TNT Biotransformation and Detoxification by a Pseudomonas Aeruginosa Strain" (2003)

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln U.S. Department of Agriculture: Agricultural Publications from USDA-ARS / UNL Faculty Research Service, Lincoln, Nebraska 9-8-2003 TNT biotransformation and detoxification yb a Pseudomonas aeruginosa strain Byung-taek Oh Seoul National University, Seoul, Korea Patrick J. Shea University of Nebraska-Lincoln, [email protected] Rhae A. Drijber University of Nebraska-Lincoln, [email protected] Galina K. Vasilyeva Russian Academy of Sciences, Pushchino, Russia Gautam Sarath University of Nebraska-Lincoln, [email protected] Follow this and additional works at: https://digitalcommons.unl.edu/usdaarsfacpub Part of the Agricultural Science Commons Oh, Byung-taek; Shea, Patrick J.; Drijber, Rhae A.; Vasilyeva, Galina K.; and Sarath, Gautam, "TNT biotransformation and detoxification by a Pseudomonas aeruginosa strain" (2003). Publications from USDA-ARS / UNL Faculty. 32. https://digitalcommons.unl.edu/usdaarsfacpub/32 This Article is brought to you for free and open access by the U.S. Department of Agriculture: Agricultural Research Service, Lincoln, Nebraska at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Publications from USDA-ARS / UNL Faculty by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Biodegradation 14: 309–319, 2003. 309 © 2003 Kluwer Academic Publishers. Printed in the Netherlands. TNT biotransformation and detoxification by a Pseudomonas aeruginosa strain Byung-Taek Oh1,∗, Patrick J. Shea2,RhaeA.Drijber3, Galina K. Vasilyeva4 & Gautam Sarath5 1School of Chemical Engineering, BK21, Seoul National University, San 56-1, Shinlim-dong, Gwanak-gu, Seoul 151-744, Korea (∗author for correspondence: e-mail: [email protected]); 2School of Natural Resources; 3Department of Agronomy and Horticulture, University of Nebraska, Lincoln, NE 68583-0915, USA; 4Institute of Physicochemical and Biological Problems in Soil Science, Russian Academy of Sciences, Pushchino, Russia 142292; 5USDA-ARS & Department of Biochemistry, University of Nebraska, Lincoln, NE 68583-0664, USA Accepted 7 May 2003 Key words: biotransformation, detoxification, pH, Pseudomonas aeruginosa,TNT Abstract Successful microbial-mediated remediation requires transformation pathways that maximize metabolism and minimize the accumulation of toxic products. Pseudomonas aeruginosa strain MX, isolated from munitions- contaminated soil, degraded 100 mg TNT L−1 in culture medium within 10 h under aerobic conditions. The major TNT products were 2-amino-4,6-dinitrotoluene (2ADNT, primarily in the supernatant) and 2,2- azoxytoluene (2,2AZT, primarily in the cell fraction), which accumulated as major products via the intermediate 2-hydroxylamino-4,6-dinitrotoluene(2HADNT). The 2HADNT and 2,2AZT were relatively less toxic to the strain than TNT and 2ADNT. Aminodinitrotoluene (ADNT) production increased when yeast extract was added to the medium. While TNT transformation rate was not affected by pH, more HADNTs accumulated at pH 5.0 than at pH 8.0 and AZTs did not accumulate at the lower pH. The appearance of 2,6-diamino-4-nitrotoluene (2,6DANT) and 2,4-diamino-6-nitrotoluene (2,4DANT); dinitrotoluene (DNT) and nitrotoluene (NT); and 3,5-dinitroaniline (3,5DNA) indicated various routes of TNT metabolism and detoxification by P. aeruginosa strain MX. Introduction (HADNTs) and nitrosodinitrotoluenes (NoDNTs) (Kaplan & Kaplan 1982; Schackmann & Mueller Contamination of soil and water from 2,4,6- 1991; Bumpus & Tatarko 1994; Alvarez et al. trinitrotoluene (TNT) has occurred during the disposal 1995; Bradley et al. 1995; Drzyzga et al. 1998; of wastewater from munitions production, loading and Esteve-Núñez & Ramos 1998; Kim & Song 2000; packing operations in many countries. The persistence Esteve-Núñez et al. 2001). Diaminonitrotoluene of TNT and its metabolites are of environmental (DANT) also has been identified (Won et al. 1974; concern because they may be toxic to fish (Osmon & Funk et al. 1993; Daniel et al. 1995; Fiorella & Klausmeier 1972; Smock et al. 1976), algae (Smock Spain 1997; Drzyzga et al. 1998; Esteve-Núñez et al. 1976; Won et al. 1976; Bennett 1994), mi- & Ramos 1998), but complete reduction of TNT croorganisms (Klausmeier et al. 1973; Won et al. to triaminotoluene (TAT) required strict anaerobic 1976; Rieger & Knackmuss 1995), higher plants conditions (Ederer et al. 1997; Drzyzga et al. 1998) (Palazzo & Leggett, 1986) and humans (Yinon 1990). and has not been observed in aerobic soils. Haidour Research has shown that the major products of aerobic & Ramos (1996) and Martin et al. (1997) revealed the TNT biotransformation by bacteria are 4-amino-2,6- capacity of some Pseudomonas strains to aerobically dinitrotoluene (4ADNT), 2-amino-4,6-dinitrotoluene denitrate TNT, producing 2,4-dinitrotoluene (2,4DNT) (2ADNT), and various azoxytoluene (AZT) conden- and 2,6-dinitrotoluene (2,6DNT). sation products from hydroxylaminodinitrotoluenes 310 Although Hankenson & Schaeffer (1991) repor- (0.1) and yeast extract (1.0). An EDTA trace element ted that TNT was 20- to 50-fold more toxic than solution (1.0 mL) was added to the culture medium, as a mixture of DANT and ADNT, toxicity may limit described by Alef & Nannipieri (1995). Culture flasks microbial degradation of TNT at highly contam- were incubated on an orbital shaker (150 strokes per inated sites. Research conducted in our labora- min) at 28 ◦C under aerobic conditions. Turbidity of tory indicated that Pseudomonas savastanoi rapidly the cultures was monitored at 560 nm using a UV-VIS transformed TNT to 2ADNT, 4ADNT, and 2,4DNT scanning spectrophotometer (Model UV-2101PC, Shi- (Martin et al. 1997). While growth was sustained madzu, Japan). Control cultures were grown without from the mid-log to stationary phases, a decline in TNT or with no inoculation under the same conditions. cell population after 24 h may be due in part to accumulation of toxic metabolites. Subsequent re- TNT transformation search indicated 2-hydroxylamino-4,6-dinitrotoluene TNT transformation was determined in culture me- (2HADNT), 2ADNT, and 4,4 ,6,6 -tetranitro-2,2 - − dium containing 100 mg TNT L 1 and inoculated azoxytoluene (2,2 AZT) were the major metabolites P. aeruginosa of aerobic TNT transformation by P. aeruginosa strain with strain MX. During the incubation, 1 mL samples were withdrawn for analysis and the MX isolated from TNT-contaminated soil (Vasilyeva cells were harvested by centrifuging for 10 min at et al. 2000). Our present objective was an in-depth , × g study of aerobic biotransformation and detoxification 14 000 . The cell pellets were extracted by vortexing three times for 1 min with 1 mL acetonitrile of TNT by the P. aeruginosa strain. and centrifuging again. TNT and its major metabolites (ADNTs, HADNTs, and AZTs) were determined Materials and methods by HPLC and changes in concentration were monitored. To detect 2,6DANT and cyclohexanone, strain Microorganism and chemicals MX was grown for 3 days as described above. After centrifugation, 500 mL of supernatant was extracted The isolate, Pseudomonas aeruginosa strain MX, was three times with 100 mL of methylene chloride. The obtained from munitions-contaminated soil (Vasilyeva extract was concentrated by rotary evaporation, H2O et al. 2000). Technical grade TNT was obtained from removed with sodium sulfate (Na2SO4), and 3 µL the Fort Detrick U.S. Biomedical Research and De- samples were analyzed by GC/MS. Controls without velopment Laboratory (Frederick, MD). Carbon-14- TNT were included in the analyses. − ring-labeled TNT (137 MBq mmol 1, radiochemical Culture medium components and their concentra- purity 98%) was custom-synthesized by NEN Re- tions were modified in the subcultures. TNT concen- search Products (Boston, MA). Analytical standards trations were 20, 40, 60, 80, and 100 mg L−1 and yeast of 2ADNT, 4ADNT, 2HADNT, 4HADNT, 2,2 AZT, extract concentration varied from 0.2 to 1.0 g L−1.The 2 ,4AZT, 4,4 AZT, and 2,6DA4NT, 2,4DA6NT and pH of the culture medium varied from 5.0 to 8.0. 2,4,6-trihydroxytoluene (THT) were provided by R. Spanggord (SRI International, Palo Alto, CA). Cy- Mass balance of 14C-TNT clohexanol, 2,4DNT, 2-amino-4-nitrotoluene Carbon utilization was tracked by adding 14C-TNT to (2A4NT), and 4-nitrotoluene (4NT) were obtained −1 from Aldrich (Milwaukee, WI). Cyclohexanone, 3,5- 30 mL of culture medium (50,000 dpm mL ). After dinitroaniline (3,5DNA), yeast extract (YE), and salts incubating for 3 d, the culture medium was centri- for preparing culture media and Tryptic Soy Agar fuged and the supernatant was extracted three times (TSA) were purchased from Sigma Chemical Co. (St. with 30 mL of methylene chloride. After acidifying Louis, MO). TAT was obtained from Chem Service to pH 1.0 with HCl, the aqueous phase was extracted (West Chester, PA). All organic solvents were HPLC with ethyl acetate as described above. The centrifuged grade. cell fraction (pellet) was extracted by vortexing and centrifuging three times with 10 mL acetonitrile. Total 14 Culture conditions and growth measurements C in the extracts and in the remaining aqueous solution was determined by mixing 0.5 mL of sample P. aeruginosa strain MX was grown in culture me- with 6 mL Ultima Gold Cocktail (Packard Instru- dium containing the following (g L−1 distilled water): ment Company, Meriden, CT) and liquid scintillation MgSO4 (0.1), K2HPO4 (3.5), KH2PO4 (1.5), TNT counting (LSC, Packard 1900TR liquid scintillation 311 counter, Packard Instrument Co., Downers Grove, IL). buffer for 1 h before dehydration in an ethanol series Unextractable 14C remaining in the pellet was determ- (50, 75, 90, and 100% for 20 min in each step) and 14 ined by combusting to CO2 in a biological oxidizer propylene oxide. Samples were left in 1 : 1 ratio of (Packard, Tri-Carb B306, Packard Instrument Co., propylene oxide and Epon 812 (Electron Microscopy 14 Downers Grove, IL). The CO2 was trapped in a mix- Sciences, Fort Washington, PA) mixture overnight ture (3 : 2, v/v) of Carbosorb and Permaflour (Packard at room temperature before embedding in pure Epon Instrument Company, Meriden, CT) and quantified 812.

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