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Journal of Environmental Quality 31:437-444 (2002)
© 2002 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America

Article
SYMPOSIUM PAPERS

Enzymatic Transformation and Binding of Labeled 2,4,6-Trinitrotoluene to Humic Substances during an Anaerobic/Aerobic Incubation

S. Thielea, E. Fernandesb and J.-M. Bollag*,c

a Institut für Bodenkunde, Universität Rostock, Justus-von-Liebig-Weg 6, D-18051 Rostock, Germany
b LabAlliance, 349 N. Science Park Rd, State College, PA 16803
c Lab. of Soil Biochemistry, The Pennsylvania State Univ., 129 Land and Water, University Park, PA 16802

* Corresponding author (jmbollag{at}psu.edu)

Received for publication June 2, 2000.

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Organic pollutants are degraded in soil and simultaneously non-extractable residues are formed. However, proof is lacking that this fixation has a detoxifying effect. We investigated the transformation and binding of 2,4,6-trinitrotoluene (TNT) with catechol or soil humic acid as cosubstrates. Carbon-14-labeled TNT and its reaction products were quantified by radiocounting; extractable compounds were identified by high performance liquid chromatography (HPLC). Bound and extractable residues of 15N-labeled TNT and metabolites were studied by 15N nuclear magnetic resonance spectroscopy (15N NMR). Since TNT is not easily transformed under oxidizing conditions an anaerobic/aerobic treatment was used. Anaerobic microorganisms from cow manure were used to reduce TNT during the anaerobic phase and subsequently, a laccase from Trametes villosa was used in the aerobic phase to oxidatively couple the metabolites to humic matter. Seventy-four percent of TNT was immobilized with catechol as cosubstrate, but only 25% with humic acid. With catechol the main extractable component was TNT, while with humic acid it was mostly the metabolite 4-aminodinitrotoluene. For both co-substrates, the spectra of immobilized metabolites obtained by solid-state 15N-cross polarization magic angle spinning (CPMAS) NMR spectroscopy showed signals in the chemical shift region for protonated aromatic amino compounds. However, in the presence of catechol, an additional signal from non-extractable nitro groups was found, which could represent sequestered TNT. The partially reduced metabolites of TNT that formed non-extractable residues in humic acid are not likely to be remobilized easily and are thus regarded as detoxified.

Abbreviations: 4ADNT, 4-amino-dinitrotoluene • CPMAS, cross polarization magic angle spinning • DMP, 2,6-dimethoxyphenol • HPLC, high performance liquid chromatography • NMR, nuclear magnetic resonance • TNT, 2,4,6-trinitrotoluene


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TNT, a major soil pollutant of former ammunition plants, and its metabolites were found to be detoxified by bacteria or fungi through aerobic degradation (e.g., Schackmann and Müller, 1991; Bruns-Nagel et al., 1996; Bhadra et al., 1999), anaerobic co-metabolism (Boopathy et al., 1994; Fiorella and Spain, 1997; Drzyzga et al., 1999) and abiotic reduction (Hofstetter et al., 1999). These detoxification processes were used in composting techniques or bioreactor systems for the bioremediation of TNT contaminated soil (e.g., Bruns-Nagel et al., 1998; Lenke et al., 1998). While complete mineralization for dinitrotoluene was determined (Nishino et al., 1999), it has not been directly achieved from TNT. Instead, the formation of soil bound residues is the major detoxification pathway for TNT (Lenke et al., 1998; Achtnich et al., 1999a,c; Drzyzga et al., 1999; Bruns-Nagel et al., 2000). Therefore, 15N-cross polarization magic angle spinning (CPMAS) nuclear magnetic resonance (NMR) is a valuable tool to determine residues from N compounds (Clinton et al., 1995; Knicker et al., 1997a) and in particular, from TNT bound to soil organic matter (Knicker et al., 1999).

However, until now, the unambiguous characterization of the end products of TNT degradation and the structural identification of fixed TNT residues has not yet been completed (Rieger and Knackmuss, 1995; Daun et al., 1998; Achtnich et al., 1999a). The analytical determination of organic nitrogen compounds bound to soil humus is hampered because of the formation of various metabolites and a large number of different modes of binding which leads to low concentrations of each single compound (Schulten and Schnitzer, 1998). It is assumed that this is also valid for nitroaromatic compounds. Well-defined phenolic compounds, representing special sites of humic substances, can serve as models to elucidate enzymatic reactions or modes of binding. These compounds are major building blocks of humic polymers (Schnitzer, 1978; Bracewell et al., 1989) and were found to polymerize to humus-like substances (Haider et al., 1975; Suflita and Bollag, 1981). Subsequently, they are referred to as humic monomers. However, to what extent results derived from work with model humic substances correspond to those found for humic substances under field conditions is still uncertain.

The objectives of this study were to quantify and identify the enzymatic transformation and fixation of TNT in the presence of model humic compounds and to compare it with results from soil organic matter. For this purpose, 14C- and 15N-labeled TNT were incubated with or without anaerobic microorganisms obtained from cow manure and a laccase from Trametes villosa which served as biocatalysts. Humic acid from Hagerstown soil or catechol, a humic monomer, were added separately as co-substrates. To obtain adequate amounts of bound residues from TNT, an anaerobic/aerobic incubation procedure was carried out. The transformation of TNT was investigated by a sequential extraction procedure, radiocounting, HPLC, and liquid and solid state 15N NMR for the analysis of nitroaromatic compounds.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals
Technical grade TNT (90% purity) and TNT completely labeled with 14C (specific radioactivity 0.1 mCi mg-1) or 15N were obtained from the U.S. Army Center of Health Promotion and Preventive Medicine (USACHPPM; Aberdeen Proving Ground, MD). Nitrogen-15 TNT was determined to be 97% pure TNT with greater than 99% 15N. Catechol was purchased from Fisher Scientific Co. (Fair Lawn, NJ). Humic acid was purified from a Hagerstown (fine, mixed, mesic, Typic Hapludalf)–Opequon silt loam (clayey, mixed, active, mesic Lithic Hapludalfs).

Biocatalysts
An experimental laccase of Trametes villosa (EC 1.10.3.2 p-phenol oxidase) was obtained from Novo Nordisk (Danbury, CT). Enzyme activity was measured with 2,6-dimethoxyphenol (DMP) as the substrate. One DMP unit was defined as the amount of enzyme that causes a change in optical density of 1.0 min-1 at 468 nm when measured at pH 4.0 and 20°C. Anaerobic microorganisms were obtained from aqueous extracts of cow manure and preserved by storing them at -18°C in the dark. Anaerobic microorganisms were quantified through the test of the dissimilatory nitrate reductase activity (Abdelmagid and Tabatabai, 1987, modified).

Reaction Incubation and Sample Preparation
TNT was dissolved in boiling 0.1 M citric-phosphate buffer at pH 6.8. After mixing 0.4 mM TNT with 40 mM catechol or 150 mg L-1 humic acid in a total volume of 5.5 mL in 15-mL Corex centrifuge tubes, anaerobic microorganisms (exhibiting a nitrate reduction of 0.09 µg nitrite N in 24 h) plus 0.4% glucose, which served as an easily degradable substrate, were added to mediate the reaction (all final concentrations). During the 4-d anaerobic phase, samples were kept under a nitrogen atmosphere at 25°C in the dark and agitated by a horizontal shaker. Switching to a subsequent 2-d aerobic phase, the laccase was added to the sample (18 DMP units mL-1). Samples, listed in Table 1 were prepared in duplicate or triplicate.


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  		Table 1. Relative amounts of extractable nitroaromatic compounds of the different samples as determined by high performance liquid chromatography (HPLC) and percentage of extractable nitroaromatics not identified.{dagger}

 
Sample Extraction and Fractionation
The reaction was stopped with the addition of 10% conc. phosphoric acid. Samples were then centrifuged (12 000 x g, 10 min) and the supernatant decanted. Turbid samples were purified by filtration through glass wool. The residue from centrifugation was sequentially extracted (Kim et al., 1998, modified). This was done three times with 1 to 3 mL citric-phosphate buffer pH 1.7. Next, the residue was dissolved in 50 µL 0.5 M NaOH and extracted three times with 1 to 3 mL ethylacetate (filtration through Whatman #1 qualitative filter) (Whatman, Maidstone, UK). This was followed by a final extraction with 1 mL methanol. Each time extraction solutions were vortexed for 1 min and centrifuged for 10 min at 12 000 x g. Ethylacetate extracts were evaporated to dryness and redissolved in methanol. Aliquots of the supernatant and the extracts were then prepared for HPLC analysis. Insoluble residues were incinerated with a OX600 biological oxidizer (R.J. Harvey Instrument Corp., Hillsdale, NJ).

Sample Analysis
Isocratic HPLC analysis was carried out on a Waters system equipped with two model 510 HPLC pumps, a 717 Plus autosampler, an automated gradient controller and a Lambda-Max 440 UV detector operated at 254 nm (Waters Corporation, Milford, MA). A 250- by 4.6-mm Hyperchrome reversed-phase Nucleosil 120-5-C18 (Supelco, Bellefonte, PA) column was used. The mobile phase consisted of water to methanol ratio of 40:60 (v/v) with a flow rate of 0.5 mL min-1.

The initial radioactivity of 14C-labeled TNT was 0.36 µCi for all incubations. Radioactivity in liquid samples was analyzed with the help of a Beta Trac 6895 liquid scintillation counter (Tracor Analytic, Elk Grove, IL).

Nitrogen-15 liquid state NMR was conducted with a Bruker AMX2-500 spectrometer operating at 50.678 MHz with a one-pulse decouple sequence, 15N 60° pulse width of 10 µs, and a pulse relaxation delay of 10 s. The number of scans collected was 4782 and the line broadening applied during data processing was 1 Hz. The sample was dissolved in MeOD and externally referenced with neat dimethylformamide (103.8 ppm versus liquid NH3 at 25°C).

Nitrogen-15-CPMAS NMR was conducted on a Doty probe with a Chemagnetics CMX-300 spectrometer operating at 30.14 MHz. A relaxation delay of 4 s, 90° 15N pulse width of 7.14 µs, contact time of 1 ms, and spinning speeds of 4100 Hz were employed for the solid-state NMR study. The number of scans collected was 9192 (Sample VI) and 18 100 (Sample V); line broadening applied was 50 Hz. Samples were externally referenced with 15N-glycine (32.6 ppm versus liquid NH3 at 25°C).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
When anaerobic microorganisms obtained from cow manure were added to 0.4 mM solutions of TNT, a subtle transformation of TNT was observed (Sample III; Fig. 1) . After four days under anaerobic conditions approximately 10% of initially added radioactivity from 14C TNT was withdrawn from the supernatant. The addition of humic acid as a co-substrate for coupling reactions did not yield any further diminution of radioactivity (Sample V). However, in the presence of catechol as the co-substrate, transformation was increased to 46% when anaerobic microorganisms were present, compared with 11% when they were absent (Samples VI and VIII).



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  		Fig. 1. Transformation of 0.4 mM TNT (T) in the presence and absence of anaerobic microorganisms (M), laccase (L, from Trametes villosa), humic acid (H, from Hagerstown soil, 100 mg L-1), and catechol (C, 40 mM) after four days incubation at anaerobic conditions and subsequent two days of aerobic conditions, respectively.

 
When incubation was extended for two more days, while maintaining aerobic conditions, significant further changes in the distribution of the radioactivity were determined. The dissolved amounts of nitroaromatics diminished to between 81 and 75% in the presence of cow manure microorganisms alone or combined with laccase and humic acid (Fig. 1; Samples III–V). It was 61, 43, and 26% when TNT was combined with catechol plus laccase, catechol plus microorganisms, and catechol plus microorganisms and laccase, respectively. Almost no transformation of TNT was observed when microorganisms were sterilized by the addition of 10% concentrated H3PO4 prior to incubation or when only TNT was incubated (Sample I and II). Transformation was also negligible when only the laccase or catechol was added to TNT (Thiele et al., 1998).

To determine the fate of the nitroaromatics withdrawn from the supernatant, a sequential extraction procedure was carried out. Corresponding with findings from Hundal et al. (1997) mobile, mobilizable, and immobilized (not short-term bioavailable) amounts can be determined through chemical extraction with aqueous solutions, organic solvents, and incineration of the insoluble residue, respectively. The radioactivity recovered in the sorbed and non-extractable fractions was approximately in a 1 to 1 ratio. The amount of radioactivity extractable by organic solvents was highest in the presence of catechol plus laccase (Samples VI and VIII; Fig. 2) . The amount of fixed residues, non-extractable from the precipitate, was highest when catechol was combined with anaerobic microorganisms (Samples VI and VII). No precipitate was found when only laccase was added at the aerobic phase in the presence of catechol. Recovery of initially added 14C-label ranged between 106 and 85% in the samples, but was insufficiently low (78%) in Sample VII.



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  		Fig. 2. Distribution of radioactivity from 14C-labeled TNT over three fractions obtained from a sequential extraction procedure (see Materials and Methods; abbreviations see Fig. 1).

 
Extractable nitroaromatics were identified by HPLC with external standards of TNT and four likely metabolites (Table 1). While in control Samples I and II nearly 100% TNT resided, it was strongly metabolized in the presence of anaerobic microorganisms. The major metabolite formed was 4-amino-dinitrotoluene (4ADNT). When catechol was added, considerably more TNT was recovered in the extracts and the amount was highest in the presence of catechol plus laccase (22%, Sample VIII). However, the percentage of nitroaromatic compounds identified by HPLC was low as was calculated from the radioactivity in the extracts (12.5–100%). In most HPLC samples, strong and nonseparated peaks eluted with a low retention time (3.5–7.0 min). These were probably attributable to dissolved organic matter which is known to sorb or bind xenobiotics and thus hide them from ultra violet detection. Therefore, selected samples were analyzed by 15N NMR.

Liquid state 15N NMR analysis of organic extracts from Sample VI confirmed that additional metabolites (unidentified by HPLC) were formed from TNT (Fig. 3A,B) . In addition to signals arising from ortho- (366 ppm) and para-nitro groups (360 ppm), resonances were seen at 320, 146, and just above the baseline at 83 ppm. These were attributed to azoxydimers, hydroxylamine, and secondary amine, respectively. Other nitro-group signals were shifted downfield to around 372 ppm due to the conversion of neighboring nitro groups to amine and hydroxylamine (Achtnich et al., 1999a; Bruns-Nagel et al., 2000).



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  		Fig. 3. Nitrogen-15-NMR spectra of (A) methanol extract from 2,4,6-15N-labeled TNT incubated for six days under anaerobic/aerobic conditions in the presence of anaerobic microorganisms and catechol (Sample VI) and (B) standard solution of 2,4,6-15N-TNT in MeOD. Upper scale referenced to NH3, lower scale referenced to CH3NO2.

 
Insoluble residues of Samples V and VI were analyzed by 15N-CPMAS NMR (Fig. 4A,B) . The signals in the range of 50 to 155 ppm were quite similar in both samples. Most peaks in the range of 110 to 130 ppm could originate from background of the humic polymers. These include a wide range of compounds like lactams, amides, and tertiary amines (Knicker et al., 1997a). Since the background of the controls could not be investigated separately, the contribution from labeled metabolites could not be determined. However, some hundred thousand scans were necessary to obtain reasonable spectra from humic material with natural 15N content (e.g., Clinton et al., 1995), while 9192 and 18 100 scans were collected from Samples V and VI. Thus, the signals can probably be attributed to residues from 15N TNT. In both spectra, broad resonances were seen at 50 to 80 ppm, with a shoulder at around 62 ppm in Fig. 4A. The 40 to 60 ppm range covers primary amines and the 60 to 85 ppm range includes secondary amines (Knicker et al., 1997b). Thorn et al. (1996) attributed signals at 74 ppm to anilinohydroquinone, while chemical shifts at 103, 112, and 117 ppm, respectively, were assigned to anilinoquinone. The signal at 134 ppm was attributed to anilide and indole (Thorn et al., 1996; Thorn, 1997). Achtnich et al. (1999a) attributed signals in the range of 120 to 170 ppm to tertiary amines and amides. For both samples, broad resonances were determined at 118 to 146 ppm. The small signals at around 150 ppm in both spectra might be due to heterocyclic N, originating from condensation reactions (Knicker et al., 1999). However, the major difference is a large amount of unreduced nitro-N in the presence of catechol, exhibiting a strong signal at 364 ppm with a shoulder downfield.



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  		Fig. 4. Nitrogen-15-CPMAS NMR spectra of insoluble residues formed from 0.4 mM 2,4,6-15N TNT within six days incubation under anaerobic/aerobic conditions in the presence of anaerobic microorganisms, laccase, and (A) 40 mM catechol and (B) 100 ppm humic acid, respectively (Samples V and VI). Upper scale referenced to NH3, lower scale referenced to CH3NO2.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A two-step transformation process of TNT was observed during the incubation. In the first step, TNT was reduced under anaerobic conditions to amino compounds which coupled to form diazo and azoxy compounds. In some samples, dinitrotoluene was identified (Table 1) which indicates denitration or desamination, as it was found by e.g., Pasti-Grigsby et al. (1996), Hawari et al. (1998), and Vorbeck et al. (1998). Also during this step, the ability of the anaerobic microorganisms to reduce TNT became evident. It has been shown that intestinal microorganisms are effective in reducing TNT through an oxygen-insensitive NAD(P)H nitroreductase (Bryant and DeLuca, 1991). After incubation in the presence of anaerobic microorganisms, 4ADNT was the major nitroaromatic compound identified by HPLC which corresponded to results from Achtnich et al. (1999b) and Bruns-Nagel et al. (2000). However, when catechol was added as a model humic substance in the presence or absence of laccase, the portion of reduced nitroaromatics was considerably lower (Table 1). During the anaerobic phase, the vast majority of the nitroaromatics resided in the solution and were completely extractable. From the distribution of 14C radioactivity, nitroaromatics were determined to be either in solution or adsorbed/sequestered to the humic substances as was shown by Li et al. (1997) and Sheremata et al. (1999). In contrast, Daun et al. (1998), Drzyzga et al. (1999), and Achtnich et al. (1999a) found major percentages of TNT to be non-extractable after anaerobic treatment, though while using much longer incubation times.

In the second step, mainly in the aerobic phase, reduced nitroaromatics became subsequently bound to reactive sites of the humic compounds probably through oxidative cross-coupling reactions. Solid state 15N-NMR spectra indicated the presence of bound residues from primary, secondary, and tertiary amines and amides, anilinohydroquinones, and anilinoquinones. Corresponding to results from Dawel et al. (1997) and Bruns-Nagel et al. (2000), the covalent binding of partially reduced TNT and azoxy compounds can be postulated which increases during an anaerobic/aerobic treatment process in the presence of oxygen (Achtnich et al., 1999c). Preferred coupling sites are quinone moieties (Thorn et al., 1996). Enzymes like laccase, catalyze a one-electron reaction generating a free radical from humic monomers like catechol, initializing the formation of quinones and their subsequent polymerization. These highly reactive products can substantially enhance the oxidative cross-coupling of recalcitrant xenobiotics (Dec and Bollag, 1990; Bollag, 1992; Scott et al., 1998). Minor signals possibly arose from 15N bound in the form of anilide and indole, indicating condensation reactions of reduced TNT with aromatic humic structures (Thorn et al., 1996). Consequently the removal of nitroaromatics from solution was strongest in the presence of catechol plus laccase. In contrast, the removal of TNT from the supernatant was significantly smaller in the presence of the microorganisms alone and with or without laccase. It was only slightly increased when humic acid was added as a co-substrate (Fig. 1, Samples III–V). It is assumed that the amount of quinone structures and their steric accessibility, respectively, was much lower in humic acid compared with catechol.

The formation of quinone from catechol appears to be also promoted by phenoloxidase enzymes of the intestinal microorganisms, yielding a strong decrease of nitroaromatics in the supernatant even under anaerobic conditions. However in these samples (VI–VIII), considerable amounts of nitroaromatics were found to be extractable by organic solvents. Since the precipitated residue, which was non-extractable by acid aqueous solutions, was dissolved prior to ethylacetate extraction therefore sequestered nitroaromatics were most probably released in the course of this treatment. Polymerization of catechol is fast and can even proceed abiotically at a basic pH. Consequently, unaltered TNT can become sequestered in the precipitating polymer (Thiele et al., 1998). Accordingly, Bruns-Nagel et al. (2000) found a complete removal of signals from nitro groups of nitroaromatics associated with soil humic substances by extraction with 0.5 M NaOH. In the absence of intestinal microorganisms able to transform TNT into reactive reduced metabolites, only sequestration but no formation of non-extractable bound residues was observed (Fig. 2; Sample VIII).

In contrast to the results obtained from humic acid (Sample V), the 15N-CPMAS NMR spectrum from non-extractable residues of Sample VI containing catechol exhibited a strong signal from nitro N (around 364 ppm). Parts of this nitro were likely to be from bound azoxy- and amino-metabolites of partially reduced TNT which moved somewhat downfield from 364 ppm due to metabolism of nitro groups within the molecule (Achtnich et al., 1999a). Bruns-Nagel et al. (2000) attributed such signals to residual free nitro groups of bound and partially reduced TNT. However, some of this could be the sequestered parent compound that even vigorous extraction procedures might not have released (Thiele and Brümmer, 1998). It is not likely that adsorbed TNT resisted sequential extractions or that TNT with unaltered nitro groups was bound to the polymer. In contrast, no evidence for the presence of nitro groups was seen under the same conditions when humic acid was present (Sample V). This indicated a lesser extent of sequestration, while complete reduction of TNT prior to binding to humic acid was unlikely. Additionally, unreacted TNT is underestimated by 15N NMR (Knicker et al., 1999).

Spectra of the humic polymers obtained by 15N-CPMAS NMR were quite similar for both of the humic co-substrates investigated. Therefore, humic monomers like catechol appear to be well-suited as model compounds for soil humic substances which was also found by Preston et al. (1982). However, reactivity of catechol was much higher, yielding a more intense transformation of TNT. In contrast, the small amount of non-extractable nitroaromatics found in humic acid was derived from metabolized TNT. It is assumed that these residues underwent covalent binding to the humic substance and are thus not to be easily remobilized. Instead, they were regarded to be detoxified.


   ACKNOWLEDGMENTS
 
This research was partially supported by the Deutsche Forschungsgemeinschaft (DFG), Bonn, Germany, and by the Office of Research and Development, USEPA (Grant no. R-823847). We thank Dr. Michael Major from USACHPP (Aberdeen Proving Ground, MD), who provided us with the labeled TNT compounds.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 




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