Biodegradation of triclosan by a wastewater microorganism

Available online xxx Keywords: Triclosan Biodegradation Sphingopyxis strain KCY1 Wastewater Meta-cleavage a b s t r a c t Triclosan, a synthetic antimicrobial agent, has been considered as an emerging environmental contaminant. Here we reported a triclosan-degrading wastewater bacterial isolate, Sphingopyxis strain KCY1, capable of dechlorinating triclosan with a stoichiometric release of chloride. The stain can degrade diphenyl ether but not 2,4,4 0 -tribromodiphenyl ether and 2,2 0 ,4,4 0 -tetrabromodiphenyl ether, despite all these three compounds are structurally similar to triclosan. While strain KCY1 was unable to grow on triclosan and catechol, it could grow with glucose, sodium succinate, sodium acetate, and phenol. When grown with complex nutrient medium containing a trace amount of triclosan (as low as 5 mg/L), the strain could retain its degradation ability toward triclosan. The maximum-specific triclosan degradation rate (q m ) and the half-velocity constant (K m ) are 0.13 mg-triclosan/mgprotein/day and 2.8 mg-triclosan/L, respectively. As triclosan degradation progressed, five metabolites were identified and these metabolites continue to transform into non-chlorinated end products, which was supported by a sharp drop in androgenic potential. The activity of catechol 2,3-dioxygenase in the cell extract was detected. No triclosan degradation was observed in the presence of 3-fluorocatechol, an inhibitor of meta-cleavage enzyme, suggesting that triclosan degradation proceed via meta-cleavage pathway. Based on all the observations, a degradation pathway for triclosan by strain KCY1 was proposed. ª 2012 Published by Elsevier Ltd. Introduction Triclosan (5-chloro-2-(2,4-dichlorophenoxy)-phenol) is a common synthetic antimicrobial agent that has been incorporated into more than 700 different industrial and personal care products. These products, including deodorants, soaps, toothpastes, and various plastic products, contain 0.1e0.3% triclosan Biodegradation of triclosan in the environment and wastewater has recently become an interesting research topic Please cite this article in press as: Lee, D. In this study, we report isolation and characterization of a wastewater triclosan-degrading bacterium, Sphingopyxis strain KCY1. This isolate showed complete dechlorination of triclosan based on stoichiometric release of chloride. We also determined triclosan degradation kinetics, proposed possible degradation pathway for triclosan, and assessed the potential significance of this isolate to triclosan biodegradation in wastewater. 2. Material and methods Chemicals Triclosan Isolation and identification of triclosan-degrading bacteria A triclosan-degrading consortium, originally inoculated with activated sludge, was used as a source for the isolation of triclosan-degrading bacteria. Briefly, a loopful of the consortium was streaked onto nitrate mineral salts (NMS)-triclosan (5 mg/L) agar plates that were incubated at 30 C (Chu and Alvarez 2.3. Determination of degradation ability toward triclosan and compounds structurally similar to triclosan The isolate was initially grown in 20% R2A medium with 5 mg/ L triclosan for three days. The cell suspension was harvested by centrifugation and the pellet was washed with 50 mM phosphate-buffered saline and then resuspended in fresh NMS medium for experimental use. The degradation tests were conducted in 250 mL flasks containing the resting cell suspension and 5 mg/L of triclosan. The flasks were incubated on a rotary shaker at 150 rpm and at 30 C, and liquid samples were collected over time for triclosan measurements. A subset of collected liquid samples was used for BLYES and BLYAS assays. Liquid samples collected from degradation experiments were also used to measure concentrations of chloride. A parallel set of experiments was conducted to determine whether the isolate could degrade compounds that are structurally similar to triclosan. Three compounds, diphenyl . The degradation experiments were conducted similarly as described above, except using resting cell suspension (OD 600 ¼ 0.6e0.8) and each of these three compounds. Resistance to other antimicrobial agents Experiments were performed to determine whether strain KCY1 could resist to three common antimicrobial agents: kanamycin, trimethoprim and ampicillin (See SI for experimental details). Determination of Monod kinetic parameters for triclosan degradation Monod degradation kinetic model q ¼ q m $S=K s þ S, was used to describe triclosan degradation by strain KCY1. The kinetic experiments were conducted in a series of 40 mL EPA glass vials containing resting cells of strain KCY1 (OD 600 ¼ 0.4) and triclosan (ranging from 0.3 to 5 mg/L) in NMS medium. The vials were incubated on a rotary shaker at 150 rpm and at 30 C for 3 h, and then used for triclosan and protein measurements. The incubation duration (3 h) was determined in the laboratory where initial degradation rates remained linear. Experimental data obtained from kinetic tests were plotted as specific triclosan degradation rates (q, mass of substrate/mass of cell protein/time) against triclosan concentrations (S, mass/ volume). The maximum specific triclosan degradation rate (q m , mass of triclosan/mass of cell protein/time) and the halfvelocity constant (K m , mass of triclosan/volume) were determined by curve fitting using Sigmaplot 8.0 (SPSS Inc.) as w a t e r r e s e a r c h x x x ( 2 0 1 2 ) 1 e9 previously described Determination of degradation/utilization ability toward other organics Experiments were performed to determine whether the isolate could grow on three common macro-organics in wastewater: glucose (300 mg/L), sodium acetate (175 mg/L), and sodium succinate (300 mg/L) (Roh and Chu, 2010). These compounds were selected for the experiments because glucose is a common carbohydrate, and sodium succinate and sodium acetate are components present inside the tricarboxylic acid cycle (TCA cycle). Cell growth expressed as optical density (OD 600 ), protein contents, and volatile suspension solids, was monitored over time. Doubling times were determined from the exponential growth phase curves. Autoclavekilled cells were used as negative controls. Effect of complex nutrients on triclosan degradation Cells grown in a complex nutrient medium without prior exposure to triclosan were tested for their ability to retain its biodegradation of triclosan. Experiments were conducted as follows. Strain KCY1 was grown in 100% R2A (fully nutrientrich) medium without or with (5 or 500 mg/L) triclosan and transferred to its respective growth medium every two days. After four consecutive transfers, the cells were harvested as described above for degradation tests. The degradation experiments were conducted in glass vials containing 5 mg/L triclosan and the resting cells in NMS medium. Bioluminescent androgenic/estrogenic screening assays To evaluate androgenic and estrogenic potential of triclosan degradation metabolites and end products, the bioluminescent androgenic and estrogenic screening (BLYES and BLYAS) assays were performed as described previously Determination of enzymes responsible for triclosan degradation The isolate was screened for the presence of catechol 2,3-dioxygenase and/or catechol 1,2-dioxygenase using a spectrophotometric method as described previously . In addition, triclosan degradation via meta-cleavage pathway was tested by adding 3-fluorocatechol (50 mg/L) or in the absence of it. 3-Fluorocatechol is an inhibitor of catechol 2,3-dioxygenase that catalyzes meta-cleavage reactions (Bartels et al., 1984; Chemical analysis Chloride concentrations were measured using a DX-80 Ion Chromatography (IC) system (Dionex, Sunnyvale, CA) equipped with an IonPac AS14A-5 mm analytical column (3  150 mm). Triclosan, tri-BDE, tetra-BDE, and DE concentrations and degradation metabolites were determined using a GC (Agilent 6890)/MS (Agilent 5973) equipped with DB-5 column. In addition, to detect possible degradation metabolites, LC/MS analysis was performed using a Surveyor HPLC system (ThermoFinnigan, San Jose, CA) interfaced with quadruple ion trap mass spectrometer (LCQ-DECA; ThermoFinnigan). Detailed description of chemical analysis is available in SI. 3. Results and discussion 3.1. Identification of a triclosan-degrading microorganism, strain KCY1 Among three presumptive triclosan-degrading colonies, one isolate (yellow-mucoid), designated strain KCY1, showed the ability to degrade triclosan in NMS medium. Strain KCY1 is a short, rod-shaped (0.5 mm  1.7 mm) Gram-negative bacterium with a flagellum a t e r r e s e a r c h x x x ( 2 0 1 2 ) 1 e9 Characteristics of strain KCY1 Since strain KCY1 can grow rapidly on R2A agar, it is expected that the strain can also grow on glucose, sodium succinate, and sodium acetate Degradation of triclosan by strain KCY1 As shown in 3.4. Androgenic and estrogenic potential of triclosan degradation metabolites and end products BYLES and BLYAS assays were used to evaluate estrogenic and androgenic potential of triclosan degradation metabolites and end products. Triclosan itself triggered weak androgenic activity in the BLYAS assay, but not estrogenic activity in the BLYES assay in this study. sensitivities between the yeast cells and human breast cancer cells. As shown in Since triclosan is a chlorinated organic compound, the decrease of androgenic potential over time might correlate to the extent of dechlorination during the triclosan degradation. The decline of androgenic potential could be explained by the decrease in initial triclosan concentration and the transformation into less-chlorinated metabolites that were detected in this study before day 1 (see identification of metabolites below). Between day 1 and day 2, the reduction rate of androgenic activity became slower than the triclosan degradation rate, suggesting that (i) other androgenic metabolites might be produced during this period or (ii) androgenic metabolites might be transformed at a slower rate than the triclosan degradation rate. Interestingly, the androgenic response (w40% of original response) was observed despite that triclosan was no longer detected after day 2. This indicated a slow transformation of triclosan metabolites with androgenicity, like 2,4-dichlorophenol. Previous studies have reported that 2,4-dichloropehnol exhibited in-vivo androgenic activity in zebrafish embryos (Sawle et al., 2010) and in human prostate cancer cells Factors affecting triclosan degradation As wastewater contains a wide range of complex organics that are readily available for microbial growth, it is important to know whether strain KCY1 would retain its ability to degrade triclosan after it has grown on complex nutrients. To determine the effects of nutrients on triclosan biodegradation by strain KCY1, the strain was initially grown in nutrient-rich medium (100% R2A) without triclosan for 8 days (4 consecutive transfers every 2-day). After grown on nutrient-rich medium without triclosan, strain KCY1 lost its degradation ability toward triclosan Degradation kinetic parameters for triclosan The results of triclosan degradation tests were used to develop the relationship between specific triclosan degradation rate (q) and triclosan concentrations 3.7. Degradation ability toward compounds which are structurally similar to triclosan Since two BDEs (tri-and tetra-BDEs) and DE are structurally similar to triclosan and strain KCY1 can degrade triclosan, we hypothesized that strain KCY1 could degrade these compounds as well. Strain KCY1 was able to degrade approximately 78% of DE (1 g/L) within 5 days, but unable to use DE as a sole carbon source (data not shown). Although earlier it was reported that CeBr and CeCl bonds are at least equally viable for enzymatic reaction (Dos Santos et al., 1999), strain KCY1 was unable to degrade tri-BDE and tetra-BDE. The inability of strain KCY1 to degrade these two BDEs could be due to a combination of various factors, including the difference in electronwithdrawing effects that would result from the difference between the halogen species (Cl vs Br) and the absence of hydroxyl group in both BDEs that could contribute to selectivity of the enzymes. The reason why strain KCY1 can degrade DE but not tri-and tetra-BDEs was unclear in this study. Enzymes involved in triclosan biodegradation Another set of experiments was conducted to examine whether 3-flurocatechol, a meta-cleavage inhibitor, would affect triclosan biodegradation. As shown in Degradation metabolites and possible degradation pathway for triclosan During the triclosan biodegradation, five metabolites were identified. These metabolites are monohydroxy-triclosan, 6-chloro-3-(2,4-dichlorophenoxy)-4-hydroxycyclohexa-3,5-diene-1,2-dione, 3-chloro-4-(5,7-dichloro-3-oxo-2,3-dihydrobenzo[1,4]dioxin-2-yl)-2-oxobut-3-enal, 3,5-dichloro-4,6-dihydroxycyclohexa-3,5-diene-1,2-dione, and 2,4-dichlorophenol ( Here we proposed a biodegradation pathway for triclosan by strain KCY

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