- Email: [email protected]
Bioremediation of tetryl-contaminated soil using sequencing batch soil slurry reactor
ARTICLE IN PRESS
International Biodeterioration & Biodegradation 55 (2005) 293–297 www.elsevier.com/locate/ibiod
Bioremediation of tetryl-contaminated soil using sequencing batch soil slurry reactor R. Boopathy Department of Biological Sciences, Nicholls State University, P.O. Box 2021, Thibodaux, LA 70310, USA
Abstract A laboratory study was conducted to determine whether tetryl (2,4,6-trinitrophenlymethylnitramine) contaminated soil could be bioremediated using a sequencing batch soil slurry reactor (SBR) operated under anoxic–aerobic sequence. The results indicated that tetryl was co-metabolically converted to aniline under anoxic conditions with molasses as the growth substrate. The gas chromatographic/mass spectrometric analysis of the soil slurry showed various metabolites, identified as trinitrobenzeneamine, dintrobenzenediamine, nitroaniline and aniline. Aniline was not metabolized further under anoxic conditions. When the soil slurry reactor was operated under aerobic conditions, the aniline concentration was reduced to below the detection limit (0.05 ppm). This metabolic conversion of tetryl is probably of value in the treatment of tetryl-contaminated soil and ground water, such as those found at the Joliet army ammunition plant site in Illinois and the Iowa army ammunition plant site in Burlington, Iowa. r 2005 Elsevier Ltd. All rights reserved. Keywords: Explosives; Tetryl; Biodegradation; Bioremediation; Sequencing batch reactor; Anaerobic treatment
1. Introduction Tetryl (2,4,6-trinitrophenylmethylnitramine), one of the major contaminants at the Joliet army ammunition plant (JAAP), is found in the JAAP soil at numerous locations (Boopathy and Manning, 1998). Tetryl (Fig. 1) has been used as a booster explosive by the military and is also used as a base charge in detonators and blasting caps (Othmer, 1980). Highly stable and slightly hygroscopic, it can withstand storage at ambient temperature for 20 years (Urbanski, 1984). The importance of tetryl has been reduced considerably with the appearance of other explosives. Tetryl is known to cause tetryl dermatitis in humans (Witkowski et al., 1942) and is mutagenic (Whong et al., 1980). A toxicity study on guinea pigs showed that tetryl can cause erythema or oedema (Gell, 1944). The known method of physical and chemical removal of tetryl in water samples is reported to be treating the Tel.: +1 985 448 4716; fax: +1 985 493 2496.
E-mail address: [email protected]. 0964-8305/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2005.03.006
waste with a 1:4 solution by weight of sodium sulfite, and the solution is heated to 80 1C to speed up the reaction. In this treatment, tetryl is completely decomposed to non-explosive water-soluble products (Urbanski, 1984). Boopathy and Manning (1998) demonstrated the anaerobic removal of tetryl in a soil slurry reactor. There are numerous reports on the biological degradation of 2,4,6-trinitrotoluene (TNT) (Williams et al., 1992; Boopathy and Kulpa, 1993; Boopathy et al., 1993; Funk et al., 1995; Boopathy and Manning, 1996; Widrig et al., 1997) by aerobic and anaerobic bacteria. The literature contains very few reports on the biological degradation of tetryl by bacteria or fungi (Boopathy and Manning, 1997, 1998). However, the present research was conducted to study the degradation of tetryl by native soil bacteria under anoxic–aerobic sequencing conditions using a sequencing batch reactor (SBR). The results suggest that the indigenous soil bacteria can effectively degrade tetryl to aniline under anoxic condition, with aniline being further degraded under aerobic condition.
ARTICLE IN PRESS R. Boopathy / International Biodeterioration & Biodegradation 55 (2005) 293–297
294
CH3 N
NO2
NO2
NO2
Fig. 2. Schematic diagram of laboratory soil slurry reactor.
NO2 Fig. 1. Structural formula of tetryl.
The tetryl concentration in the soil was monitored periodically.
2. Materials and methods
2.3. Analytical methods
2.1. Soil
Soil bound tetryl was analyzed by using a soilextraction procedure developed by Jenkins and Walsh (1987). Soil slurry was dried in an oven and 1 g soil was extracted with 10 ml acetonitrile. The whole mixture was agitated for 18 h using a solid-state ultrasonic cleaning system (Model FS 7652, Fisher Scientific, Itasca, IL). After agitation, 5 ml soil-slurry extract was mixed with 5 ml calcium chloride ð5 g L1 Þ. Then, the whole mixture was filtered through a 0:45 mm filter (Millipore, Bradford, MA). Tetryl and other contaminants in the filtrate were analyzed by high-performance liquid chromatography with a Waters (Milford, MA) liquid chromatograph equipped with two Model 6000A solvent pumps, a Model 490E programmable multiwavelength detector set at 254 nm, a data module, and a Model 600E system controller. The mobile phase was methanol–water (50:50). Aliquots ð50 mLÞ were injected into a Supelco column (Model C-18, Supelco Inc., Bellefont, PA). The flow rate of the solvent was 1:5 mL min1 . DO in the soil slurry was monitored weekly by using an oxygen analyzer (Gilson Oxygraph, Model oxy-5, Gilson Electronics Inc., Middleton, WI). A slurry sample from the reactor was added to the cell, and the DO concentration in this sample was measured and expressed in mg L1 . The instrument was calibrated with deionized water saturated with oxygen. The pH in the slurry sample was measured using a pH meter (Model 50, Accumet, Denver, CO). At several stages of the experiment, the bacteria growing in the slurry reactor were enumerated. Slurry samples taken from the reactor were serially diluted with phosphate buffer. Standard methods for total plate counts on tryptic soy agar (TSA) plates, as described by Brown (2002) were followed, and the anaerobic plates were incubated in an anaerobic jar.
Tetryl-contaminated soil was collected from JAAP. The soil had a tetryl concentration that ranged from 2000 to 7000 mg kg1 soil. A homogenous mixture of soil was prepared using a tumbler. The homogenized soil, with a tetryl concentration of 4216 mg kg1 , was used for this study. 2.2. Soil-slurry reactor Three 2-L laboratory-scale soil-slurry reactors (Fig. 2) were designed and operated under co-metabolic conditions using molasses as the co-substrate. The working volume of each reactor was 1.5 L. The reactors were started with 20% (w/v on a dry weight basis) tetrylcontaminated soil in sterile tap water. One reactor was operated aerobically and the second reactor was operated under anoxic conditions through out the experiment. The third reactor was operated in a sequencing mode in which the reactor was operated anoxically for the first 60 days, then it was switched to aerobic mode. All three reactors received molasses (Grandma’s Molasses, Mott’s Cadbury Beverages Inc., Stamford, CT) at 0.3% (v/v) as growth substrate at the beginning and once every 2 weeks thereafter. Additionally, two control soil slurry reactors (one under aerobic condition and another under anoxic condition) were operated without co-substrate (molasses). All reactors were mixed continuously by using a magnetic stirrer (100 rpm). The aerobic reactors were aerated throughout the study. The dissolved oxygen (DO) in this reactor was around 5 mg L1 . The headspace of the anoxic reactors was purged with 100% helium in order to maintain the anoxic condition and this was achieved by constantly bubbling helium gas into all reactors.
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2.4. Chemicals Tetryl was obtained from the Naval Surface Warfare Center, Indian Head, MD. Aniline was purchased from Sigma Chemical Co., St. Louis, MO. The metabolic standards were obtained from Ultra Scientific Analytical and Environmental Standards (North Kingstown, RI). All other chemicals were reagent grade.
3. Results and discussion 3.1. Performance of soil slurry reactors The present study was designed for co-metabolic conditions with molasses as a co-substrate based on an earlier report (Boopathy et al., 1994a) showing that the removal of explosive chemicals was accomplished under such conditions with molasses as the growth substrate. Fig. 3 shows the concentration of tetryl in the soil. Under anoxic conditions, the initial tertyl concentration of 4216 mg kg1 was gradually reduced to below the detection limit (0:05 mg kg1 soil) within 60 days. The soil slurry reactor operated under aerobic conditions did not perform very well and the tetryl concentration dropped only slightly from around 4200 to 3800 mg kg1 . In anoxic and aerobic control reactors, operated with no additions of co-substrate, the tetryl concentration remained at the initial concentration ca. 4200 mg kg1 soil, indicating that tetryl removal in soil is a co-metabolic process. Bacterial growth, pH, and DO were maintained in all reactors including the control reactors throughout the study. At day 30 of the reactor operation (Table 1) the bacterial counts were high in reactors operated with molasses as co-substrate under both aerobic and anoxic
5000
4000 Tetryl (mg.kg-1 of soil)
The intermediates produced during tetryl metabolism by soil bacteria were analyzed by gas chromatographymass spectrometry (GC-MS) methods. Concentrated samples were prepared by drying the slurry sample using a roto-evaporator and were reconstituted with methylene chloride. The GC-MS analyses were performed in the electron-ionization mode on a Finnigan model INCOS 50 System (Finnigan, Cincinnati, OH). The samples were chromatographed with a gradient temperature program: initial temperature 80 1C for 1 min; temperature increased to 250 1C at 20 C min1 and held at 250 1C for 10 min. An SPB-5 (Supelco, Supelcopark, PA), (30 m 0:25 mm, 0:25 mm film) was used. The injection temperature was 250 1C; transport line was maintained at 200 1C; the helium flow was 25 cm s1 and injection volume 2 mL. Peaks of interest were analyzed for mass spectra, and the spectra of metabolites were compared with known standard spectra of individual metabolites.
295
3000
2000
1000
0 0
20
40 Time (days)
60
80
Fig. 3. Concentration of tetryl in various soil slurry reactors. Data points represent the average value of triplicate analysis at every sampling event. Symbols: , aerobic control reactor; ––, anaerobic control reactor; n, reactor operated under aerobic condition with molasses as co-substrate; m, reactor operated under anaerobic condition with molasses as co-substrate; &, SBR. Table 1 Bacterial count, pH, and DO in soil slurry reactors on day 30 of the experiment Reactor
Bacterial count
pH
DO ðmg L1 Þ
Aerobic control Anaerobic control Aerobic Anaerobic SBR
45 2 102 32 3 102 126 6 109 198 9 109 202 7 109
6:8 0:2 7:1 0:3 6:4 0:25 5:8 0:22 5:9 0:31
5:4 0:4 0:1 0:33 5:1 0:28 0:08 0:01 0:07 0:01
The data represents mean and standard deviation of triplicate samples. Anaerobic plates were incubated in an anaerobic jar.
conditions, indicating the presence of aerobic and anoxic bacteria in the contaminated soil. In the control reactors operated without molasses, the bacterial counts were significantly lower; this indicates that tetryl which was the sole source of carbon, was not used for growth. The pH of the experimental reactors was acidic (pH 5.8–6.4), whereas, the pH in the control reactors was close to neutral (pH 6.8–7.1). The DO in the aerobic reactors was ca. 5 mg L1 and in the anoxic reactor the DO was p0:1 mg L1 throughout the experiment. 3.2. Identification of tetryl metabolites Tetryl was metabolized in the reactors operated under anoxic conditions and the SBR operated in anoxic–aerobic sequence (Fig. 3). Various metabolites were produced in these reactors (chromatogram not shown). Mass-spectral analysis of the peak that eluted at 34 min showed a fragmentation profile similar to that of
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trinitrobenzeneamine. The mass spectrum of standard dinitrobenzenediamine perfectly matched that of the peak that eluted at 36 min on GC total ion chromatogram. The next compound identified was nitroaniline, which had a retention time of 36.5 min under the chromatographic conditions used. The mass spectrum of this compound matched the standard nitroaniline. The last compound identified in the GC-MS analysis was aniline. This compound eluted at 24 min on the GC chromatogram. The major ions at m=z at 39, 66, and 93 a.m. showed 100% similarity with the standard aniline. There were no other major metabolites identified in this study. The metabolites trinitrobenzeneamine, dinitrobenzenediamine, and nitroaniline did not persist more than a week after they appeared in the reactor system. Aniline did not undergo further metabolism under anoxic conditions.
3.3. Degradation of aniline The concentration of aniline increased and accumulated in the reactors operated under anoxic conditions (Fig. 4). However, in the SBR, when the reactor was switched to aerobic mode on day 60 of the experiment, the aniline concentration was reduced below the detection limit ð0:05 mg L1 Þ within 3 weeks. To further demonstrate that the aerobic consortium in the SBR reactor uses aniline as the sole source of carbon, aniline ð100 mg L1 Þ was spiked to the SBR reactor on day 95 without any addition of molasses. The added aniline concentration dropped to 0:05 mg L1 within 1 week of its addition (data not shown). 800
Aniline (mg.kg-1 of soil)
SBR operated aerobically 600
400
200
This study showed that an anoxic/aerobic soil slurry reactor can effectively remediate tetryl contaminated soil. Under anoxic condition, the tetryl is converted to aniline and the aerobic soil microbes metabolize aniline, further resulting in complete degradation of the tetryl. The initial step is a reduction process, in which the nitro group is reduced and the methyl group is demethylated, resulting in the production of trinitrobenzeneamine. The nitro group present in the para position is reduced to an amino group to form dinitrobenzenediamine. Later, two nitro groups are removed from the benzene ring by reduction, followed by reductive deamination; in this process, nitroaniline is formed, similarly to the formation of nitrobenzene from trinitrobenzene as described by Boopathy et al. (1994b). The nitro group present in the sixth position is also eventually removed by bacterial activity, either by reduction and reductive deamination (Boopathy et al., 1993) or by the removal of the nitro group as such (Dickel and Knackmuss, 1991), resulting in the production of aniline in the reactor. This probable metabolic pathway is based only on GC-MS identification of the compounds in the soil slurry system. Further detailed study is needed to identify each step of the pathway, the enzymes involved in the metabolism, and the soil bacteria responsible for the action. This study demonstrated that soil bacteria, by employing various metabolic enzymes and reactions, including demethylation, reduction, and reductive deamination can metabolize tetryl in the soil. An aspect of this project requiring further investigation in a detailed radiolabelled experiment is the mineralization of tetryl. Previous studies by various investigators have been focused on the bioremediation of TNT (Williams et al., 1992; Bossert and Compeau, 1995; Funk et al., 1995; Boopathy and Manning, 1996; Widrig et al., 1997), and very few reports on partial degradation of tetryl are available (Boopathy and Manning, 1998; Boopathy, 2000). To our knowledge, this is the first report on the complete biological removal of tetryl from contaminated soil by indigenous soil bacteria. The sequencing batch soil slurry reactor system operated under anoxic/ aerobic sequence seems to be promising. The advantage of the slurry reactor is its simple operating conditions: the method needs only mixing, aeration, and a carbon source. Molasses is an inexpensive carbon source that could be used in a large-scale operation at low cost. The other treatment costs include excavation of soil and dewatering of soil after treatment.
0 0
20
40 60 Time (days)
80
100
Fig. 4. Concentration of aniline in the anaerobic and SBR. Data points represent the average value of triplicate analysis at every sampling event. Symbols: , anaerobic reactor; –m–, SBR. The SBR was operated under anaerobic condition from day 0 to day 60 and operated aerobically from day 61 until the end of the experiment.
Acknowledgements This work was supported in part by a grant from Nicholls Research Council. The author would like to thank Steve Kent and Yifan Psai for the GC-MS analyses.
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References Boopathy, R., 2000. Formation of aniline as a transient metabolite during the metabolism of tetryl by a sulfate-reducing bacterial consortium. Current Microbiology 40, 190–193. Boopathy, R., Kulpa, C.F., 1993. Nitroaromatic compounds serve as nitrogen source for Desulfovibrio sp. (B strain). Canadian Journal of Microbiology 39, 430–433. Boopathy, R., Manning, J.F., 1996. Characterization of partial anaerobic metabolic pathway for 2,4,6-trinitrotoluene degradation by a sulfate-reducing bacterial consortium. Canadian Journal of Microbiology 42, 1203–1208. Boopathy, R., Manning, J.F., 1997. Biodegradation of tetryl in a soil slurry reactor with molasses as co-substrate. Abstract Q-354, 97th General Meeting of the American Society for Microbiology (ASM), Miami, FL. Boopathy, R., Manning, J.F., 1998. Biodegradation of Tetryl (2,4,6trinitrophenylmethylnitramine) in a soil slurry reactor. Water Environment Research 70, 1049–1055. Boopathy, R., Wilson, M., Kulpa, C.F., 1993. Anaerobic removal of 2,4,6-trinitrotoluene (TNT) under different electron accepting conditions: laboratory study. Water Environment Research 65, 271–275. Boopathy, R., Kulpa, C.F., Manning, J., Montemagno, C., 1994a. Biotransformation of 2,4,6-trinitrotoluene (TNT) by co-metabolism with various co-substrates: A Laboratory scale study. Bioresource Technology 47, 205–208. Boopathy, R., Manning, J., Montemagno, C., Rimkus, K., 1994b. Metabolism of trinitrobenzene by a Pseudomonas consortium. Canadian Journal of Microbiology 40, 787–790. Bossert, I.D., Compeau, G.C., 1995. Clean up of petroleum hydrocarbon contamination in soil. In: Young, L.Y., Cerniglia, C.E. (Eds.), Microbial Transformation and Degradation of Toxic Organic Chemicals. Wiley-Liss Inc., New York, pp. 77–125.
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Brown, A.P., 2002. Benson’s Microbiological Applications, eighth ed. McGraw-Hill, Boston, MA. Dickel, O., Knackmuss, H.J., 1991. Catabolism of 1,3-dinitrobenzene by Rhodococcus sp. QT-1. Archives of Microbiology 157, 76–81. Funk, S.B., Crawford, D.L., Crawford, R.L., Mead, G., DavisHoover, W., 1995. Full scale anaerobic bioremediation of trinitrotoluene (TNT) contaminated soil—a US EPA SITE program demonstration. Applied Biochemistry and Biotechnology 51, 625–633. Gell, P.G.H., 1944. Sensitization of tetryl. British Journal of Experimental Pathology 25, 174–178. Jenkins, T.T., Walsh, M.E., 1987. Development of an analytical method for explosive residues in soil. CRREL Report 87-7, US Army Toxic Hazards Materials Agency, AMXTH-TE-FR 861602, US Army, Aberdeen Proving Ground, MD. Othmer, K., 1980. Encyclopaedia of Chemical Technology, vol. 9, third ed. Wiley, New York. Urbanski, T., 1984. Chemistry and Technology of Explosives, vol. 4. Pergamon Press, Oxford, UK, pp. 69–80. Whong, W.Z., Speciner, N.D., Edwards, G.S., 1980. Mutagenic activity of tetryl, a nitroaromatic explosive, in three microbial test systems. Toxicology Letters 5, 11–15. Widrig, D.L., Boopathy, R., Manning, J.F., 1997. Bioremediation of TNT-contaminated soil: a laboratory study. Environmental Toxicology and Chemistry 16, 1141–1148. Williams, R.T., Ziegenfuss, P.S., Sisk, W.E., 1992. Composting of explosives and propellant contaminated soils under thermophilic and mesophilic conditions. Journal of Industrial Microbiology 9, 137–144. Witkowski, L.J., Fischer, C.N., Murdock, H.D., 1942. Industrial illness due to tetryl. Journal of American Medical Association 119, 1406–1410.
International Biodeterioration & Biodegradation 55 (2005) 293–297 www.elsevier.com/locate/ibiod
Bioremediation of tetryl-contaminated soil using sequencing batch soil slurry reactor R. Boopathy Department of Biological Sciences, Nicholls State University, P.O. Box 2021, Thibodaux, LA 70310, USA
Abstract A laboratory study was conducted to determine whether tetryl (2,4,6-trinitrophenlymethylnitramine) contaminated soil could be bioremediated using a sequencing batch soil slurry reactor (SBR) operated under anoxic–aerobic sequence. The results indicated that tetryl was co-metabolically converted to aniline under anoxic conditions with molasses as the growth substrate. The gas chromatographic/mass spectrometric analysis of the soil slurry showed various metabolites, identified as trinitrobenzeneamine, dintrobenzenediamine, nitroaniline and aniline. Aniline was not metabolized further under anoxic conditions. When the soil slurry reactor was operated under aerobic conditions, the aniline concentration was reduced to below the detection limit (0.05 ppm). This metabolic conversion of tetryl is probably of value in the treatment of tetryl-contaminated soil and ground water, such as those found at the Joliet army ammunition plant site in Illinois and the Iowa army ammunition plant site in Burlington, Iowa. r 2005 Elsevier Ltd. All rights reserved. Keywords: Explosives; Tetryl; Biodegradation; Bioremediation; Sequencing batch reactor; Anaerobic treatment
1. Introduction Tetryl (2,4,6-trinitrophenylmethylnitramine), one of the major contaminants at the Joliet army ammunition plant (JAAP), is found in the JAAP soil at numerous locations (Boopathy and Manning, 1998). Tetryl (Fig. 1) has been used as a booster explosive by the military and is also used as a base charge in detonators and blasting caps (Othmer, 1980). Highly stable and slightly hygroscopic, it can withstand storage at ambient temperature for 20 years (Urbanski, 1984). The importance of tetryl has been reduced considerably with the appearance of other explosives. Tetryl is known to cause tetryl dermatitis in humans (Witkowski et al., 1942) and is mutagenic (Whong et al., 1980). A toxicity study on guinea pigs showed that tetryl can cause erythema or oedema (Gell, 1944). The known method of physical and chemical removal of tetryl in water samples is reported to be treating the Tel.: +1 985 448 4716; fax: +1 985 493 2496.
E-mail address: [email protected]. 0964-8305/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2005.03.006
waste with a 1:4 solution by weight of sodium sulfite, and the solution is heated to 80 1C to speed up the reaction. In this treatment, tetryl is completely decomposed to non-explosive water-soluble products (Urbanski, 1984). Boopathy and Manning (1998) demonstrated the anaerobic removal of tetryl in a soil slurry reactor. There are numerous reports on the biological degradation of 2,4,6-trinitrotoluene (TNT) (Williams et al., 1992; Boopathy and Kulpa, 1993; Boopathy et al., 1993; Funk et al., 1995; Boopathy and Manning, 1996; Widrig et al., 1997) by aerobic and anaerobic bacteria. The literature contains very few reports on the biological degradation of tetryl by bacteria or fungi (Boopathy and Manning, 1997, 1998). However, the present research was conducted to study the degradation of tetryl by native soil bacteria under anoxic–aerobic sequencing conditions using a sequencing batch reactor (SBR). The results suggest that the indigenous soil bacteria can effectively degrade tetryl to aniline under anoxic condition, with aniline being further degraded under aerobic condition.
ARTICLE IN PRESS R. Boopathy / International Biodeterioration & Biodegradation 55 (2005) 293–297
294
CH3 N
NO2
NO2
NO2
Fig. 2. Schematic diagram of laboratory soil slurry reactor.
NO2 Fig. 1. Structural formula of tetryl.
The tetryl concentration in the soil was monitored periodically.
2. Materials and methods
2.3. Analytical methods
2.1. Soil
Soil bound tetryl was analyzed by using a soilextraction procedure developed by Jenkins and Walsh (1987). Soil slurry was dried in an oven and 1 g soil was extracted with 10 ml acetonitrile. The whole mixture was agitated for 18 h using a solid-state ultrasonic cleaning system (Model FS 7652, Fisher Scientific, Itasca, IL). After agitation, 5 ml soil-slurry extract was mixed with 5 ml calcium chloride ð5 g L1 Þ. Then, the whole mixture was filtered through a 0:45 mm filter (Millipore, Bradford, MA). Tetryl and other contaminants in the filtrate were analyzed by high-performance liquid chromatography with a Waters (Milford, MA) liquid chromatograph equipped with two Model 6000A solvent pumps, a Model 490E programmable multiwavelength detector set at 254 nm, a data module, and a Model 600E system controller. The mobile phase was methanol–water (50:50). Aliquots ð50 mLÞ were injected into a Supelco column (Model C-18, Supelco Inc., Bellefont, PA). The flow rate of the solvent was 1:5 mL min1 . DO in the soil slurry was monitored weekly by using an oxygen analyzer (Gilson Oxygraph, Model oxy-5, Gilson Electronics Inc., Middleton, WI). A slurry sample from the reactor was added to the cell, and the DO concentration in this sample was measured and expressed in mg L1 . The instrument was calibrated with deionized water saturated with oxygen. The pH in the slurry sample was measured using a pH meter (Model 50, Accumet, Denver, CO). At several stages of the experiment, the bacteria growing in the slurry reactor were enumerated. Slurry samples taken from the reactor were serially diluted with phosphate buffer. Standard methods for total plate counts on tryptic soy agar (TSA) plates, as described by Brown (2002) were followed, and the anaerobic plates were incubated in an anaerobic jar.
Tetryl-contaminated soil was collected from JAAP. The soil had a tetryl concentration that ranged from 2000 to 7000 mg kg1 soil. A homogenous mixture of soil was prepared using a tumbler. The homogenized soil, with a tetryl concentration of 4216 mg kg1 , was used for this study. 2.2. Soil-slurry reactor Three 2-L laboratory-scale soil-slurry reactors (Fig. 2) were designed and operated under co-metabolic conditions using molasses as the co-substrate. The working volume of each reactor was 1.5 L. The reactors were started with 20% (w/v on a dry weight basis) tetrylcontaminated soil in sterile tap water. One reactor was operated aerobically and the second reactor was operated under anoxic conditions through out the experiment. The third reactor was operated in a sequencing mode in which the reactor was operated anoxically for the first 60 days, then it was switched to aerobic mode. All three reactors received molasses (Grandma’s Molasses, Mott’s Cadbury Beverages Inc., Stamford, CT) at 0.3% (v/v) as growth substrate at the beginning and once every 2 weeks thereafter. Additionally, two control soil slurry reactors (one under aerobic condition and another under anoxic condition) were operated without co-substrate (molasses). All reactors were mixed continuously by using a magnetic stirrer (100 rpm). The aerobic reactors were aerated throughout the study. The dissolved oxygen (DO) in this reactor was around 5 mg L1 . The headspace of the anoxic reactors was purged with 100% helium in order to maintain the anoxic condition and this was achieved by constantly bubbling helium gas into all reactors.
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2.4. Chemicals Tetryl was obtained from the Naval Surface Warfare Center, Indian Head, MD. Aniline was purchased from Sigma Chemical Co., St. Louis, MO. The metabolic standards were obtained from Ultra Scientific Analytical and Environmental Standards (North Kingstown, RI). All other chemicals were reagent grade.
3. Results and discussion 3.1. Performance of soil slurry reactors The present study was designed for co-metabolic conditions with molasses as a co-substrate based on an earlier report (Boopathy et al., 1994a) showing that the removal of explosive chemicals was accomplished under such conditions with molasses as the growth substrate. Fig. 3 shows the concentration of tetryl in the soil. Under anoxic conditions, the initial tertyl concentration of 4216 mg kg1 was gradually reduced to below the detection limit (0:05 mg kg1 soil) within 60 days. The soil slurry reactor operated under aerobic conditions did not perform very well and the tetryl concentration dropped only slightly from around 4200 to 3800 mg kg1 . In anoxic and aerobic control reactors, operated with no additions of co-substrate, the tetryl concentration remained at the initial concentration ca. 4200 mg kg1 soil, indicating that tetryl removal in soil is a co-metabolic process. Bacterial growth, pH, and DO were maintained in all reactors including the control reactors throughout the study. At day 30 of the reactor operation (Table 1) the bacterial counts were high in reactors operated with molasses as co-substrate under both aerobic and anoxic
5000
4000 Tetryl (mg.kg-1 of soil)
The intermediates produced during tetryl metabolism by soil bacteria were analyzed by gas chromatographymass spectrometry (GC-MS) methods. Concentrated samples were prepared by drying the slurry sample using a roto-evaporator and were reconstituted with methylene chloride. The GC-MS analyses were performed in the electron-ionization mode on a Finnigan model INCOS 50 System (Finnigan, Cincinnati, OH). The samples were chromatographed with a gradient temperature program: initial temperature 80 1C for 1 min; temperature increased to 250 1C at 20 C min1 and held at 250 1C for 10 min. An SPB-5 (Supelco, Supelcopark, PA), (30 m 0:25 mm, 0:25 mm film) was used. The injection temperature was 250 1C; transport line was maintained at 200 1C; the helium flow was 25 cm s1 and injection volume 2 mL. Peaks of interest were analyzed for mass spectra, and the spectra of metabolites were compared with known standard spectra of individual metabolites.
295
3000
2000
1000
0 0
20
40 Time (days)
60
80
Fig. 3. Concentration of tetryl in various soil slurry reactors. Data points represent the average value of triplicate analysis at every sampling event. Symbols: , aerobic control reactor; ––, anaerobic control reactor; n, reactor operated under aerobic condition with molasses as co-substrate; m, reactor operated under anaerobic condition with molasses as co-substrate; &, SBR. Table 1 Bacterial count, pH, and DO in soil slurry reactors on day 30 of the experiment Reactor
Bacterial count
pH
DO ðmg L1 Þ
Aerobic control Anaerobic control Aerobic Anaerobic SBR
45 2 102 32 3 102 126 6 109 198 9 109 202 7 109
6:8 0:2 7:1 0:3 6:4 0:25 5:8 0:22 5:9 0:31
5:4 0:4 0:1 0:33 5:1 0:28 0:08 0:01 0:07 0:01
The data represents mean and standard deviation of triplicate samples. Anaerobic plates were incubated in an anaerobic jar.
conditions, indicating the presence of aerobic and anoxic bacteria in the contaminated soil. In the control reactors operated without molasses, the bacterial counts were significantly lower; this indicates that tetryl which was the sole source of carbon, was not used for growth. The pH of the experimental reactors was acidic (pH 5.8–6.4), whereas, the pH in the control reactors was close to neutral (pH 6.8–7.1). The DO in the aerobic reactors was ca. 5 mg L1 and in the anoxic reactor the DO was p0:1 mg L1 throughout the experiment. 3.2. Identification of tetryl metabolites Tetryl was metabolized in the reactors operated under anoxic conditions and the SBR operated in anoxic–aerobic sequence (Fig. 3). Various metabolites were produced in these reactors (chromatogram not shown). Mass-spectral analysis of the peak that eluted at 34 min showed a fragmentation profile similar to that of
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trinitrobenzeneamine. The mass spectrum of standard dinitrobenzenediamine perfectly matched that of the peak that eluted at 36 min on GC total ion chromatogram. The next compound identified was nitroaniline, which had a retention time of 36.5 min under the chromatographic conditions used. The mass spectrum of this compound matched the standard nitroaniline. The last compound identified in the GC-MS analysis was aniline. This compound eluted at 24 min on the GC chromatogram. The major ions at m=z at 39, 66, and 93 a.m. showed 100% similarity with the standard aniline. There were no other major metabolites identified in this study. The metabolites trinitrobenzeneamine, dinitrobenzenediamine, and nitroaniline did not persist more than a week after they appeared in the reactor system. Aniline did not undergo further metabolism under anoxic conditions.
3.3. Degradation of aniline The concentration of aniline increased and accumulated in the reactors operated under anoxic conditions (Fig. 4). However, in the SBR, when the reactor was switched to aerobic mode on day 60 of the experiment, the aniline concentration was reduced below the detection limit ð0:05 mg L1 Þ within 3 weeks. To further demonstrate that the aerobic consortium in the SBR reactor uses aniline as the sole source of carbon, aniline ð100 mg L1 Þ was spiked to the SBR reactor on day 95 without any addition of molasses. The added aniline concentration dropped to 0:05 mg L1 within 1 week of its addition (data not shown). 800
Aniline (mg.kg-1 of soil)
SBR operated aerobically 600
400
200
This study showed that an anoxic/aerobic soil slurry reactor can effectively remediate tetryl contaminated soil. Under anoxic condition, the tetryl is converted to aniline and the aerobic soil microbes metabolize aniline, further resulting in complete degradation of the tetryl. The initial step is a reduction process, in which the nitro group is reduced and the methyl group is demethylated, resulting in the production of trinitrobenzeneamine. The nitro group present in the para position is reduced to an amino group to form dinitrobenzenediamine. Later, two nitro groups are removed from the benzene ring by reduction, followed by reductive deamination; in this process, nitroaniline is formed, similarly to the formation of nitrobenzene from trinitrobenzene as described by Boopathy et al. (1994b). The nitro group present in the sixth position is also eventually removed by bacterial activity, either by reduction and reductive deamination (Boopathy et al., 1993) or by the removal of the nitro group as such (Dickel and Knackmuss, 1991), resulting in the production of aniline in the reactor. This probable metabolic pathway is based only on GC-MS identification of the compounds in the soil slurry system. Further detailed study is needed to identify each step of the pathway, the enzymes involved in the metabolism, and the soil bacteria responsible for the action. This study demonstrated that soil bacteria, by employing various metabolic enzymes and reactions, including demethylation, reduction, and reductive deamination can metabolize tetryl in the soil. An aspect of this project requiring further investigation in a detailed radiolabelled experiment is the mineralization of tetryl. Previous studies by various investigators have been focused on the bioremediation of TNT (Williams et al., 1992; Bossert and Compeau, 1995; Funk et al., 1995; Boopathy and Manning, 1996; Widrig et al., 1997), and very few reports on partial degradation of tetryl are available (Boopathy and Manning, 1998; Boopathy, 2000). To our knowledge, this is the first report on the complete biological removal of tetryl from contaminated soil by indigenous soil bacteria. The sequencing batch soil slurry reactor system operated under anoxic/ aerobic sequence seems to be promising. The advantage of the slurry reactor is its simple operating conditions: the method needs only mixing, aeration, and a carbon source. Molasses is an inexpensive carbon source that could be used in a large-scale operation at low cost. The other treatment costs include excavation of soil and dewatering of soil after treatment.
0 0
20
40 60 Time (days)
80
100
Fig. 4. Concentration of aniline in the anaerobic and SBR. Data points represent the average value of triplicate analysis at every sampling event. Symbols: , anaerobic reactor; –m–, SBR. The SBR was operated under anaerobic condition from day 0 to day 60 and operated aerobically from day 61 until the end of the experiment.
Acknowledgements This work was supported in part by a grant from Nicholls Research Council. The author would like to thank Steve Kent and Yifan Psai for the GC-MS analyses.
ARTICLE IN PRESS R. Boopathy / International Biodeterioration & Biodegradation 55 (2005) 293–297
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