Environ. Sci. Technol. 2005, 39, 4540-4549
Oxidative Microbial Degradation of 2,4,6-Trinitrotoluene via 3-Methyl-4,6-dinitrocatechol JACQUELINE M. TRONT AND JOSEPH B. HUGHES* School of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332
A novel pathway for biodegradation of 2,4,6-trinitrotoluene (TNT) was investigated where TNT was the sole carbon, nitrogen, and energy source. Results showed the ability of microorganisms to metabolize TNT through removal of a nitrogroup, oxygenation of the aromatic ring, and production of a metabolite that is typically a precursor to oxygenolytic ring cleavage. Nitrite production was observed in active systems, and TNT degradation activity was repeatable and transferable. The metabolic intermediate, 3-methyl-4,6dinitrocatechol, was positively identified through stable isotope mass spectrometry and tandem mass spectrometry. Experimentation with 14C-TNT showed >3% 14C-labeled CO2 in active systems after 30 d exposure to microorganisms. An increasing fraction of 14C-labeled material was associated with biomass with time, where 11.41 ( 2.91% and 17.09 ( 1.49% of 14C was associated with biomass in active systems after 20 and 30 d, respectively, as compared with 5.68 ( 1.33% and 6.08 ( 1.27% in inactive systems. Parallel degradation of TNT and production of organic metabolites and nitrite were observed in shake flasks constructed with soil from historically contaminated sites, indicating that the novel pathway identified herein is disturbed in the environment. Therefore, results presented provide evidence of a previously unreported pathway for oxidative degradation of TNT.
Introduction 2,4,6-Trinitrotoluene (TNT) is a stable, persistent, and toxic contaminant that has been widely produced and distributed in the environment through military production, testing, and dissemination (1). Investigation of biological treatment strategies for TNT contamination in soils and groundwater has been ongoing for several decades (1, 2). Studies have demonstrated that TNT can be biotransformed under aerobic and anaerobic conditions. The most often studied biotransformation pathways for TNT involve the reduction of one or more aryl nitro groups forming arylamines (3, 4). Aminodinitrotoluenes (ADNTs) and diaminonitrotoluenes (DANTs) are commonly cited metabolic products of TNT, and formation of triaminotoluene has been observed under strong reducing conditions (3-9). Additional products from condensation, ring reduction, and ring substitution have been identified (7, 10-12). Despite the numerous intermediates and initial products of TNT biotransformation that have been observed, the ultimate fate of TNT in bioremediation systems remains unclear. The final products of TNT biodegradation * Corresponding author phone: (404)894-2201; fax: (404)894-2278; e-mail:
[email protected]. 4540
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are best described as either reversibly bound to soil or undefined polar metabolites that remain in the aqueous phase. To date, naturally occurring microbial populations have not been identified that have the ability to utilize TNT as a primary energy or carbon source, and evidence for biological mineralization of TNT has been limited to very small percentages of the total contaminant in the system (9, 13-15). Pseudomonoas sp. strain JLR11 has been shown to release nitrogen from TNT as nitrite or ammonia and thereby uses TNT as a primary nitrogen source (16, 17). Further studies have investigated the ability of strain JLR11 to use TNT as a terminal electron acceptor under anoxic conditions (18). These findings indicated that the energy coupling in reduction of the nitro-group is similar to energy coupling in reduction of nitrate in denitrification, a process that results in microbial energy production, thus implicating TNT as the terminal electron acceptor for some microbial processes. A fundamental microbial strategy for aerobic degradation of aromatic hydrocarbons is oxygenation of the ring through mono- and dioxygenase attack to activate the ring for further oxygenolytic ring cleavage (2). Degradation of mono- and di-substituted nitrotoluenes, nitrobenzenes, nitrophenols, and other nitroaromatics has been demonstrated to proceed through initial hydroxylation followed by ring cleavage and finally, mineralization (1). White-rot fungi have the ability to co-metabolically mineralize TNT through extracellular oxidative attack. Several fungal species have been studied extensively in this capacity, and it has been established that certain fungi have the ability to use carbohydrates as growth substrates and fortuitously degrade TNT and other recalcitrant organic contaminants (19). Oxygenolytic degradation of nitroaromatics and fungi-mediated radical attack on the aromatic ring after reduction of an aryl nitro group are the primary mechanisms observed leading to mineralization of nitroaromatics. Previous studies have not provided any evidence for oxygenase attack on TNT citing high redox potential, steric hindrance, and strong electron deficiency of the π electron system as factors that do not favor oxidative attack (2). This study investigated a novel pathway for TNT metabolism, where TNT served as the sole carbon, nitrogen, and energy source under aerobic conditions. Results showed the ability of microorganisms to oxidize TNT directly through removal of a nitro-group and oxygenation of the aromatic ring. The metabolic intermediate, 3-methyl-4,6-dinitrocatechol, was positively identified through stable isotope mass spectrometry and tandem mass spectrometry. Radiolabeled tracers were used to demonstrate that TNT-derived carbon was incorporated into biomass and that metabolic products included CO2 and NO2-, thus demonstrating an alternative aerobic metabolic pathway that leads to mineralization of TNT.
Materials and Methods Chemical Sources. All chemicals used in media preparation were reagent grade or better and were purchased from the Sigma-Aldrich Company. Aminodinitrotoluenes and diaminonitrotoluenes were supplied by Dr. Ronald Spanggord of SRI International, and radioisotopes were from American Radiolabeled Chemicals, Inc. Stable isotopes were purchased from the Sigma-Aldrich Company, and organic trapping fluid was supplied by the R. J. Harvey Instrument Company. Synthesis of 13C/15N TNT. Stable isotope-labeled TNT was synthesized after Kroger and Fels (20) using 13C-labeled 10.1021/es048014i CCC: $30.25
2005 American Chemical Society Published on Web 05/17/2005
toluene (99 atom % 13C) and 40% (wt) H15NO3 (98 atom % 15N). A reaction mixture of 195 mg of 13C-toluene dissolved in 2 mL of concentrated H2SO4 was maintained at -80 °C for 1 h. An acid mixture (2.0 g of 40% H15NO3 and 4 mL of concentrated H2SO4) was then added dropwise to the reaction mixture at 0 °C. The reaction mixture was stirred for 30 min at 0 °C and was then brought to room temperature and stirred for an additional 30 min. HPLC analysis (UV/vis) of a sample of the reaction mixture after incubation at room temperature indicated that 2,4-DNT and 2,6-DNT were the primary products and the molar ratio of 2,4-DNT to 2,6-DNT was 8.3:1. The reaction mixture was then extracted four times with 3 mL of methylene chloride, and the aqueous phase was discarded. The solvent was removed by drying with N2 gas, and the product was dissolved in 1.5 mL of H2SO4 and maintained at 0 °C. An acid mixture (2 mL of 70% unlableledHNO3 and 4 mL of H2SO4) was added dropwise to the reaction mixture at 0 °C. The reaction mixture was subsequently incubated at 90 °C for 5 h. The reaction mixture was allowed to cool to room temperature and then poured over ice. The crystals were separated from the acid mixture by vacuum filtration. LC/MS analysis demonstrated that the 2,4,6-TNT produced was >99% pure. Batch Reactor Protocol. A mixed 2,4-DNT degrading culture enriched from contaminated soil described by Fortner et al. (22) was maintained in a continuously stirred, draw and fill reactor in the laboratory for approximately 5 years. The hydraulic retention time (HRT) was approximately 5.5 d, and the culture consumed approximately 3 mM 2,4-DNT every 5 d. Biomass from the 2,4-DNT degrading culture was used to seed a TNT degrading culture that was maintained as a continuously stirred, draw and fill reactor with a 10 d HRT. Influent for the TNT-reactor was 40 mg/L TNT and 8 mg/L 2,4-DNT dissolved in media (100 mL). Media did not contain organic carbon or any source of nitrogen as was described previously (22). Aerobic batch reactors containing TNT as the only carbon and nitrogen source were used to assess degradation of TNT by microorganisms in the TNT-reactor. Batch reactors contained nominally 10 mg/L TNT and 100 mL of media, with variations noted with specific results. Biomass for initial batch reactors showing TNT degradation, exposure to aminodinitrotoluenes, and radioactive tracer experiments was from the TNT-reactor. Biomass for stable isotope experiments and demonstration of transferable TNT degradation activity was from batch experiments that contained only TNT as a carbon and nitrogen source. Biomass was washed three times with media before use in batch reactors. Radioactive Tracer Experimentation. Experimental reactors consisted of 140 mL serum bottles fitted with a PTFElined butyl rubber septa. A total of 25 µL of 14C-labeled TNT stock solution (50 µCi/mL in 50% ethanol, >99% pure TNT) was added to empty reactors where total 14C activity was 1.25 µCi/reactor. The carrier solvent was purged from the reactors using N2 gas until reactors were completely dry. Next, 75 mL of media (pH ) 8.5) at a concentration of 44 µM TNT was added to each reactor, and reactors were stirred for 30 min to ensure dissolution of 14C-labeled TNT. 14C-Labeled material comprised 4.5% of total contaminant in reactors. Reactors were sealed and incubated at 30 °C. Mass of media in reactors was recorded at each sampling event, and mass balance calculations were based on mass measurements. Active organism reactors and sodium azide inhibited controls (1 g/L) were run in triplicate to demonstrate repeatability of results. All data are shown as an average value and one standard deviation for the triplicate. To assess the fate of the 14C-tracer after a given time and to ensure that reactors were aerobic, the headspace was periodically purged with air for 5-10 min. Tubing coming from the outflow line of an aquarium air pump was fitted
with a 0.2 µm filter and a 1.5 in. needle that penetrated the septa of the reactor. A second needle was fitted to the outflow line of the reactor and was connected to three organic traps in series. Organic traps contained 5 mL of R. J. Harvey trapping solution (a xylenes based scintillation cocktail designed for trapping gaseous CO2), and no radioactive material was ever observed in the second or third trap in the series, thus demonstrating trap efficiency. After purging the headspace, 1 mL of media was extracted from the reactor via the sampling syringe and filtered with a 0.2 µm PTFE syringe filter. Scintillation measurements were made for filtered and unfiltered samples to assess the amount of 14C-labeled material associated with the biomass. To assess mineralization of TNT, aqueous samples were acidified, and inorganic carbon evolved was counted. A known volume (2 mL) of aqueous sample was pipetted into the first of three traps in series. The second two traps contained organic trapping solution designed to capture 14CO2. Aqueous samples were acidified (2.5% acetic acid), and air was rapidly bubbled through the sample for 5 min. The trapping solution and original aqueous sample were counted to determine quantity of 14C material that was volatile after acidification. Aqueous samples were filtered and then injected into an HPLC equipped with a standard bond C18 column to generally characterize degradation of TNT and hydrophobicity of metabolic products. Mass recoveries for fractionation were 95.7-107.6% of total 14C injected, demonstrating that all radiolabeled tracer material injected was captured during fractionation. Sample Collection from Historically Contaminated Sites. Contaminated soil samples were gathered from a former munitions manufacturing facility in Barksdale, WI, that has been contaminated with TNT for over a century. Samples were collected from five distinct contaminated areas (labeled A-E) with high levels of TNT contamination ranging from solid TNT particles mixed in the soil (C-E) to levels near solubility of TNT in water (A, B). Sites C and D contained only TNT, sites A and B contained TNT and trace quantities of 2,4-DNT, and site E contained a mixture of TNT and 2,4DNT with trace amounts of 2,6-DNT. Samples were collected in sterile bags and stored in the refrigerator until use. Shake flasks to assess degradation of TNT in soil from sites A-E were assembled using sterile technique, and no external TNT was added. Shake flasks contained approximately 10 g of soil and 100 mL of media titrated to pH 8.5 using 1 N NaOH. After 4 d of incubation, 2% v/v was transferred into flasks containing 10 mg/L TNT to assess potential for degradation of TNT by organisms found at sites A-E. Aqueous samples were taken periodically to monitor for TNT concentration, nitrite concentration, and the presence of metabolites. Analytical Methods. Aqueous samples were filtered through a 0.2 µm PTFE syringe filter, and nitroaromatics were separated using reverse-phase liquid chromatography (HP 1100 and Supelco C18 columns) and quantified with a diode array UV/vis detector (λ ) 230 nm). Mass spectrometry was performed using an HP 1100 HPLC-MSD. The elution protocol consisted of a 3 min hold at 70:30 A/B after injection, 13 min linear gradient to 65% B, 5 min hold 100% B, where A and B were 0.1% acetic acid in water and acetonitrile, respectively. All spectra were gathered using electrospray ionization in negative ion mode (ESI-NEG); therefore, m/z values represent negatively charged ions produced with the loss of a proton. Tandem mass spectrometry (MS/MS) experiments were performed on a PE Sciex API 4000 hybrid quadrupole-ion trap tandem mass spectrometer equipped with a turbo V ionspray source. The ionspray needle was held at -4500 V while the declustering potential was kept low (-50 V) to minimize collisional decomposition of molecular ions prior VOL. 39, NO. 12, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. TNT degradation by mixed aerobic microbial community shown in (A) active microbial systems is compared with inactive systems. A small volume (6 mL) was taken from reactor shown in panel A at time ) 4 d and used to inoculate the system shown in panel B. Nonstoichiometric nitrite was observed in both systems. to entry into the first quadrupole. Nitrogen was used as the curtain, nebulizing, and drying gas with the latter heated to 300 °C. A linear elution gradient of methanol and 5 mM ammonium acetate in water was used with a Supelco C18 column. The elution protocol consisted of a 5 min hold at 95:5 A/B after injection, 30 min linear gradient to 100% B, 5 min hold 100% B, where A and B were 5 mM ammonium acetate in H2O and CH3OH, respectively. Enhanced product ion spectra were acquired by setting Q1 to pass a 1 amu wide window around the precursor ion of interest. Nitrogen was used to collisionally activate precursor ion dissociation in Q2, which was offset from Q1 by 30 eV to maximize formation of molecularly distinctive product ions. Subsequently formed product ions were accumulated in the linear ion trap for 20 ms then scanned out to the detector at 1000 amu/s to yield the resulting product ion spectrum. Radioactive materials were quantified via liquid scintillation on a Beckman LS II. The lumex indicator was below 0.5% in all samples; therefore, corrections for quenching were not required. Nitrite concentration was monitored using Method 4500-B (21).
Results Degradation of TNT by an Aerobic Microbial Community. Degradation of TNT was carried out by an aerobic microbial community without an additional carbon or nitrogen source. TNT was degraded by microorganisms in active systems, and minimal loss of TNT was observed in noninoculated and killed controls. Organic metabolites and nitrite were repeatedly produced in active reactors. Organic metabolites were not observed in control reactors and nitrite concentration increased minimally. Example data demonstrating degradation of TNT are shown in Figure 1A for batch reactors containing 54 µM TNT. In Figure 1A, the active system contained microorganisms from the TNT reactor, and the inactive control system was not inoculated with microorganisms. TNT degradation followed first-order kinetics in active microbial systems, and >95% of TNT was depleted from the active system in Figure 1A after 14 d. Minimal losses of TNT occurred in inactive control systems, where 95% TNT depletion in the original flask after 14 d (Figure 1A). Results demonstrating TNT loss and NO2- production paralleling those shown in Figure 1 were observed repeatedly in batch reactors and transfer-flasks. Reaction kinetics and nitrite production stoichiometry were not consistent between batch experiments; however, TNT degradation and nitrite production were reliably observed in active systems, and activity was reliably transferred multiple times. Therefore, it was clear that degradation of TNT occurred via aerobic microbial processes and that degradation activity was repeatable and transferable. However, degradation was not observed to occur rapidly, and further study is needed to characterize microorganisms responsible for degradation activity to enhance metabolism of TNT. Characterization of Metabolic Products of TNT. Batch reactors repeatedly showed steady losses of TNT and consistent production of multiple metabolic products as verified by UV spectra. An example UV chromatogram (λ ) 230 nm) of an aqueous phase sample of TNT exposed to the mixed microbial community is presented in Figure 2. TNT eluted at 13.68 min, and multiple metabolites with considerable UV adsorption eluted with retention times ranging from 2.1 to 11.1 min. Products observed at 10.9 and 11.1 min were identified as 2-amino-4,6-dinitrotoluene (2ADNT) and 4-amino-2,6-dinitrotoluene (4ADNT), respectively, through comparison of UV spectra and retention time with standard material. The aminodinitrotoluenes were repeatedly observed in aqueous samples, but did not accumulate to high concentrations and only comprised a small fraction of the TNT initially in the reactor. 2,4-Diamino-6-nitrotoluene, 2,4dinitrotoluene, and 2,6-dinitrotoluene were not observed in any of the UV specta gathered for microbial exposure to TNT. Parallel experimentation demonstrated that degradation of 4ADNT or 2ADNT did not occur upon exposure to aerobic culture used herein, where ADNT concentrations did not decrease with time and metabolic products were not observed. These results demonstrated that the ADNTs were dead-end products that were likely produced by nonspecific reductase enzymes and were not precursors for additional metabolites. Additional unidentified metabolic products of TNT existed in batch exposures to TNT, and the compound that eluted at 4.87 min (TNT-C) in Figure 2 was examined extensively. TNT-C was consistently observed in HPLC chromatograms
FIGURE 2. HPLC-UV chromatogram for aqueous sample from aerobic community exposure to TNT. 2-Amino-4,6-dinitrotoluene and 4-amino2,6-dinitrotoluene (ADNTs) are present along with multiple unidentified metabolic products. The UV profile for the metabolite observed at 4.874 (TNT-C) is shown in the upper left-hand corner. with relatively small UV absorbance. TNT-C was analyzed on an HPLC-MS using an identical solvent profile and column, with peak identity confirmed using UV spectroscopy (Figure 2). TNT-C had a mass-to-charge ratio (m/z) of 213.1 when analyzed using ESI-NEG mass spectrometry, which potentially corresponded with a methyldinitrocatechol. The metabolic product TNT-C was further investigated to (i) determine if TNT-C was a metabolic product of TNT or a derivative of routine microbial processes and (ii) to identify the chemical structure of the metabolite. Stable isotopelabeled TNT was used to determine that TNT-C was derived from TNT, and chemical analysis using mass spectrometry (i.e., single and tandem mass spectra) was used to identify the chemical structure. The structure of TNT-C was examined to determine if it was a metabolic product of TNT. Microorganisms were exposed to stable isotope labeled TNT in conjunction with natural abundance isotope TNT. It was hypothesized that, in this mixed isotope system, mass spectral analysis of microbial products of TNT would provide spectra with two molecular ions with the difference in mass corresponding to the number of labeled atoms. Therefore, for any mass spectrum that represented a derivative of TNT, the molecular ion with the smaller m/z value corresponded to the natural abundance isotope compound and the molecular ion with the greater m/z value corresponded to the 13C/15N labeled compound. For example, the ESI-NEG spectrum of a TNT sample with equimolar quantities of labeled and unlabeled TNT showed m/z values of 226.1 and 235.1 with equal relative abundance. It is important to note that the stable isotope labeled TNT discussed herein contained seven 13C-labeled carbon atoms and two 15N-labeled nitrogen atoms (molecular weight ) 236). The stable isotope TNT contained 15N label
in the 2 and 4 positions for 92% of TNT, and the 2 and 6 positions were 15N-labeled for 8% of TNT, as described above. Microorganisms were exposed to TNT in a batch reactor that contained 44 µM 12C/14N-TNT and 44 µM 13C/15N-TNT. Greater than 50% of TNT was degraded by 30 d, and the presence of previously observed metabolic products was verified by UV spectrometry. Examination of the ESI-NEG spectrum of the 12C-13C exposure showed several peaks with recognizable stable isotope signatures, one of which contained a UV spectrum identical to that of TNT-C. The ESINEG spectrum for TNT-C produced in the reactor dosed with equimolar quantities of 12C/14N-TNT and 13C/15N-TNT is presented in Figure 3. The mass spectrum contained a peak with an m/z value of 213.1 (100% relative abundance), which was assigned to be the molecular ion (M) for the 12C/ 14N-TNT-C. The mass spectrum for the TNT-C peak also contained two corresponding stable isotope labeled molecular ion peaks with m/z values of 221.1 (71.7% relative abundance) and 222.1 (53.6% relative abundance), which were assigned to be the molecular ion for 13C/15N-TNT-C. Therefore, the mass of the stable isotope TNT-C was 8 and 9 amu greater than natural abundance TNT-C (∆MTNT-C ) 8 and 9 amu). Peaks at 221.1 or 222.1 were not observed in mass spectra for systems fed only natural abundance isotope TNT. Therefore, a clear stable isotope pattern existed in the mass spectrum for the 12C-13C system and thus demonstrated that TNT-C was a microbial product of TNT. Further examination of the mass spectrum generated from the 12C-13C reactor system provided additional information regarding the structure of TNT-C. Through application of the nitrogen rule to the molecular ion for the natural isotope abundance peak, it was clear that TNT-C contained an even number of nitrogen atoms (i.e., odd molecular ion in ESIVOL. 39, NO. 12, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 3. API-ES NEG mass spectrum of TNT-C when produced in a system exposed to a system with equimolar amounts of 12C/14N-TNT and 13C/15N-TNT. NEG mode dictates an even number of nitrogen atoms). Therefore, one or three N were removed from TNT in the production of TNT-C. The molecular ion for 13C/15N TNT was 9 amu greater than that of 12C/14N TNT (∆MTNT ) 9), and the molecular ions for 13C/15N-TNT-C were 8 and 9 amu greater than the 12C/14N-TNT-C (∆MTNT-C ) 8 and 9). If one or more carbon atoms were removed in the formation of TNT-C, generation of a single molecular ion for 13C/15NTNT-C and ∆MTNT-C < 9 would be expected because TNT was uniformly 13C-labeled. If a nitrogen atom was removed in formation of TNT-C, generation of multiple molecular ions for 13C/15N-TNT-C was probable because the 15N labeling was not uniform. In addition, if three nitrogen atoms were removed from TNT in formation of TNT-C, ∆MTNT-C would be e 6. Therefore, formation of TNT-C occurred through removal of a single nitrogen, and no removal of carbon occurred. Further analysis of the molecular ions generated for 13C/ 15N-TNT-C provided evidence for removal of nitrogen from the ortho position. The relative abundance of the 221.1 peak compared to 222.1 peaks indicated that a 15N atom was removed in 57% of TNT metabolized in the production of TNT-C. Probability calculations were conducted that estimated the expected relative abundance of 221.1 versus 222.1 (i.e., the probability of 15N removal) for nitrogen removal from either the ortho or para positions. In synthesis of 13C/ 15N-TNT only two of the nitro groups were labeled with 15N. The para position and one ortho position contained 15N for 92% of the labeled-TNT, and the remaining 8% of labeledTNT contained 15N in both of the ortho positions and not in the para position. Calculations indicated that if para-N was removed the probability of heavy isotope removal was 92% (i.e., 221.1 peak would have encompassed 92% of the stable isotope peak). If the ortho-N was removed, the probability of 15N removal was 54%. A comparison of these calculations with the actual observation of 57% of 15N removal (i.e., relative abundances of 221.1 and 222.1 were 71.7% and 53.6%, respectively) indicated that the N was removed from the ortho position for production of TNT-C. Examination of fragmentation patterns derived from tandem mass spectrometry was used to identify the base molecule structure and functional groups of TNT-C (Figure 4). The parent ion (213.0) was fragmented under ESI-NEG conditions, and prominent daughter ions observed were m/z 183, 153, and 95. Minor daughter ions observed were m/z 167, 151, 138, 136, 125, 109, and 63. Typical fragmentation patterns observed for nitroaromatics include neutral losses of 30, 46, and 47 corresponding to loss of NO, NO2, and HNO2 (23, 24). Formation of m/z 183 was assigned as a neutral loss of NO, a typical rearrangement observed in nitroaromatics 4544
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which results in the formation of the phenoxy cation. The m/z 153 daughter ion was assigned to be a loss of two NO groups, and it is important to note that no peak was observed at m/z 123, which would have been indicative of a third nitrogroup loss. The m/z 167 daughter ion was assigned as loss of a NO2 group, and m/z 136 was considered to be a loss of NO and HNO2. The ion observed at m/z 63 was considered to be a rearrangement product of the aromatic ring and a signature daughter ion indicating that TNT-C contained an intact aromatic ring. Additional small daughter ions (m/z 151, 138, 125, 109, 95) were not assigned due to the high level of uncertainty associated with assigning fragmentation patterns when multiple parent ions are present. A daughter ion resulting from a water loss from the molecular ion (M H2O) was not observed in the spectrum. Typically, a hydroxyl group located next to a free proton would generate a considerable M - H2O daughter ion resulting from a rearrangement that cleaves the hydroxyl group and adjacent proton. The fact that no M - H2O ions were observed indicates that hydroxyl groups were likely not present adjacent to free protons. The information supplied by mass spectrometry in stable and natural isotope systems coupled with tandem mass spectrometry results provided a means of assigning a chemical structure to TNT-C. A mass calculator was used to generate all the possible empirical formulas for TNT-C (28 possibilities) with inputs of molecular weight of 213.0 amu and an error range of 200 ppm (parameters set for negative ion mode). Bounds were placed on elements present based on the stable isotope mass spectra as discussed previously. Specifically, only two nitrogen atoms were allowed, a minimum of seven carbon atoms was required, and any possible combination of oxygen and hydrogen was permitted. Additional boundaries placed on mass calculator results involved the index of hydrogen deficiency (IHD). Results with an IHD of less than 6 were eliminated (i.e., aromatic ring ) 4; two nitro groups ) 2 as observed in tandem mass spectrum in Figure 4). Therefore, two possible empirical formulas (C7H5N2O6, IHD ) 6, and C10HN2O4, IHD ) 11) remained after all boundaries were applied. The C10HN2O4 was eliminated as an option because of the large number of carbon atoms and the high level of unsaturation. Therefore C7H5N2O6 was assigned as the empirical formula for TNT-C. Coupling the assigned empirical formula with information from stable isotope experimentation and tandem mass spectrometry, the chemical structure of 3-methyl-4,6-dinitrocatechol was assigned to TNT-C as shown in Figure 7. Given the empirical formula (C7H5N2O6) and the presence of an aromatic ring and two nitro groups, it was clear that TNT-C contained two nitro groups, two hydroxyl groups, and a
FIGURE 4. Tandem mass spectrum for parent ion 213.0 (TNT-C). Multiple daughter ions were observed, and peak assignments are indicated. methyl group on an aromatic ring. Exact structure assignment was achieved by incorporating information from the stable isotope experimentation, which demonstrated that the nitro group was removed from TNT in the ortho position as described above. This conclusion was confirmed with evidence from tandem mass spectrometry, which showed that hydroxyl groups were not present adjacent to free protons (i.e., introduction of hydroxyl groups in the para and meta positions would have resulted in facile loss of water as a result of hydroxyl group adjacent to a free proton in the meta position). This observation provided additional evidence for dioxygenase attack ortho to the methyl group on TNT. Therefore, 3-methyl-4,6-dinitrocatechol was positively identified as an intermediate in oxidative microbial metabolism of TNT in microbial systems. Mineralization of TNT by an Aerobic Microbial Community. Radiolabled tracer studies were conducted to demonstrate that aerobic mixed cultures both mineralized TNT and incorporated TNT-derived carbon into biomass. Microorganisms were incubated with 10 mg/L TNT as the sole source of organic carbon, where 4.5% of the total TNT in the reactor was 14C-labeled. Data for radiolabled tracer studies are presented in Table 1, where data for 14C in headspace, associated with biomass and volatile after acidification, are provided with corresponding time and TNT removal data. Results showed that 0.02 ( 0.00% and 0.04 ( 0.00% of total 14C in active reactors was contained in the headspace at 20 and 30 d (Table 1). 14C was not detected in the headspace of inactive reactors during any sampling event. Detection of this small, quantifiable amount of 14C in the headspace of active reactors at each sampling time point was indicative of mineralization of TNT occurring in aqueous phase. It is important to note that the pH of the media in this system was buffered to a value greater than 8; therefore, a relatively small fraction of total inorganic carbon in the system was expected in the gaseous form. To further demonstrate that quantifiable mineralization of TNT occurred in aerobic reactors, aqueous samples were acidified and inorganic
carbon evolved was counted. After acidification of aqueous samples, 3.38 ( 0.29% of 14C in aqueous samples for active reactors was captured in the trap. In inactive reactors, 0.55 ( 0.06% of 14C in aqueous samples was volatile after acidification. Appreciably more 14C in active reactors was volatile after acidification (3.38 ( 0.29% after acidification vs 0.02 ( 0.00% prior to acidification) further indicating that the 14C captured in headspace traps was inorganic carbon. Significantly more 14C was CO2 in active reactors than in inactive reactors (p < 0.001), demonstrating that active microbial processes were necessary to mineralize TNT. Therefore, results indicated that a portion of the TNT in the aqueous phase of active reactors was CO2 after exposure to active organisms. Aqueous samples were taken at each sampling event to fully describe the distribution of TNT-derived carbon in the reactor. Aqueous samples were filtered with a 0.2 µm PTFE filter immediately after removal from reactors, and a comparison of radioactivity before and after filtration was made to assess quantity of TNT-derived carbon associated with biomass. Quantity of radiolabeled tracer associated with biomass increased significantly with time in active systems and significantly more tracer material was associated with biomass in active systems than in sodium azide controls (Figure 6). Average values of filterable 14C material in active reactors were 11.41 ( 2.91% and 17.09 ( 1.49% after 20 and 30 d, respectively. In contrast, 5.68 ( 1.33% and 6.08 ( 1.27% of total 14C in aqueous phase was associated with the filter after 20 and 30 d in sodium azide control reactors. Therefore, results indicated that TNT-derived carbon was increasingly associated with biomass with time. Evidence for the degradation of TNT by aerobic microorganisms was expanded through separation and fractionation of 14C-labeled metabolites via HPLC. In active systems, the radiolabeled tracer material associated the TNT peak decreased with time. As exposure to active microorganisms extended, the 14C was increasingly associated with the polar front. A small quantity of 14C was associated with the ADNTs VOL. 39, NO. 12, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 5. UV profile (A) at λ ) 246 nm with corresponding 14C profile (B) for an example active microbial system. (∼10%) and 14C associated with several metabolites, which were less hydrophobic than TNT was also quantifiable (i.e., rt ) 3-6 min). An example chromatograph for a sample taken from an active microbial system after 20 d where HPLC eluent was fractionated at 0.5 min intervals is presented in Figure 5. In the example shown in Figure 5 (t ) 20 d), 93.2% of 14C material was recovered, where 54.6% was in the polar solvent front, 9.9% was associated with the ADNTs (rt ) 10-11 min), 5.1% was associated with other metabolites that were less hydrophobic than TNT, and 23.6% remained as TNT. In inactive systems, effectively all radiolabled tracer material (>97%) eluted with the TNT peak. These results demonstrated that metabolic degradation of TNT resulted in metabolites that were more polar than TNT, and a large portion of the TNT metabolites were not retained on a C18 column after long-term exposure to microbial systems. 4546
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Metabolic Products Observed in Shake Flasks from Historically Contaminated Sites. Degradation of TNT was observed in reactors inoculated with organisms from shake flasks assembled with soil from a former munitions manufacturing facility in Barksdale, WI. Rate of TNT degradation varied among the field sites and was generally a slow process. Samples taken after 30 d exposure showed that nitrite was produced in all reactors, where greater losses of TNT corresponded to greater production of nitrite (i.e., sites A, B, E). In addition, 3-methyl-4,6-dinitrocatechol was observed in reactor D after 1 month of exposure in shake flasks. After 3 months of exposure in the shake flasks, 3-methyl-4,6dinitrocatechol was present in reactors from all sites. Degradation kinetics were not quantifiable due to the long duration of the shake-flasks; however, the presence of nitrite and 3-methyl-4,6-dinitrocatechol demonstrated that organ-
sampling event
102.21 ( 1.52 90.59 ( 3.93 0.55 ( 0.06 3.38 ( 0.29 99.35 ( 0.71 98.01 ( 0.99 97.63 ( 4.38 97.53 ( 4.89 5.68 ( 1.33 6.08 ( 1.27 11.41 ( 2.91 17.09 ( 1.49 0.00 ( 0.00 0.00 ( 0.00 0.02 ( 0.00 0.04 ( 0.00 100.41 ( 2.67 81.21 ( 2.59 24.14 ( 5.44 8.98 ( 0.90 20 d 30 d
reactor mass balance active control
% 14C volatile after acidification active control
acidificationvolatilization mass balance active control
TNT was degraded by an aerobic microbial community as the sole carbon, nitrogen and energy source. Multiple TNT metabolites, including nitrite, were present in active systems and did not appear in inactive systems. Actively degrading organisms retained the ability to degrade TNT through a series of small volume transfers into fresh media, demonstrating that TNT degradation was a result of active microbial systems and not a product of overexpression of 2,4-DNT degrading enzymes. Degradation of TNT occurred via active aerobic microbial processes, and that activity was repeatable and transferable. However, degradation was not observed to occur rapidly in any studies attempted to date, and further study is needed to characterize microorganisms responsible for degradation activity to enhance the metabolism and mineralization of TNT. Multiple metabolites were identified in the TNT reactors. Two metabolic products indicative of active, aerobic microbial degradation of TNT (nitrite and 3-methyl-4,6-dinitrocatechol) were repeatedly observed in experimental systems. Nitrite concentration increased as TNT concentration decreased, demonstrating that the microorganisms had the ability to cleave a nitro-group from the aromatic ring of TNT. Nitrite production and TNT degradation were not observed in a constant stoichiometric relationship. However, competing reductive processes were occurring in reactors (e.g., dead-end production of ADNTs) so stoichiometric production of nitrite was not expected. The presence of nitrite was taken as an indicator of nitro-substituent cleavage, and further process refinement is necessary to more fully characterize TNT degradation. Formation of a substituted catechol through oxygenation of the aromatic ring is a fundamental microbial strategy for aerobic degradation of aromatic hydrocarbons. Formation of a catechol is an energy investment, where activation of the aromatic ring through oxygenation requires energy input. However, catechol formation facilitates further dehydrogenation and hydroxylation reactions that yield energy and ultimately mineralize the
% 14C associated with biomass active control
Discussion
cumulative % 14C in headspace active control
isms capable of aerobic degradation of TNT were present in field samples.
% 14C remaining as aqueous TNT (HPLC analysis) active control
FIGURE 7. Chemical structure of TNT-C (3-methyl-4,6-dinitrocatechol), an intermediate of microbial metabolism of TNT.
TABLE 1. Experimental Results for Microorganisms Exposed to 14C-TNT in Sealed, Batch Reactors
FIGURE 6. Percentage of radiolabeled tracer that was retained on a 0.2 µM PTFE filter for active systems and sodium azide control systems.
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catechol (25) providing an energy and carbon source for microorganisms. Therefore, microbial formation of a substituted catechol from TNT is indicative of a possible microbial strategy for the aerobic metabolic degradation of TNT and use of TNT as a carbon and energy source. Commonly reported metabolites (i.e., ADNTs) were observed in active systems. However, experiments that used the ADNTs as the primary substrate demonstrated that cultures used herein did not metabolize the ADNTs, and production of nitrite or 3-methyl-4,6-dinitrocatechol was not observed in ADNT systems. Therefore, ADNTs appeared to be dead-end products of reductase enzymes, and a production of ADNTs was a process that was competing with active metabolic degradation of TNT. Results which demonstrated that substituted-catechol production did not stem from ADNTs indicated that initial reduction of the nitro-groups was not an initial step in this novel pathway for degradation of TNT. Results gathered with radiolabeled tracer material indicated that >3% of TNT in active reactors was mineralized to CO2 after 20 d. Data from control reactors indicated 30 microbial species present; data not shown). Therefore, many competing processes were underway in TNT reactors which resulted in production of ADNTs by nonspecific reductase enzymes, anabolism by microorganisms, and other competing processes. This variety of competing processes played an important role in fate of TNT in microbial reactors and potentially reduced the overall quantity of TNT available for mineralization. Results with radiolabeled tracers demonstrated that a significant amount of carbon derived from TNT was associated with biomass. The quantity of TNT sorbed to biomass in the inactive controls was estimated to be the quantity of filterable TNT in inactive controls (i.e., 5-6% of TNT in inactive reactors was filterable and was assumed to be sorbed to biomass). The quantity of TNT incorporated into cell components could be estimated as the amount of filterable 14C-labeled material in active systems less the quantity filterable in inactive systems. Synthesis of results presented in Table 1 indicates that 5.1 and 11.0% of TNT in reactors had been incorporated into cell components by 10 and 20 d, respectively. In addition, it is clear from radiolabeled tracer fractionation assays that the hydrophobicity of the contaminant decreased as TNT was metabolized by cells. Thus, the capacity for sorption to biomass generally decreased as metabolism proceeded, and we can assume that taking the difference between filterable 14C in active and inactive systems was a conservative estimate of TNT incorporated into biomass. However, this estimate of TNT sorption to biomass is limited by the fact that all of the metabolic products produced in this system are not known. It is possible that metabolites that have not been fully characterized in this study have an unexpected affinity for cellular material and thus are associated with biomass rather than being incorporated into biomass. While extensive mineralization was not observed within the time frame of this study, the unique observation of nitrite in TNT-only systems in addition to significant and increasing incorporation of 14C-labeled TNT into biomass is indicates the presence of a previously unreported pathway for oxidative metabolism of TNT. In addition, this study demonstrated that an oxidative attack on TNT is possible through identification of a novel oxidative metabolite of TNT (3-methyl4548
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4,6-dinitrocatechol). Observation of this novel metabolic product of TNT in slurries seeded with soil from a historically contaminated site suggests that this activity is distributed in the environment. Therefore, oxidative metabolism is a viable strategy for microbial degradation of TNT. The extent of oxidative metabolism of TNT in field sites remains unknown, however identification of a TNT-derived catechol possibly allows for monitoring productive metabolic activity at field sites. The ability to harness this novel TNT degradation pathway for bioremediation and natural attenuation remains an important unanswered question.
Acknowledgments This work was supported by the DuPont Corporate Remediation Group. We thank Cameron Sullards, Director of the Mass Spectrometry Center at Georgia Institute of Technology, for his help with generation and interpretation of the tandem mass spectrum presented.
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Received for review December 15, 2004. Revised manuscript received April 4, 2005. Accepted April 5, 2005. ES048014I
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