Environ. Sci. Technol. 1998, 32, 1676-1679
Anaerobic/Aerobic Composting of 2,4,6-Trinitrotoluene-Contaminated Soil in a Reactor System D . B R U N S - N A G E L , * ,† O . D R Z Y Z G A , † K. STEINBACH,‡ T. C. SCHMIDT,‡ E. VON LO ¨ W,† T. GORONTZY,§ K.-H. BLOTEVOGEL,§ AND D. GEMSA† Institute of Immunology and Environmental Hygiene, Philipps University, Marburg, Pilgrimstein 2, D-35037 Marburg, Germany, Department of Chemistry, Philipps University Marburg, Hans-Meerwein-Strasse, D-35032 Marburg, Germany, and Department of Microbiology, Carl-von-Ossietzky University, P.O. Box 2503, D-26111 Oldenburg, Germany
A compost mixture consisting of 50% 2,4,6-trinitrotoluene (TNT)-contaminated soil, 30% chopped sugar beet, and 20% straw was anaerobically percolated for 19 days with tap water. Thereafter, the water was drained off, and the mixture was aerated for 58 days. The anoxic conditions led to the transformation of 90% of the TNT to monoaminodinitrotoluenes and diaminomononitrotoluenes. In addition to well-known reduction products, three substances with identical UV spectra were generated during this phase. They were identified as 4-acetylamino-2-hydroxylamino-6-nitrotoluene (4-N-AcOHANT), 4-formamido-2amino-6-nitrotoluene (4-N-FAmANT), and 4-acetylamino-2amino-6-nitrotoluene (4-N-AcANT). The first two metabolites were degraded under aerobic conditions by 100% and 99.6%, respectively, whereas the concentration of 4-N-AcANT increased during the first 15 days of the aeration. Thereafter, the compound was also degraded by 99.6%. In conclusion, an efficient TNT remediation system is presented that led to the identification of three conjugated TNT metabolites.
Introduction 2,4,6-Trinitrotoluene (TNT) is frequently a heavy contaminant of soil and groundwater of former ammunition plants all over the world. Since it is toxic (1-4), causes mutations (5), and might be a cause of cancer (6), a decontamination is needed. For this purpose, the development of remediation techniques is of great interest. Besides incineration, biological cleanup methods have attracted great attention. A microbial mineralization of TNT has been shown for different fungi (7-9) and naturally occurring soil microorganisms (10-12). Furthermore, a degradation of TNT to fatty acids by a consortium of Desulfovibrio sp. (13) and an anaerobic transformation of TNT to methylphloroglucinol and p-cresol (14) were reported. Lately, Fiorella and Spain (15) published a novel aerobic degradation pathway for TNT via 2,4-dihydroxylamino-6-nitrotoluene (2,4-DHANT) and * Corresponding author telephone: 0049-6421-285495; fax: 00496421-282309; e-mail:
[email protected]. † Institute of Immunology and Environmental Hygiene, Philipps University. ‡ Department of Chemistry, Philipps University. § Carl-von-Ossietzky University. 1676
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2-hydroxylamino-4-amino-6-nitrotoluene (2HA4ANT) by Pseudomonas pseudoalcaligenes JS52 and proposed an aerobic ring fission. Although the reductive transformation of TNT and the elimination of nitro or amino groups was investigated elaborately (16, 17), little is kown about the conjugation of TNT transformation products. The acetylation of 2,4diamino-6-nitrotoluene (2,4-DANT) has been reported three times yet. Two reports described the resulting 4-N-acetylamino-2-aminonitrotoluene (4-N-AcANT) as a dead-end product (18, 19); whereas, we found a further degradation of the metabolite in specific percolation experiments (20). Michels and Gottschalk (21) were able to identify 4-formamido-2,6-dinitrotoluene (4-N-FAmDNT) and 4-formamido2-amino-6-nitrotoluene (4-N-FAmANT) as intermediate compounds during mineralization of TNT by Phanerochaete chrysosporium. At present, composting (22-24) is investigated intensively as one possible bioremediation method for TNT-contaminated soil. We previously demonstrated that composting can be improved by using a two-step system in which the compost is kept anoxic during a first phase and is aerated during a second phase (22, 25). Under these conditions, the explosive was reduced to amino- and diaminonitrotoluenes during the anoxic phase, and the subsequent aerated phase led to an elimination of the major amount of the transformation products. The key aspect of the present study was to optimize the two-step composting system. We demonstrate a simple technique to drastically shorten the anoxic phase. Attention was also focused on the identification of new TNT metabolites.
Methods All experiments were performed with TNT-contaminated surface soil from the former ammunition plant Tanne near Clausthal-Zellerfeld, Lower Saxony, Germany. The sampling procedure and soil characterization has been published previously (20). A schematic drawing of the anaerobic/aerobic reactor is shown in Figure 1. The reactor was filled with 4 l (2.5 kg dry weight) of a mixture of 50% TNT-contaminated soil, 30% chopped sugar beet, and 20% straw (vol/vol/vol). Previous composting experiments showed that this mixture is suitable for an effective bioremediation of TNT-contaminated soil via an anaerobic/aerobic composting process in static piles (25). During the anaerobic phase, the reactor was percolated with 2.5 L of tap water (1.5 L in the reservoir and 1 L in the reactor) with a flow rate of 12 mL/h. During the percolation process, the pH of the percolation fluid was regulated online to a value of 7 ( 0.2 (HWS pH-Mess-und Regelgera¨t PhR 1400, Mainz, Germany). This was essential since prior experiments showed that without pH regulation an acidification occurred that negatively affected the number of viable cells, inhibited the TNT transformation in the system, and prolonged the time to achieve anaerobic conditions (26). The redox potential in the percolation solution reservoir was determined regularly with an electrode. After 19 days, the tap water was drained off and filled in a 10 L biofermenter (Janke & Kunkel, Staufen, Germany) where it was treated without the addition of nutrients for 48 days. During this time, the fermenter was aerated (25 L/h) and stirred continuously (150 rpm) at 22 °C. After the percolation solution had been withdrawn, the soil-sugarbeet-straw mixture in the anaerobic/aerobic reactor was aerated from the bottom (25 L/h) for 58 days. S0013-936X(97)00757-8 CCC: $15.00
1998 American Chemical Society Published on Web 04/18/1998
FIGURE 2. Concentration of nitroaromatics during the anaerobic/ aerobic composting in the compost material. Data represent the mean ( standard deviation of five parallel compost extracts.
FIGURE 1. Experimental setup of the compost reactor. Before the aerobic phase began, the stagnant water was drained off. Air was supplied than through the outlet at the bottom of the reactor. Samples of approximately 25 g were withdrawn from the compost at intervals ranging from 2 to 15 days. Five aliquots of 5 g of each sample were extracted with methanol for analysis of nitroaromatics. The extraction procedure, the analysis of aqueous samples, and the analytical method (HPLC/DAD) have been described in detail elsewhere (20). For separation of the nitroaromatics, a Nucleosil 120-3 C18 column (3 × 250 mm; CS-Chromatographie Service, Langerwehe, Germany) and a solvent gradient program with water (A) and methanol (B) as mobile phase was used. The gradient progam was as follows: 0-10 min 70% A-30% B; 10-15 min linear gradient to 50% A-50% B; 15-35 min 50% A-50% B; 35-70 min linear gradient to 100% B, which was maintained for 10 min. HPLC/MS analysis was performed with a Gynkotek system (Germering, Germany) consisting of a M 480 pump, an online degasser GT-103, and a GINA 50 autosampler combined with a Hewlett-Packard HP 5989B mass spectrometer with electrospray ionization (Analytika of Branford, Branford, CT). The same gradient as for HPLC/DAD analysis was used, except that 0.15% formic acid (pH 2.3) was used instead of water. TNT was purchased from Fluka (Buchs, Switzerland) and recrystallized five times in ethanol. All TNT transformation products were provided by the Department of Chemistry, Philipps University, Marburg, Germany. The purity of each chemical used as a standard was 99% or more. All other chemicals used were of reagent grade.
Results and Discussion The anaerobic percolation led to an extremely rapid decrease of the redox potential in the aqueous phase to below -200 mV within 2 days. In comparable anaerobic/aerobic static compost piles without a pH regulation, 60-80 days were required to achieve redox potentials between -100 and -170 mV (22, 26). This finding indicates that the anaerobic
percolation with on-line pH regulation is a highly efficient method to shorten the anaerobic phase in two-step compost systems. Analysis of the soil mixture showed that the anaerobic treatment (19 days) caused an almost 90% transformation of TNT to 4-amino-2,6-dinitrotoluene (4-ADNT) and 2,4diamino-6-nitrotoluene (2,4-DANT) (Figure 2). Only minor amounts of 2-amino-4,6-dinitrotoluene (2-ADNT), 2,6-diamino-4-nitrotoluene (2,6-DANT), and 4,4′-azoxy-2,2′,6,6′tetranitrotoluene (4-Az) could be detected. Interestingly, 4-ADNT and 2,4-DANT seemed to be generated simultaneously. This could be due to a fast reduction of 4-ADNT to 2,4-DANT, which corresponds to the presently prevailing notion of a successive reduction pathway (27, 28). A further possible explanation for this finding might be the recently published new transformation pathway of TNT to 4-hydroxylamino-2,6-nitrotoluene, 2,4-dihydroxylamino-6-nitrotoluene, and finally 2-hydroxylamino-4-amino-6-nitrotoluene (2HA4ANT) (15). A similar mechanism could be responsible for the formation of 2,4-DANT in the compost, with the only difference that the generated 2HA4ANT was further reduced to 2,4-DANT. The aeration of the reactor led to an elimination of most of the remaining TNT and reduction products (Figure 2). At day 77, only 65.2 ( 56.5 mg of TNT, 34.9 ( 8.1 mg of ADNT, and 10.3 ( 3.4 mg of DANT per kg dry sample were detectable. A total of 18.9% of the initial 18 mmol of nitroaromatics in the compost was washed out by the percolation process. The 89.5% of the nitroaromatics in the compost were not recoverable after the anaerobic/aerobic treatment. Fractionation experiments of compost material with concentrated hydrochloric acid (25%) and sodium hydroxide solution (30%) did not lead to a mobilization of TNT or any reduction products. Also different solvents (dichloromethane, ethyl acetate) did not yield a higher recovery rate (data not shown). The disappearance of partially reduced TNT metabolites in soil under aerobic conditions is a well-kown phenomenon that is still not understood. It might be caused by an oxygendepending covalent binding of aromatic amines to the soilhumus matrix (2, 29). Dawel et al. published the first experimental evidence for this hypohesis (30). We recently did experiments with anarobic/aerobic soil treatment systems using [14C]TNT (31). Mass balance analysis showed that about 84% of the radioactivity was bound to humic compounds and no [14C]CO2 was generated. Currently experiments with VOL. 32, NO. 11, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. List of Identified Acetylated and Formylated TNT Metabolites in Anaerobic/Aerobic Compost
FIGURE 4. TNT metabolites in drained off stagnant water from the anaerobic/aerobic compost during an aerobic treatment in a bioreactor. For 4-N-AcOHANT, 4-N-FAmANT, and 4-N-AcANT, the area of the corresponding peaks is shown.
FIGURE 3. Time course of the formation and degradation of conjugated TNT metabolites during anaerobic/aerobic composting. Data represent the mean ( standard deviation of five parallel compost extracts. For peak identification see Table 1. [15N]TNT are under way to characterize the binding types via nuclear magnetic resonance (NMR). In addition to well-known TNT metabolites, three peaks with identical UV spectra but different retention times appeared in most of the HPLC chromatogramms of the soil extracts (Table 1). The time course of the generation and degradation of these substances is plotted in Figure 3. The substance represented by peak 3 was produced under anaerobic conditions and during the first 15 days of the aerobic phase. Thereafter, the molecule was degraded to unknown substances. The metabolites represented by peaks 1 and 2 were only formed under anaerobic conditions. The aeration of the soil mixture led to a complete elimination of these metabolites. Peaks 2 and 3 could be identified as 4-NFAmANT and 4-N-AcANT, respectively, by comparison of retention time and UV spectra to authentical standards (Table 1). HPLC/MS analysis confirmed the identification of peak 3 (Table 1). For unknown reasons, it was not possible 1678
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to obtain a mass spectra of peak 2. Peak 1 could be identified by HPLC/MS as the novel TNT metabolite 4-N-acetylamino2-hydroxylamino-6-nitrotoluene (4-N-AcOHANT) (Table 1). A quantification of the acetylated and formylated metabolites was not yet possible since sufficient amounts of authentical standards were not available. Since high amounts of aromatic amines were detectable in the percolation solution after draining it off (Figure 4), the solution was aerobically treated in a bioreactor. This process led to the generation of significant amounts of 4-N-Ac-ANT during the first 14 days, which was accompanied by a decline of the 2,4-DANT concentration (Figure 4). During the following 14 days the acetylated metabolite was completely eliminated, and the concentration of 2,4-DANT increased almost to the initial value of about 170 mg/L. To our knowledge, this is the first experimental evidence for the reversibility of the acetylation of 2,4-DANT. The aerobic treatment led also to a complete elimination of 4-NAcOHANT and 4-N-FAmANT. The acetylation and formylation of aromatic amines has been reported for different microbe species (18-20). On the other hand, there is no proof for abiotic reactions causing these conjugations. Therefore, it is very likely that the acetylation and formylation in the anaerobic/aerobic reactor and bioreactor were also catalyzed by microbes. To date, little is kown about the underlying mechanisms causing acetylation and formylation of TNT reduction products by microorganisms. In the case of the white rot fungus P. chrysosporium, 4-N-FAmANT is an intermediate for the formation of 2,4-DANT (21). This reduction pathway might not be restricted to the fungus but may also be pertinent to anaerobic and facultative anaerobic soil organisms. Further investigations are needed to confirm this assumption. Furthermore, the formylation and acetylation reaction could possibly serve as a detoxification mechanism as postulated for aniline (32). The fact that the acetylation is easily reversible supports this hypothesis. If the acetylated product was a metabolite in a catabolic pathway, a transformation to further unkown metabolites would be expected. Since the formation of acetylated and formylated TNT metabolites seems to be a general ability of the soil microflora, in the future, these TNT metabolites have to be included in concepts directed toward the improvement of TNT bioremediation processes.
Acknowledgments This work was supported by the Federal Ministry of Education, Science, Research and Technology (BMBF), by the state Lower Saxony, and by the Industrieverwaltungsgesellschaft AG (IVG), Bonn, Germany. We would like to thank L. Kaminski and M. Mu ¨ ller for technical assistance. We also thank Dr. J. Michels, DECHEMA, Frankfurt, Germany, for providing a standard of 4-N-FAmANT.
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Received for review August 26, 1997. Revised manuscript received January 26, 1998. Accepted February 25, 1998. ES970757Z
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