Microbial and Photolytic Degradation of 3,5,6-Trichloro-2-pyridinol

Chapter 2

Microbial and Photolytic Degradation of 3,5,6Trichloro-2-pyridinol

Downloaded by COLUMBIA UNIV on July 17, 2012 | http://pubs.acs.org Publication Date: October 10, 2003 | doi: 10.1021/bk-2004-0863.ch002

Yucheng Feng Department of Agronomy and Soils, Auburn University, Auburn, A L 36849

3,5,6-Trichloro-2-pyridinol (TCP) is a primary degradation product of the insecticide chlorpyrifos and the herbicide triclopyr. A bacterium, Pseudomonas sp. strain ATCC 700113, capable of using TCP as a sole source of carbon and energy, was isolated from a soil treated repeatedly with chlorpyrifos. TCP was metabolized to C O , chloride, and unidentified polar metabolites. Pseudomonas sp. ATCC 700113 immobilized on diatomaceous earth beads also mineralized [2,6- C]TCP rapidly; about 75% of the initial radioactivity was recovered as CO . Immobilized cells effectively removed TCP from wastewater generated from a chlorpyrifos-manufacturing plant; however, degradation of TCP was inhibited by high concentrations of NaCl. Photolysis of TCP occurred rapidly upon UV irradiation and released CO , chloride, dichlorodihydroxypyridine isomers, and reductive dechlorination products. Resting cell cultures of Pseudomonas sp. ATCC 700113 can only degrade the reductive dechlorination products in the mixture of photodegradation products, suggesting TCP degradation by this organism involves a reductive dechlorination pathway. 2

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© 2004 American Chemical Society In Pesticide Decontamination and Detoxification; Gan, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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16 Halogenated heterocyclic aromatic compounds are widely used for the production of pesticides, pharmaceuticals, and dyes, but much less is known regarding their metabolism by microorganisms compared with their homocyclic analogs. 3,5,6-Trichloro-2-pyridinol (TCP) is a major metabolite of the insecticide chlorpyrifos and the herbicide triclopyr. It has been detected in environments where chlorpyrifos and triclopyr were previously applied (1-6). TCP can be mineralized in soil, and its half-life varies with soil type, ranging from 10 to 325 days (5, 7). The mineralization of TCP in soil is microbially mediated, but isolation of the degradative microorganisms has rarely been attempted. Chlorpyrifos and triclopyr readily degrade to TCP in the environment via photolysis and hydrolysis. Although relatively non-toxic to mammals, TCP exhibits low-to-moderate toxicity to aquatic and terrestrial biota (8). In addition, TCP has displayed some potential to affect microorganisms; soil concentrations higher than 100 ppm have been reported to retard the microbial degradation of several insecticides (5, 9,10). In addition to the environmental matrices, TCP is also present in the raw, untreated wastewater originating from a chlorpyrifosproducing facility. Chemical oxidation is the conventional method to treat TCPcontaining industrial wastewater. Biological methods (e.g., immobilized cell systems) are emerging as effective alternative treatment strategies, due to costeffectiveness and lack of toxic by-product formation. Like its parent compounds, TCP undergoes photodecomposition, but little is known about the nature of the photodegradation products. Several researchers suggested that the combined use of photolysis and microbial metabolism might provide an alternative method for the treatment of chemical wastes (11-13). The research presented in this paper deals with the isolation and characterization of a pure culture of TCP-degrading bacteria, treatment of TCPcontaining wastewater using an immobilized cell system, photodegradation of TCP, and metabolism of TCP photolysis products by TCP-degrading bacteria.

Experimental Methods Enrichment culture techniques were used to obtain TCP-degrading bacteria from an agricultural soil that had been treated repeatedly with chlorpyrifos (14). Identification of the TCP-degrading isolate was based on phenotypic characterization, substrate utilization pattern (Biolog, Inc.), and fatty acid profile analysis (MIDI, Inc.). The TCP-degrading isolate was grown in a mineral salt medium (14); 0.01% (w/v) yeast extract and 0.018% (w/v) glucose were added when large amounts of cells were needed. Mineralization of [2,6- C]TCP by the TCP-degrading bacterium was determined with growing, resting, and immobilized cells in batch cultures (14, 15). Diatomaceous earth pellets served as solid supports for immobilized cells. Glass columns (40 cm long and 4.9 cm ,4

In Pesticide Decontamination and Detoxification; Gan, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

17 in diameter) containing immobilized bacteria were used to evaluate the removal of TCP from industrial wastewater (15). For photodegradation experiments, TCP solutions were irradiated with an ultraviolet (UV) light source (254 nm) at room temperature for various periods of time depending on the purpose of each experiment (16). The photodegradation products were extracted with ethyl acetate and analyzed by thin-layer chromatography (TLC) and gas chromatography-mass spectrometry (GC-MS). The samples derivatized with bis(trimethylsilyl)trifluoroacetamide (BSTFA) were also analyzed by GC-MS. [2,6- C]TCP was used in some experiments to monitor C 0 evolution and product formation. Unknown products with high radioactivity on TLC plates were isolated and characterized using mass spectrometry and proton nuclear magnetic resonance analyses. Metabolism of TCP photolysis products by resting cells of TCP-degrading bacteria was monitored by analyzing ethyl acetate extracts of the reaction solution using GC (16). 14

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Results and Discussion Isolation and characterization of TCP-degrading bacteria A bacterium capable of using TCP as a sole source of carbon and energy was isolated from a soil with previous exposure to chlorpyrifos, using enrichment culture techniques. The organism was identified as a Pseudomonas sp., deposited in the general collection of the American Type Culture Collection, and assigned the accession number ATCC 700113. Pseudomonas sp. ATCC700113 was a Gram-negative rod; colonies formed on agar plates were circular with an entire margin and smooth surface. Colonies appeared yellow on nutrient agar and yeast-extract/glucose agar, but a brown pigment was produced on tryptic soy and King's Β agar media. The pigment was non-fluorescent and diffusible on King's Β plates. The isolate was catalase positive, cytochrome oxidase positive, and arginine dihydrolase negative. It reduced nitrate to nitrite, used glucose oxidatively, and hydrolyzed gelatin but not starch. Cells grown on TCP did not cleave the aromatic ring of catechol at either ortho or meta position according to the method described by Smibert and Krieg (17). Pseudomonas sp. ATCC 700113 was able to mineralize [2,6- C]TCP readily. About 72.4% of the initial radioactivity was recovered as C 0 , 9% remained in the medium, and 4.1% in biomass. Chloride was released stoichiometrically and concurrently with the disappearance of TCP. Manipulation of experimental conditions revealed that TCP was most rapidly degraded at a temperature of 28°C and pH in the 6.2 to 7.8 range. TCP at 14

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18 concentrations of 100 mg/L or less was degraded rapidly, but at 200 mg/L no transformation occurred, perhaps due to its toxic effect on the bacteria. Transformation of TCP was also affected by the presence of NaCl; degradation rates decreased with increasing concentrations of NaCl and degradation was completely inhibited at a NaCl concentration of 10 g/L. Supplementation with a second carbon source such as glucose, maleic acid, and succinic acid was shown to stimulate bacterial growth as well as TCP degradation. This suggests that cells were carbon starved while growing on TCP alone. Resting cells of Pseudomonas sp. ATCC 700113 metabolized TCP (40 mg/L) within 70 hours at 28°C. About 87% of the initial radioactivity was recovered as C 0 , 3% remained in the medium, and 1.6% was associated with the biomass (Figure 1). HPLC chromatograms revealed the presence of some polar metabolites; however, the amounts were insufficient for further characterization. Resting cells, however, did not metabolize 2-, 3-, and 4hydroxypyridines, 2,4,5-trichlorophenol, and 2,4,5-trichloroaniline. Currently, Pseudomonas sp. ATCC700113 is the only known pure culture of bacteria capable of mineralizing TCP and using it as a sole source of carbon and energy. I4


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Treatment of TCP-containing industrial wastewater Immobilized bacteria have the potential to degrade chemical wastes faster than conventional wastewater treatment systems since high densities of bacteria are used in immobilized cell systems (18-20). Diatomaceous earth pellets are good support materials for the immobilization of bacteria due to their large surface area, high thermal and chemical stability, and mechanical strength. Pseudomonas sp. ATCC 700113 immobilized on diatomaceous earth pellets degraded [2,6- C]TCP (40 mg/L) in seven days. At the end of the experiment, 75.6% of initial radioactivity was recovered in the form of C 0 and 2.5% of initial radioactivity remained in the medium that can be attributed to the polar metabolites formed during the degradation process. Chemical oxidation is the most common method used to remove TCP from industrial wastewater. Numerous organic by-products form during the chemical treatment process. Results from this study indicate that the metabolites remaining after microbial degradation of TCP were negligible. Using columns containing immobilized bacteria, TCP removal of 80 to 100% was achieved with gradual increases of flow rates (from 0.12 to 0.73 ml/min) and TCP concentrations (from 60 to 140 mg/L) in the influent. The column inoculated with growing cells showed immediate removal of TCP after the inoculation while there was a lag phase for the column inoculated with resting cells. Figure 2 indicates that 100% removal of TCP (70 mg/L) was achieved when salt concentrations remained at 0.3%; however, reduced TCP ,4

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Figure 1. Metabolism of TCP by resting cells of Pseudomonas sp. ATCC 700113. (Reproduced with permissionfromreference 14. Copyright 1997 American Society for Microbiology.)

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Figure 2. Effect of sodium chloride concentration on the removal of TCP by immobilized Pseudomonas ATCC 700113. Theflowrates were gradually increased to ImL/min. (Reproduced with permission from reference 17. Copyright 1997 Springer-Verlag.)


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removal was observed when sodium chloride concentration was increased. TCP removal was completely inhibited when sodium chloride concentration reached 1.8%. These results showed that Pseudomonas sp. ATCC 700113 immobilized on diatomaceous earth pellets effectively removed TCP ftom wastewater at low salt concentrations. Growing cell cultures were found to be better inocula for immobilization than resting cells. Since large amounts of salts are present in wastewater generated from cMo^yrifos-manufacturing plants, the tolerance of microorganisms to salts is important for a microbial treatment process. Pseudomonas sp. ATCC 700113 cannot tolerate a sodium chloride concentration of 10 g/L and addition of 1 mM osmoprotectants (i.e., betaine and proline) did not reduce the adverse effect of salt. Further research is needed to develop TCPdegrading bacteria that tolerate high salt concentrations.

Photodegradation of T C P and biodégradation of TCP photolysis products TCP has been shown to undergo rapid photodegradation in aqueous solutions and on natural and synthetic surfaces (8). Although the complete pathway of TCP photolysis has not been established, photodehalogenation and C 0 production have been observed (21). Upon exposure to U V light, TCP (80 mg/L) disappeared to nondetectable levels within 2 hours and the amount of chloride released was 85% of the calculated stoichiometric amount (Figure 3). The color of the reaction solution changed from colorless to reddish brown. Approximately 25% of the initial radioactivity was recovered as C 0 , and 67% remained in the reaction solution (16). Several products resulting from photodegradation of TCP were identified; they are isomers of dichlorodihydroxypyridines (I), isomers of chlorodihydro-2-pyridone (II), tetrahydro-2-pyridone (III), maleamide semialdehyde (IV), and isomers of dichlorocyanopropene. Formation of these products suggests that both reductive dechlorination and hydrolytic dechlorination mechanisms were involved in TCP photolysis. A proposed photodegradation pathway is shown in Figure 4. Mixtures of photodegradation products were used as substrates for resting cells of Pseudomonas sp. ATCC 700113. After 4-day incubation, most TCP photodegradation products were degraded, with the exception of dichlorodihydroxypyridines. This observation suggests that Pseudomonas sp. ATCC 700113 used the reductive dechlorination pathway to degrade TCP. This finding could also explain why Pseudomonas sp. ATCC 700113 gave a negative result for the aromaticringcleavage test. Although TCP was degraded rapidly upon U V irradiation, the extent of mineralization was much less compared to microbial degradation. A combined use of photolysis and microbial metabolism has been suggested as an alternative 2

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Figure 3. Photodegradation of 3,5 6-trichloro-2-pyridinol upon exposure to UV light. (Reproduced with permissionfromreference 18. Copyright 1998 Society of Environmental Toxicology and Chemistry.) t


In Pesticide Decontamination and Detoxification; Gan, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003. f

Figure 4. Proposed photodegradation pathway of 3,5 6~trichloro-2-pyridinol. (Reproduced with permissionfromreference 18. Copyright 1998 Society of Environmental Toxicology and Chemistry.)


23 method for the treatment of chemical wastes (11-13). Due to the free radical nature of photolysis, products of the reaction can be complex. A consortium of microorganisms may be needed to achieve a complete degradation in the treatment process combining microbial and photolytic activity.

Conclusions Pseudomonas sp. ATCC 700113, capable of using TCP as a sole source of carbon and energy, was isolated from a soil with previous exposure to chlorpyrifos. This organism appeared to utilize a reductive dechlorination mechanism to degrade TCP and produce C 0 , chloride, and water as end products. Immobilized cells of Pseudomonas sp. ATCC 700113 effectively removed TCP from industrial wastewater at low salt concentrations. The salt inhibition effect must be overcome before a biological treatment method can be implemented in treating wastewater generated from chlorpyrifos-manufacturing plants.


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24 12. Amador, J. Α.; Taylor, B. F. Appl. Environ. Microbiol. 1990, 56, 13521356. 13. Somich, C. J.; Kearney, P. C.; Muldoon, M. T.; Elsasser, S. J. Agric. Food Chem. 1988, 36, 1322-1326. 14. Feng, Y.; Racke, K. D.; Bollag, J.-M. Appl. Environ. Microbiol. 1997, 63, 4096-4098. 15. Feng, Y.; Racke, K. D.; Bollag, J.-M. Appl. Microbiol. Biotechnol. 1997, 47, 73-77. 16. Feng, Y. Environ. Toxicol. Chem. 1998, 17, 814-819. 17. Smibert, R. M . ; Krieg, N. R. In Methods for general and molecular bacteriology, Krieg, N. R., Ed.; American Society for Microbiology: Washington, DC, 1994; pp 607-654. 18. Hallas, L.; Adams, W. J.; Heitkamp, M . A. Appl. Environ. Microbiol. 1992, 58, 1215-1219. 19. Heitkamp, Μ. Α.; Adams, W. J. Can. J. Microbiol. 1992, 38, 921-928. 20. Rothenburger, S.; Atlas, R. M . Appl. Environ. Microbiol. 1993, 59, 21392144. 21. Smith, G. N. J. Econ. Entomol. 1968, 61, 793-799.


Microbial and Photolytic Degradation of 3,5,6-Trichloro-2-pyridinol

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