Identification of Products Resulting from the Biological Reduction of 2

Molecular an Cellular Biology of Plants, Apdo Correos 419,. 18008 Granada, Spain. Pseudomonas sp. clone A is able to use 2,4,6- trinitrotoluene (TNT),...
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Environ. Sci. Technol. 1996, 30, 2365-2370

Identification of Products Resulting from the Biological Reduction of 2,4,6-Trinitrotoluene, 2,4-Dinitrotoluene, and 2,6-Dinitrotoluene by Pseudomonas sp. A L IÄ H A ¨I D O U R A N D J U A N L . R A M O S * Consejo Superior de Investigaciones Cientı´ficas, Estacio´n Experimental del Zaidı´n, Department of Biochemistry and Molecular an Cellular Biology of Plants, Apdo Correos 419, 18008 Granada, Spain

Pseudomonas sp. clone A is able to use 2,4,6trinitrotoluene (TNT), 2,4-dinitrotoluene, and 2,6dinitrotoluene as an N-source after the enzymatic removal of nitro groups from the aromatic ring. We have identified in culture supernatants a hydrideTNT Meisenheimer complex, which is an intermediate in the removal of the first nitro group from the TNT aromatic ring. It has been shown in vitro that the hydride-TNT Meisenheimer complex is transformed to 2,4-dinitrotoluene and an unidentified compound, the molecular mass of which is 235. As side metabolites, reduced TNT derivativesswhich cannot be used as an N-source by this bacteriumsappeared in culture supernatants. 2-Hydroxylamino-4,6-dinitrotoluene, 4-hydroxylamino-2,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene, 2-amino-4,6-dinitrotoluene, and 2,4-diamino6-nitrotoluene were found and chemically characterized. Spontaneous condensation of partially reduced TNT forms leads to the production of azoxytoluenes, and a possible condensation mechanism is discussed. For novel compounds, 1H NMR, UV, and infrared data are provided. Therefore, Pseudomonas sp. clone A carries out two different initial reductive reactions in the metabolism of 2,4,6-trinitrotoluene; one process removes a nitro group from the aromatic ring and allows it to be used as an N-source, while the other gives rise to side products. The biological elimination of xenobiotic compounds is an area of current interest. Many synthetic organic compounds can be used by microbes as the sole source of carbon and energy. Among such compounds, benzene, xylenes, and other aromatic compounds are mineralized by microbes, i.e., transformed into CO2 and H2O (1). Aromatics * Corresponding author telephone: +34-58-121011; fax: +34-58129600.

S0013-936X(95)00824-8 CCC: $12.00

 1996 American Chemical Society

substituted with nitro or amino groups can be used not only as a C-source but also as the sole N-source. A number of microorganisms able to use nitrobenzoates and monoand dinitrotoluenes have been described (2-7). 2,4,6Trinitrotoluene (TNT) is a compound relatively refractory to biological degradation, although it can be metabolized by microbes (8-14). Reduction of nitro groups on the aromatic ring and subsequent condensation of the reduced forms have been reported when microbes are exposed to TNT (15-18). These side products appear even in culture fluids of microbes able to use TNT as the sole C- and N-sources (5). Pseudomonas sp. clone A was originally isolated as able to use TNT as the sole N-source, although the bacterium could not use it as the sole C-source (9). Identification of dinitrotoluene (DNT), mononitrotoluene, and toluene in the culture fluid of this bacterium after growth with TNT as an N-source and fructose as a C-source revealed that the microorganism progressively removed nitro groups from the aromatic ring, although the enzymatic mechanism(s) behind these reactions has not been elucidated. The transference to Pseudomonas sp. clone A of the TOL plasmid derivative pWW0-Km (19), which encodes the enzymes for the mineralization of toluene to CO2 and H2O via benzoate and catechol and carries a kanamycin marker, conferred to the host bacterium the ability to grow on TNT as the sole N-, C-, and energy source (9). However, quantitative analyses revealed that at least 30% of the total TNT was misrouted to reduced forms of TNT, some of which formed condensation products (9). The use of microbes as decontamination agents requires the complete conversion of the target compound to CO2 and water. To isolate new microbes able to use the side products resulting from metabolism of TNT by Pseudomonas sp. clone A, we analyzed the products resulting from the metabolism of TNT and DNTs by this bacterium. These products were isolated and are being used to isolate bacteria able to further metabolize them.

Materials and Methods Microorganism, Media, and Culture Conditions. Pseudomonas sp. clone A was grown at 30 °C in M8 minimal medium with fructose (0.5% [wt/vol]) (9). As nitrogen sources 0.1-2 g/L TNT or 2,4-DNT and 2,6-DNT supplied as crystals in excess of their solubility in water were used. The microbe can be obtained from Dr. Jose´-Miguel Oliva, UEE, Avd del Parteno´n 16, Madrid, Spain. Chemicals. TNT was obtained from the Unio´n Espan ˜ ola de Explosivos (Madrid, Spain) and was more than 99% pure. 2,4- and 2,6-dinitrotoluene, 4-methyl-3-nitroaniline, and 2-methyl-5-nitroaniline were purchased from Aldrich Chemicals. Isolation of Metabolites. Cells were grown for 1-7 days under aerobic conditions at 30 °C and were removed by centrifugation at 5000g for 10 min. Culture fluids were extracted twice with ethyl acetate. The extracts were dried over anhydrous sodium sulfate, and excess solvent was usually removed by evaporation under reduced pressure at 35 °C. The extracted metabolites were then dissolved in a small volume of acetonitrile or deuterated acetone. When required, the extracts were fractionated on a silica gel

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column (0.040-0.063 mm [230-400 mesh ASTM]) under pressure (0.2 kg/cm2), using mixtures of hexane-CH2Cl2 and CH2Cl2-ethyl acetate as eluents in order of increasing polarity. Analytical Methods. High-performance liquid chromatography (HPLC) was done on a Hypersil ODS-C-18 column (10 × 0.46 cm) with acetonitrile:water (60:40) as the mobile phase, at a flow rate of 0.7 mL/min. Aromatic compounds were detected at 254 nm with a HewlettPackard visible-UV detector. The TNT-Meisenheimer complex was monitored at 475 nm. Gas chromatography/mass spectrometry (GC/MS) analyses were done at the University of Granada Technical Services Center with an HP-5890 GC/MS system. Mass spectra were recorded at an ionization energy of 70 eV, and methane gas was used for chemical ionization. 1H nuclear magnetic resonance (1H NMR) spectra were recorded with Bruker ARX400 (400-MHz) and Bruker AM300 (300-MHz) spectrometers on solutions prepared in deuterated acetone. Tetramethylsilane was used as an internal standard. 13C nuclear magnetic resonance (13C NMR) were recorded with a Bruker 300-MHz spectrometer on solutions prepared in deuterated acetone. Elemental analyses were done in a Perkin Elmer 240C thermal conductivity apparatus. Cell-Free Extracts. Pseudomonas sp. clone A was grown on minimal medium with TNT as the sole N-source. Cells were harvested by centrifugation (5000g 10 min), washed once in 50 mM phosphate buffer, resuspended in 5 mL of the same buffer, and disrupted in a French Press at 120 MPa. Cell debris was removed by centrifugation (10000g 10 min), and the clear supernatant was used as a source of cell-free extract. Chemical Synthesis of Azoxytoluenes Derived from DNT. The three possible azoxytoluenes derived from 2,4DNT were synthesized from mononitromonoaminotoluenes after oxidation of these compounds with metachloroperbenzoic acid, as described by McCormick and Stizmann (16, 20). Chemical Synthesis of Hydride-TNT Meisenheimer Complexes. Hydride-TNT complexes were prepared basically as described by Taylor (21) and Kaplan and Siedle (13) except that either tetramethylammonium borohydride or potassium borohydride were used. The 1H NMR data were in agreement with Kaplan and Siedle (22) and Vorbeck et al. (23). The maximum peak was found at 475 nm (log  4.39). Spectrophotometric and Infrared Data of Compounds Isolated in This Study. 2,4-Diamino-6-nitrotoluene: UV λACN nm, 214 (log  4.5); λACN nm: 242 (log  4.5); IR νKBrmax cm-1: 3540, 3350, 1640, 1510, 1350, 1160, 850, 750. 2-Hydroxylamino-4,6-dinitrotoluene: UV λACN nm, 221 (log  4.23); IR, νKBrmax cm-1: 3633, 3492, 3315, 3106, 2924, 1702, 1618, 1533, 1434. 2-Amino-4,6-dinitrotoluene: UV λACNmax nm, 233 (log :4.41); 318 (log  4.17). 2,2′,6,6′-Tetranitro4,4′-azoxytoluene, UV λACNmax nm, 238 (log  4.34); IR νKBrmax cm-1: 3100, 1620, 1540, 1490, 1350, 910, 865, 830, 730. 4,4′-Dinitro-2,2′-azoxytoluene: λACNmax nm, 271 (log  4.39); IR νKBrmax cm-1: 3160, 3120, 2940, 1620, 1540, 1490, 1465, 1360, 1270, 1150, 1090, 910, 890, 870, 840, 830, 750. 6,6′-Dinitro-2,2′-azoxytoluene: UV λACNmax nm, 238 (log  4.17); IR νKBrmax cm-1: 3130, 2970, 1640, 1540, 1470, 1440, 1385, 1360, 1060, 920, 890, 830, 790, 765, 740, 700, 680.

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FIGURE 1. New unidentified products resulting from the reduction of TNT in culture supernatants of Pseudomonas sp. clone A.

Results Reduced Forms Resulting from TNT Metabolism by a Pseudomonas Strain. Culture supernatants of Pseudomonas sp. clone A grown on TNT were extracted with ethyl acetate. The extract was evaporated, and compounds soluble in ethyl ether and methylene chloride were removed. The remaining products of the ethyl acetate extract were dissolved in dimethyl sulfoxide-d6, and 1H NMR spectra were recorded with a Bruker ARX400 spectrometer at 400 MHz. We identified a compound whose spectroscopic characteristics (2 H, 3.92 ppm, br s; H, 8.31 ppm, t (2.25 Hz); CH3, 2.49 ppm) coincided with those of the chemically synthesized H--TNT Meisenheimer complex. Products from ethyl acetate extraction were fractionated in silica columns and analyzed by IR, GC/MS, and 1H NMR. The following compounds were identified in the culture supernatants: 2-amino-4,6-dinitrotoluene; 4-amino-2,6dinitrotoluene; 4,6-dinitro-2-hydroxylaminotoluene; 2,6dinitro-4-hydroxylaminotoluene, 2,2′,6,6′-tetranitro-4,4′azoxytoluene, 4,4′,6,6′-tetranitro-2,2′-azoxytoluene, and 2′,4,6,6′-tetranitro-2,4′-azoxytoluene. These compounds had been isolated before from culture supernatants of other microorganisms, and our IR, 1H NMR, and mass spectrometric data were in agreement with those reported by Stizmann (20), Kaplan and Siedle (15), and Nielsen et al. (24). We also identified 2,4-diamino-6-nitrotoluene (compound I in Figure 1 and Table 1), which was reported as a side metabolite in cultures of Pseudomonas fluorescens but had not been fully characterized. In addition, we identified a new product, namely, 2,4′,6,6′-tetranitro-2′,4azoxytoluene (compound II in Figure 1 and Table 1). Ultraviolet spectra of the last two pure compounds were recorded, and their  value at the wavelength at which they exhibited a maximum are given in Materials and Methods. The amount of the above compounds recovered from a culture supplemented with 2 mg of TNT/mL are given in Table 2. About 25% (wt/wt) of the initial TNT load was recovered as azoxytoluenes. Most of these products accumulated linearly with time (not shown). The spectroscopic characteristics of compounds I and II are described below. We have now identified 2,4diamino-6-nitrotoluene in the culture fluids and provided

TABLE 1 1H NMR Data

of 2,4-Diamino-6-nitrotoluene and Azoxytoluenes Derived from TNT or DNT Metabolism by Pseudomonas sp. clone Aa compound analyzed

H-3 H-4 H-5 H-6 H-7 H′-3 H′-4 H′-5 H′-6 H′-7 -NH2 a

I

II

6.32; d (2.5 Hz)

9.14; d (3.30 Hz)

8.88; d (2 Hz)

8.81; d (2 Hz)

8.94; d (2 Hz)

6.44; d (2.5 Hz)

8.94; d (2.30 Hz)

2.10; s

2.53; s 8.75; s

8.33; dd (2; 8 Hz) 7.70; d (8 Hz) 2.70; s 8.93; d (2 Hz)

8.28; dd (2; 8 Hz) 7.75; d (8 Hz) 2.60; s 9.14; d (2 Hz)

8.25; dd (2; 8 Hz) 7.82; d (8 Hz) 2.90; s 9.17; d (2 Hz)

8.57; dd (2;8 Hz) 7.70; d (8 Hz) 2.75; s

8.43; dd (2; 8 Hz) 7.75; d (8 Hz) 2.75; s

8.62; dd (2; 8 Hz) 7.82; d (8 Hz) 3.02; s

8.75; s 2.55; s

IV

V

VI

VII 8.02; dd (2;8 Hz) 7.45; t (8 Hz) 7.86; dd (2; 8 Hz) 2.47; s 8.28; dd (2; 8 Hz) 7.52; t (8 Hz) 7.86; dd (2; 8 Hz) 2.56; s

3.95; m

s, singlet; d, doublet; dd, double doublet; t, triplet; m, multiplet.

TABLE 2

Compounds Recovered from Culture Supernatants of Pseudomonas sp. Clone A Growing with TNTa compound 4,4′,6,6′-tetranitro-2,2′-azoxytoluene 2′,4,6,6′-tetranitro-2,4′-azoxytoluene plus 2,4′,6,6′-tetranitro-2′,4-azoxytoluene 2,2′,6,6′-tetranitro-4,4′-azoxytoluene 2-hydroxylamino-4,6-dinitrotoluene plus 4-hydroxylamino-2,6-dinitrotoluene 2-amino-4,6-dinitrotoluene plus 4-amino-2,6-dinitrotoluene 2,4-diamino-6-nitrotoluene

biotransformation (% of total TNT) 13.2 8.7 3.2 5.5 4 0.8

a30

mL of M9 minimal medium with 60 mg of TNT and 0.5% (wt/vol) fructose was inoculated with Pseudomonas sp. clone A to an initial cell density of about 0.05 unit at 660 nm. The culture was then incubated at 30 °C until the TNT was exhausted. At that time, the density of the culture at 660 nm was about 1.2 units. The proportion of products resulting from TNT reduction is given. In similar assays, the same products were recovered at similar ratios.

the first available 1H NMR data for this compound (Table 1). The spectroscopic data were confirmed by comparison with chemically synthesized product by reduction of TNT with S(NH4)2. The molecular mass of the product isolated from the culture and the chemically synthesized product (168) were determined by chemical ionization mass spectrometry, and its UV spectra was recorded with a Shimadzu spectrophotometer. It showed a peak at 257 nm with 1mM1cm ) 15 000. Azoxytoluenes derived from the reduction of TNT have been reported before (16, 21). We found four TNT-derived azoxytoluenes in the culture supernatant of Pseudomonas sp. clone A; three of them 2,2′,6,6′-tetranitro-4,4′-azoxytoluene; 4,4′,6,6′-tetranitro-2,2′-azoxytoluene, and 2′,4,6,6′tetranitro-2,4′-azoxytoluene (compound III in Figure 1) have been described before (25). The isomer 2,4′,6,6′-tetranitro2′,4-azoxytoluene (II in Figure 1) is described for the first time. Table 1 shows its 1H NMR data, which are in agreement with Sitzmann’s proposal for the chemically equivalent protons H′-3 and H′-5 in the substance II. These protons appeared as a single chemical shift at δ 8.75 ppm. In contrast, protons H-3 and H-5 in compound III appeared at 9.07 ppm. The difference might be explained by the proximity of oxygen to the phenyl ring bearing these protons, in agreement with Freeman’s observation (25).

Molecular mass (406) was determined by chemical ionization mass spectrometry and elemental analysis. In Vitro Metabolism of the Meisenheimer Complex. Pseudomonas sp. clone A was grown on minimal medium with TNT, and cell-free extracts were prepared from cultures in the exponential growth phase. The appearance of TNTMeisenheimer complex products was followed by gas chromatography after extraction with ethyl acetate, as described in Materials and Methods. Two products were found whose retention times were 10.5 and 10.65 min (Figure 2). The retention time of the former product was identical to that of 2,4-dinitrotoluene, suggesting the removal of a nitro group from the TNT-Meisenheimer complex. The nature of this product was further confirmed by mass spectrometry (not shown). The molecular mass of the second product was found to be 235. The pattern of molecular fragmentation of this product is consistent with a linear form, namely, 1,3,5-trinitroheptane or 4-methyl-1,3,5-trinitrohexane (Figure 3), although further analysis needs to be done to identify unequivocally the compound. TNT was also identified, suggesting oxidation of the TNTMeisenheimer complex. Reduction of TNT in Vitro. To determine whether the appearance of the azoxyderivatives was spontaneous, TNT was partially reduced in vitro under anaerobic and aerobic conditions with S(NH4)2. Under a nitrogen atmosphere, the addition of less than stochiometric amounts of S(NH4)2 to reduce one nitro group of TNT resulted in the corresponding hydroxylamino- and monoaminodinitrotoluenes, as monitored after separation of products by HPLC with UV detection (λ ) 254 nm), and no axozytoluenes were detected (Table 3). When S(NH4)2 was added to TNT under air, at least two azoxytoluenes appeared (Table 3) in addition to the above monomers; this further suggested their spontaneous generation in the presence of molecular oxygen. Reduction of Dinitrotoluene by Pseudomonas sp. Clone A. Pseudomonas sp. clone A was able to grow with 2,4- and 2,6-DNT as an N-source in addition to TNT. Bacteria grew with a generation time of 12 h. From cultures grown with 2,4-DNT, we isolated the two possible monoamino derivatives, namely, 2-amino-4-nitrotoluene and 4-amino-2nitrotoluene. These compounds were characterized by comparison with authentic samples, and the 1H NMR and IR data were identical to those published in the Aldrich

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TABLE 3

In Vitro Reduction of TNT under Anaerobiosis and Aerobiosisa concentration (µM) products identified

anaerobiosis

aerobiosis

monohydroxylaminodinitrotoluene monoaminodinitrotoluene azoxytoluenes TNT (unreacted)

164 25 0 58

35 47 174 57

a TNT (220-250 µM) disssolved in methanol was bubbled with air or maintained under N2 for 30 min. Then S(NH4)2 was added to reach concentration of 140 µM. The reaction was allowed to proceed for 2 h at 75 °C, after which time an aliquot of the reaction mixture was taken and immediately injected into a high-performance liquid chromatography apparatus as described in Materials and Methods. The amount of each product formed was estimated from the area of the corresponding peak in the chromatograms.

FIGURE 2. Products resulting from H--TNT metabolism in vitro. The reaction mixture consisted of 100 mM phosphate buffer, pH 7.0, 1 mM H--TNT, and 15 mg of protein/mL. The reaction was allowed to proceed for 20 min, and then the whole reaction was extracted twice with ethyl acetate as described in Materials and Methods. Products were separated by GC with an HP-1 column (cross-linked methylsilicone, 25 m × 0.32 mm × 0.52 µm film thickness) with a gradient from 100 to 250 °C using temperature increases of 5 °C/min, and N2 as the carrier gas. The injector temperature was 250 °C, and the FID detector temperature was 250 °C. Retention times were 1.6, 1.7, and 2.0 min for solvent; 10.5 min for 2,4-dinitrotoluene; 10.65 min for an unidentified compound that may be either 1,3,5-trinitroheptane or 4-methyl-1,3,5-trinitrohexane; and 12.0 min for TNT.

FIGURE 3. Mass spectrum of 1,3,5-trinitroheptane or 4-methyl-1,3,5trinitrohexane resulting from hydride-TNT metabolism in vitro.

Library (26, 27). From these cultures, we isolated and identified only three azoxytoluenes derived from 2,4-DNT metabolism, namely, 4,4′-dinitro-2,2′-azoxytoluene (compound IV in Table 1), 2,2′-dinitro-4,4′-azoxytoluene (compound V in Table 1), and 2,4′-dinitro-2′,4-azoxytoluene (compound VI in Table 1). Their 1H NMR data, which were unknown, are now reported in Table 1. Chemical synthesis

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of these azoxy derivatives as described in Materials and Methods allowed us to attribute each spectrum to the corresponding isomer. The molecular mass of these compounds (316) was determined by chemical ionization mass spectrometry (CH4). From the culture supernatant of bacteria grown with 2,6-DNT, we isolated 2-amino-6-nitrotoluene, which was identified by comparison with an authentic sample. Its 1H NMR and IR data were identical to those published in the Aldrich Library. 6,6′-Dinitro-2,2′-azoxytoluene (compound VII, see Table 1), the sole possible azoxytoluene expected, was also found and isolated, and its 1H NMR data are reported for the first time in Table 1. Chemical ionization mass spectrometry of this dimer showed [M + 1]+ at m/z 317.

Discussion Pseudomonas sp. clone A seems to perform two different types of reduction processes with TNT. One of the processes leads to the removal of a nitro group from the aromatic ring and allows its utilization as an N-source, while the other leads to unproductive compounds that accumulate in culture medium. We previously proposed that removal of the first nitro group from TNT to yield DNTs might occur via a Meisenheimer complex (9). However, no biochemical or chemical evidence for this proposal was provided. Given that we have now identified in culture supernatants trace amounts of a compound with the Meisenheimer structure of the complex for TNT and that the hydride-TNT complex was a substrate for the removal of a nitro group in in vitro assays with cell-free extracts prepared from Pseudomonas sp. clone A, we suggest that the hydride-TNT complex is a true intermediate in metabolism of TNT. In addition, we found a second product in vitro and in vivo that resulted from the metabolism of the hydride-TNT Meisenheimer complex. The molecular mass of the product was 235. This product may have been formed after reduction of the TNTMeisenheimer complex and redistribution of the electrons to yield 1,3,5-trinitroheptane or 4-methyl-1,3,5-trinitrohexane. Regardless of the mechanism by which this product is produced, its appearance in vivo is evidence that the TNT-Meisenheimer complex was generated in vivo. Reduction of TNT to hydroxylaminodinitrotoluenes and monoaminodinitrotoluene derivatives was found. It has been proposed that the reduction of aromatic nitro groups to amino groups proceeds through nitroso and hydroxy-

lamino intermediates. Whereas the isolation and characterization of nitroso intermediates have not been reported except for the reduction of 2,4-dinitrotoluene by Liu et al. (28), we isolated and characterized the possible hydroxylamino intermediates in the reduction of TNT, namely, 2-hydroxylamino-4,6-dinitrotoluene and 4-hydroxylamino2,6-dinitrotoluene. Further reduction of these compounds rendered 2-amino-4,6-dinitrotoluene and 4-amino-2,6dinitrotoluene, which were also found in the bacterial culture supernatant. Because the amount of 2-amino-4,6dinitrotoluene was similar to that of 4-amino-2,6-dinitrotoluene, it seems that either the ortho or the para nitro group can be reduced. Trace amounts of 2,4-diamino-6nitrotoluene were found, suggesting that reduction of the second nitro group does not start until the first one is fully reduced. Biological degradation of certain nitroaromatic compounds such as 4-nitrobenzoate involves the initial reduction of nitro groups to hydroxylamino groups, then the release of hydroxylamino, and the concomitant dihydroxylation of the aromatic ring (4, 5). This yields 3,4-dihydroxybenzoate, which was the substrate for ring cleavage. In the degradative pathway of nitrobenzene by P. pseudoalcaligenes, the initial product hydroxylaminobenzene is further converted to 2-aminophenol by an enzyme-assisted Bamberger-like rearrangement. 2-Aminophenol was shown to be the substrate for ring cleavage (29). Hydroxylaminodinitrotoluenes are therefore compounds of interest for the isolation of microbes able to use reduced TNT derivatives. The production of azoxytoluenes in the metabolism of TNT by microbes has frequently been observed (9, 13, 14, 17, 18, 20, 25). Two findings suggest that this is a spontaneous process in which O2 is involved: (i) after partial reduction of TNT with S(NH4)2 in vitro, no azoxytoluenes appeared when the reaction occurred anaerobically (Table 3), and (ii) azoxytoluenes appeared when TNT was reduced in air. We suggest a possible mechanism for the production of azoxytoluenes in vitro: In the presence of O2, R-NHOH intermediates are oxidized to R-nitroso intermediates (as suggested in step 1), which react with R-NHOH compounds (as suggested in step 2), leading to the production of azoxytoluenes: 2R

NHOH + O2

2R

NHOH + 2R

N

O

2R N O

O + 2H2O

(step 1)

2R N

N R + 2H2O

(step 2)

O

4R

NHOH + O2

2R N

N R + 4H2O

We do not have in vivo evidence for the production of RsNdO intermediates, but they could be formed after reduction of a nitro group (2 e-) on the TNT aromatic ring. If this were the case, RsNdO could be a source of azoxytoluenes; otherwise they could be formed as suggested above for the in vitro process. Of the four possible TNTderived azoxytoluenes, the spectroscopic characteristics of three of them have been determined previously (20); in this study, we determined the spectroscopic characteristics of 2,4′,6,6′-tetranitro-2′,4-azoxytoluene (see Table 1).

In agreement with previous observations, the use of DNTs as an N-source by Pseudomonas sp. clone A involves the removal of one or both nitro groups (9). In addition to removing nitro groups, this bacterium can also reduce DNTs to amino derivatives, which are not used as N-sources. In the present study, we found all possible monoaminomononitrotoluenes derived from 2,4-DNT and 2,6-DNT in culture supernatants. The reduction of these probably occurs via hydroxylamino intermediates, since under aerobiosis all possible azoxytoluenes resulting from the condensation of two rings were also found in culture supernants. The 1H NMR data of these azoxytoluenes were determined in this study and are given in Table 1. The complete mineralization of TNT and DNTs to CO2, NO2-, and H2O is a sine qua non for the bioremediation of polluted sites. The biological reduction of TNT and DNTs to recalcitrant amino derivatives and the production of more recalcitrant azoxytoluenes represent a serious obstacle to the process.

Acknowledgments This work was supported by a grant from the Unio´n Espan ˜ ola de Explosivos. We thank Alejandro Ferna´ndezBarrero for laboratory facilities at the Department of Organic Chemistry of the University of Granada, and we thank the University of Granada Technical Services for assistance with 1H NMR and GC/MS. We also express our appreciation to Jose´ Miguel-Oliva for support and critical reading of the manuscript.

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Received for review November 2, 1995. Revised manuscript received February 28, 1996. Accepted March 18, 1996.X ES950824U X

Abstract published in Advance ACS Abstracts, May 15, 1996.