 226, 227, 228, and 229 were run additionally. Condensation Reactions of Reactive Intermediates. In principle, the interaction between the two reactive intermediates of TNT catabolism, of the 4-hydroxylaminodinitrotoluene (4HADNT) and of the Meisenheimer dihydride complex of TNT was performed as previously described (12). Here, the authentic standard of 0.47 mM 4HADNT was mixed with an equivalent amount of the Meisenheimer dihydride complex obtained from 15N3-TNT and the reaction followed by HPLC which was complete after about 3 h at 25 °C.
Results and Discussion To determine the mechanism of the condensation reaction, the following reactions had to be performed in the absence of biocatalysts. This is due to the unavailability or inexistence of type II OYE hydride transferases which catalyze exclusively the reduction of the TNT’s nitroarene ring but not of its nitro groups. Furthermore, as either type I or type II hydride transferases generally furnish a mixture of 2-hydroxylaminoand 4-hydroxylaminodinitrotoluenes (16, 17), a chemical model system as already established previously (12) had to be used. 2774
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The progress of the reaction of 4-hydroxylaminodinitrotoluene with the Meisenheimer dihydride complex derived from 15N3-TNT (97% purity) was nearly identical to the previously reported reaction kinetics with 2-hydroxylaminodinitrotoluene ((12), not shown). However, we found clear evidence for the generation of both of the structural isomers of the earlier reported diarylamines; the relative retention times and UV-vis spectra obtained on the HPLC-DAD system of N,N-bis(3,5-dinitrotolyl) amine (RT ) 17.5 min) and N-(2methyl-3,5-dinitrophenyl)-4-methyl-3,5-dinitroaniline (RT ) 15.8 min) were identical to the previously reported data. Their re-evaluation on the HPLC-MS system confirmed this finding (Figure 2). This result can only be explained by a reaction involving both the equivalent ortho and para nitro groups of the Meisenheimer dihydride complex of TNT which, indeed, should be available in equal amounts within their equilibrium (21), although often in the literature only the protonated nitro group in para position is depicted in the respective figures. Furthermore, the analysis of the reaction products by HPLC-MS in the negative ionization mode (Figure 2B and C), presenting pseudo molecular masses of 378 [M-H]-, clearly showed a difference of two mass units when masses of the found products were compared to those of the final products obtained from the condensation of nonlabeled
FIGURE 2. Ion stream chromatogram (TIC, upper A) of the separation of both structural isomers of secondary diarylamines obtained from the condensation of 14N3 hydroxylaminodinitrotoluenes and the Meisenheimer dihydride complex of 15N3-TNT. Below (B, C), the two mass spectra of the respective peaks at retention times of 15.8 and 17.5 min are given. parent compounds (12). This explains unambiguously that the nitrite released during the condensation of both classes of compounds originated from the Meisenheimer dihydride complex of the 15N3-TNT, and not from the reactive hydroxylamine. The obtained mass spectra were in full accordance with the mixed isotopic pattern of the two synthons. This result allows the proposal of the reaction sequence shown in Figure 3, which permits the following interpretation: (1) the two secondary diarylamines of identical molecular masses are obtained by elimination of nitrite and water from the protonated dihydride complex in a concerted action in the protic (aqueous) solvent system, and (2) the two isomeric forms are furnished by condensation of 4HADNT with equivalent isoforms of the Meisenheimer dihydride complex. As a consequence, the previously established model needs to be modified insofar as the formation of the two reported secondary diarylamines (12) does not necessarily require the simultaneous presence of 2HADNT and 4HADNT to furnish the symmetrical N,N-bis(3,5-dinitro-
tolyl) amine and the asymmetrical N-(2-methyl-3,5dinitrophenyl)-4-methyl-3,5-dinitroaniline: we now show that only 4HADNT is required for the condensation with both isoforms of the dihydride complex of TNT to form the two structural isomers (Figure 3). Theoretically, an additional product from the condensation of 2HDNT with the nitro group in the ortho position of the Meisenheimer dihydride complex could be expected but we were not able to detect it which may be due to sterical hindrance within the condensation reaction or its generation at only minute amounts. The current study of the condensation of reactive intermediates of TNT biotransformation highlights the importance of the formation of Meisenheimer dihydride complexes for the release of nitrite from TNT. Although bacteria which use TNT as a nitrogen source might employ other catalytic routes to obtain nitrogen from this xenobiotic compound (Figure 1) the mechanism described is the only fully proven pathway initiated by nitroreductases. Since most microorganisms exhibit the cometabolic or VOL. 43, NO. 8, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 3. Suggestion for the mechanism of condensation of 14N3 4-hydroxyl-aminodinitrotoluene and two isoforms of the 15 N3-labeled Meisenheimer dihydride complex of TNT to secondary diarylamines and the concomitant liberation of nitrite. fortuitous capacity to reduce the nitro groups of nitroaromatics and many may harbor enzymes capable of producing Meisenheimer complexes (16), the subsequent chemical condensation of biologically activated TNT may constitute a key reaction in TNT-polluted environments. However, the importance of diarylamine formation for TNT degradation in the environment remains unknown as these hitherto unknown products or their further reduced derivatives have not yet been identified in TNT-contaminated sites.
Acknowledgments
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(9)
(10)
(11)
We thank Patricia Godoy for GC-MS analysis and Lourdes Sa´nchez of the Servicio de Instrumentacio´n Cientı´fica of the EEZ for running the HPLC-MS system. (12)
Supporting Information Available Figure S1, plot of an HPLC-MS chromatogram (TIC) showing the separation of the synthesized 15N3-TNT and the corresponding mass spectrum recorded under negative ionization, confirming its identity. Figure S2, HPLC-MS chromatogram of the separated diarylamines in four selected channels of the SIM mode confirming their masses. This material is available free of charge via the Internet at http://pubs.acs. org.
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