Biodegradation and metabolic pathway of sulfamethoxazole by Sphingobacterium mizutaii

Sulfamethoxazole (SMX) is the most commonly used antibiotic in worldwide for inhibiting aquatic animal diseases. However, the residues of SMX are difficult to eliminate and may enter the food chain, leading to considerable threats on human health. The bacterial strain Sphingobacterium mizutaii LLE5 was isolated from activated sludge. This strain could utilize SMX as its sole carbon source and degrade it efficiently. Under optimal degradation conditions (30.8 °C, pH 7.2, and inoculum amount of 3.5 × 107 cfu/mL), S. mizutaii LLE5 could degrade 93.87% of 50 mg/L SMX within 7 days. Four intermediate products from the degradation of SMX were identified and a possible degradation pathway based on these findings was proposed. Furthermore, S. mizutaii LLE5 could also degrade other sulfonamides. This study is the first report on (1) degradation of SMX and other sulfonamides by S. mizutaii, (2) optimization of biodegradation conditions via response surface methodology, and (3) identification of sulfanilamide, 4-aminothiophenol, 5-amino-3-methylisoxazole, and aniline as metabolites in the degradation pathway of SMX in a microorganism. This strain might be useful for the bioremediation of SMX-contaminated environment.

www.nature.com/scientificreports/ to degrade SMX. When the initial concentration is 25, 50, 100, 150, and 200 mg/ L, the removal rates of SMX by SRB sludge through adsorption and biodegradation are 3.9, 5.6, 13.2, 15.9, and 21.3 mg/L/d 15 . Other bacteria, including Achromobacter denitrificans PR1 16 , P. chrysosporium 17 , Pseudomonas stutzeri 18 , and Acinetobacter sp. 19 , could serve as resources for the bioremediation of SMX from the polluted environment. Nevertheless, further studies are needed. Previous studies mainly isolated SMX-degrading bacteria through traditional methods, and the degradation efficiency was relatively low. The degradation mechanism of SMX and the metabolic pathway involved are also unclear. The aims of this study were: (1) to isolate a bacterial strain that can highly degrade SMX by a novel method; (2) to optimize the environmental parameters to improve degradation efficiency; and (3) to deduce the possible degradation pathway of degrading strain and examine the mechanism underlying SMX degradation.

Results
Community changes and diversity of SMX-degrading enrichment cultures. A total of 5978, 6012, 6048, 6005, and 6003 16S rRNA gene sequences of the distinct V3-V4 regions were obtained from sludge sample (SMXY) and four generations of enrichment cultures (SMX1-4) by high-throughput sequencing, respectively. After statistical analysis and annotation, the results ( Fig. 1) showed that 5978 sequences in sludge sample SMXY were clustered into 166 OTUs. The highest abundance of OTUs belonged to bsv13, Lentimicrobium, Flaviolibacter, Ramlibacter, and Rhodoferax. After enrichment, the bacterial diversity of SMX1 rapidly decreased, 6012 sequences were clustered into only 23 OTUs. The main genera were Pseudomonas, Sphingobacterium, Escherichia-Shigella, Alcaligenes, and Sediminibacter. Pseudomonas became the highest abundance genus in SMX2. After the second passage, with the increase in the SMX concentration, the bacterial diversity of SMX2 decreased continuously, and 6048 sequences were clustered into 20 OTUs. The main genera were Sphingobacterium, Escherichia-Shigella, Alcaligenes, and Sediminibacter, Sphingobacterium replaced Pseudomonas as the genus with the highest abundance, and the proportion of Sphingobacterium reached 85.84%. The result indicates that Sphingobacterium can tolerate higher concentrations of SMX than the other bacteria. As a result, the diversity in the enrichment gradually declined with the increase in SMX concentrations and passage generations.   Fig. 3a), with an optimum temperature of 30 °C. Although the temperature decreased to 15 °C, the degradation efficiency was more than 42.32%. When the temperature rose to 40 ℃, the degradation efficiency was more than 58.65%, and these results showed that S. mizutaii LLE5 has an obvious temperature adaptability. pH is another key factor affecting the degradation efficiency of the strain. The results of different pH on the degradation of the strain (Fig. 3b) show that LLE5 can degrade SMX in the pH range 4.0-9.0. At the optimal pH of 7.0, the degradation of SMX was 91.77%. In addition, the degradation efficiency of LLE5 was more than 50% in the pH range 5.0-8.0, which indicated that the degradation efficiency of strain LLE5 was higher under neutral conditions. When the pH was reduced to 4.0, the degradation efficiency was still 48.64%, indicating that the strain was tolerant to acid conditions. When the pH was 9.0, the degradation efficiency was 38.70%, which indicated that LLE5 could be applied under neutral and acid conditions. The different initial inoculum amount on the degradation efficiency was analyzed (Fig. 3c). When the inoculation amount was 5 × 10 7 cfu/mL, the degradation efficiency of S. mizutaii LLE5 was the highest, which was 92.89%. However, when the initial inoculation amount was 2.0 × 10 7 -8.0 × 10 7 cfu/mL, the degradation efficiency was more than 90%, indicating that the degradation efficiency of S. mizutaii LLE5 did not change significantly with the change in inoculum amount. The initial concentration of SMX also influenced the degradation efficiency of S. mizutaii LLE5 (Fig. 3d). The degrada- The temperature, pH levels and inoculation size were further designed through the response surface method in accordance with the results of single-factor experiments, and 17 degradation tests were carried out (Table 1). By the statistical analysis of data, the following second-degree polynomial equation was obtained to explain SMX biodegradation by S. mizutaii LLE5: where Y 1 represents the SMX degradation efficiency, and A, B, and C are the coded values for temperature, pH level, and inoculum amount respectively. Analysis of variance (ANOVA) for the fitted quadratic polynomial model is shown in Table 2. The model was signifcant (P < 0.05) with R 2 = 0.9831 and Adj R 2 = 0.9613. The results of regression analysis indicated that A, B, AB, A 2 and B 2 are significant model terms, whereas the C, AC, BC, C 2 are nonsignificant model terms. The same was confirmed from the Pareto chart ( Fig. 4) in which higher effects were presented in the upper portion and then progress down to the lower effects. It directly shows that the most import factors influencing biodegradation efficiency were A, B, AB, A 2 and B 2 . The three-dimensional response surface was plotted to directly display the effects of the temperature and pH level on SMX biodegradation. At the theoretical maximum point of response surface (Fig. 5), the optimum conditions for SMX degradation by S. mizutaii LLE5 were 30.8 °C, pH 7.2, and inoculum amount of 3.5 × 10 7 cfu/mL. Degradation of SMX by strain LLE5. The degradation characteristics of SMX by S. mizutaii LLE5 under the optimal conditions were studied. The results indicated that the most efficient degradation was obtained during the first 3 days. On the third day, the degradation efficiency was 84.02%, and the cell concentration was 68.95% × 10 7 cfu/mL (Fig. 6). The degradation efficiency was positively correlated with the cell growth density. At 5-7 days, the degradation efficiency of SMX gradually decreased and was accompanied by no further increase in S. mizutaii LLE5 cell density. Finally, the degradation efficiency of SMX with initial concentration of 50 mg/L was 93.87% after 7 days. This is the first report that Sphingobacterium mizutaii has a good degradation effect on SMX. S. mizutaii LLE5 can also degrade other sulfonamides, and the degradation efficiencies of strain LLE5 for sulfadiazine, sulfaguanidine, sulfamisoxazole, and sulfadimidine were 59.85%, 51.68%, 46.95%, and 37.42%, www.nature.com/scientificreports/ respectively (Fig. 7). To elucidate the degradation ability of S. mizutaii LLE-5 against various sulfonamides, the degradation constant (k) and half-life (t 1/2 ) were determined by using the first-order kinetic model. Table 3 presents the kinetics parameters calculated from the model. The coefficient of determination R 2 varied from 0.9572 to 0.9924 indicating that the degradation data reliably fitted with the first-order kinetic model. Degradation rate constants (k) varied from 0.0620 to 0.4247 d −1 that characterized the degradation process of various sulfonamides by strain LLE-5. Theoretical half-life (t 1/2 ) of SMX, sulfaguanidine, sulfamisoxazole, and sulfadimidine was noted as 1.63, 5.22, 6.80, 7.96, and 11.18 days, respectively. These results show that S. mizutaii LLE5 has a broad specificity for the degradation of sulfonamides and has considerable potential for processing sulfonamide pollution in the environment.
Metabolic pathways of SMX degradation by S. mizutaii LLE5. The metabolites of SMX degraded by S. mizutaii LLE5 in MSM liquid medium were detected by HPLC/MS. According to the chemical structure of SMX and the mass spectrum, four candidate products were identified. These products were sulfanilamide (171 m/z), 4-aminothiophenol (124 m/z), 5-amino-3-methylisoxazole (99 m/z), and aniline (92 m/z). None of these products were detected when the culture medium only contained SMX and without S. mizutaii LLE5, indicating that they are SMX biodegradation metabolites. A possible metabolic pathway for SMX biodegradation by LLE5 was proposed (Fig. 8). SMX is first transformed into sulfanilamide and 5-amino-3-methylisoxazole through hydrogenation. Then, sulfanilamide is degraded via desulfurization into aniline and via deamination into 4-aminothiophenol. Although the ring-opening products of hydroquinone were not detected, it is still the first report of the pathway of SMX degradation by a Sphingobacterium strain. The candidate products were iden-

Discussion
Sphingobacterium strains widely exist in natural environment 20 . Sphingobacterium is a kind of gram-negative bacteria and does not produce spores. Since the genus Sphingobacterium was proposed originally by Yabuuchi et al. in 1983 21 , new species have been discovered from a variety of environments, such as soil, plants, animals, and even clinical samples of ventricular fluid and urine. Among them, Sphingobacterum thalpohilum and Sphingobacterum multivarum can degrade petroleum hydrocarbons. For example, S. multivorum SWH-2 has a strong ability to degrade petroleum 22 . After ensuring optimal conditions, the normal growth and enzyme secretion and activity of S. multivorum SWH-2 will also change, which can further improve the oil degradation of the strain. In addition, most Sphingobacterium found thus far are resistant to a variety of antibiotics from microorganisms. However, there is no report on the direct degradation of antibiotics by Sphingobacterium. Sphingobacterium   www.nature.com/scientificreports/ mizutaii isolated in this study is the first Sphingobacterium reported to degrade SMX. The discovery of this bacterium not only expanded the functional range of Sphingobacterium but also provided valuable microbial resources for the remediation of SMX contaminated aquaculture environment.
In recent years, a number of strains with good SMX degradation ability, such as Planococcus kocurii O516, Achromobacter sp. JL9, Phanerochaete chrysosporium, and Acinetobacter sp., have been isolated from soil, activated sludge, and aquaculture water. Compared with the reported strain, the strain LLE-5 had a better environmental range and tolerance. The study on the influence of environmental factors on the degradation effect indicates that strain LLE-5 can degrade SMX at the temperature range 15-40 ℃ and at pH 4-9. This finding provides a basis for the practical application of the strain in the future. The physiological and biochemical results Table 3. Kinetic parameters of various sulfonamides degradation by S. mizutaii LLE-5. k represents degradation constant (d −1 ); t 1/2 represents half-time (d); R 2 represents correlation coeffificient; Ct is the concentration (mg/L) of sulfonamides at time t.

Sulfonamides
Regression equation k (d −1 ) t 1/2 (d) www.nature.com/scientificreports/ showed that LLE-5 could use a variety of carbon sources. The bacteria have strong adaptability to the environment, rich types of nutrition metabolism, and potential to degrade other complex compounds. The existing reports show that most strains of Sphingobacterium cannot produce antibiotics but are resistant to antibiotics, which is an important factor for the Sphingobacterium to adapt to extreme environments 23 . Antibiotics are compounds that inhibit the growth of microorganisms. Thus, degrading bacteria need to have antibiotic resistance. Some studies have shown that Klebsiella pneumoniae can use chloramphenicol as the sole carbon source for growth 24 . However, the drug sensitivity test showed that these strains were sensitive to chloramphenicol, which indicated that the drug resistance and drug degradation processes were two independent pathways. Drug-resistant bacteria can only resist antibiotics and avoid growth inhibition. By contrast, antibiotic-degrading bacteria resist antibiotics and produce degrading enzymes such as monooxygenase, esterase, or hydrolase to degrade the drug. We sequenced the whole genome of strain LLE5 and found some genes that might be involved in SMX degradation (data not shown). Their functions will be identified in our future work. According to the degradation products of SMX by S. mizutaii LLE-5, the strain mainly transformed SMX into sulfanilamide and 5-amino-3-methylisoxazole through hydrogenation. Then, sulfanilamide is degraded via desulfurization into aniline and via deamination into 4-aminothiophenol. The results showed that the strain had a clear degradation function of sulfamethoxazole and had a good tolerance. However, SMX could not be completely mineralized in terms of degradation products. This work is the first to report the pathway of SMX degradation by a Sphingobacterium strain and provided a certain reference for the study of microbial degradation of SMX.

Methods
Chemicals and media. SMX (analytical standard, > 99.5%), HPLC-grade methanol, and acetonitrile were purchased from Sinopharm Chemical Reagent Beijing Co., Ltd. All other reagents used in the present study were of analytical grade. Mineral salt medium (MSM) and Luria-Bertani (LB) medium were used as described by Song et al 25 . Agar plates were prepared by adding 1.5% (w/v) agar into the liquid media.
Enrichment culture and high-throughput sequencing. Activated sludge samples were collected from the wastewater treatment pond of a pharmaceutical factory in Liaoyang City, Liaoning Province, China. 10.0 g of sample was suspended in 100 mL of MSM supplemented with 100 mg/L SMX. Then, the sample was incubated on a rotary incubator shaker at 30 °C and 100 r/min in the dark. After 7 days, 10 mL of the sample was transferred into fresh MSM supplemented with 200 mg/L of SMX and incubated for another 7 days. After repeating this process four times, the SMX concentration in the fourth generation increased to 500 mg/L. The activated sludge sample was designated as SMXY, and the four generations of SMX enrichment cultures were designated as SMX1, SMX2, SMX3, and SMX4. The total DNA of SMXY and SMX1-4 were extracted using a FastDNA SPIN kit for soil (MP, Biomedicals, USA). After purification, the 16S rRNA distinct regions V3-V4 were amplified with the following primers: 338F (5′-ACT CCT ACG GGA GGC AGC A-3′) and 806R (5′-GGA CTA CHVGGG TWT CTAAT-3′). The polymerase chain reaction (PCR) system was designed as described by Li et al 26 . The PCR product was sent to Biomark Gene Technology Company for library construction and high-throughput sequencing. The obtained sequence data were analyzed using Arch software (http:// www. drive5. com/ usear ch/). The identity of sequences ≥ 97% was assigned to an operational taxonomic unit (OTU), and each OTU was considered to represent a species. The relative abundance of OTUs in the samples was statistically analyzed using R software (https:// www.R-proje ct. org) 27 .

Isolation and identification of SMX-degrading bacteria.
According to the results of high-throughput sequencing, the fourth-generation enrichment sample SMX4 containing the most Sphingobacterium cells was used for isolation of SMX-degrading bacteria. Then, 100 μL of SMX4 was spread on the MSM solid plate containing 100 mg/L SMX and incubated at 30 °C. The rapidly growing colonies on the plate with different morphologies were selected and restreaked three times to obtain pure cultures. The degradation efficiency of the isolates was determined by a high-performance liquid chromatography (HPLC) system (1260, Agilent, Santa Clara, CA) equipped with an eclipse Plus C 18 column. 10 mL of the culture and equal volume of ethyl acetate were added into a 50 mL centrifuge tube and mixed using a vortex mixer for 1 min. Then, the centrifuge tube was placed in a shaker, mixed at 220 r/min for 30 min, and centrifuged at 5000 g for 8 min. The upper phase was filtered with a 0.22 μm membrane for HPLC analysis. The elution comprised a mixture of methanol, formic acid (85/15/0.1, v/v/v), and distilled water, running at the flow rate of 1.0 mL/min. The injection volume was 10 μL, and the column temperature was 30°C 28 . The isolate with the highest degradation efficiency was selected for further analysis. Morphology was investigated using a light microscope (BX-51; Olympus, Japan). The carbohydrate assimilation of isolate was conducted with Biolog GEN3 plates following the analytical methods described by Hobbie et al 29 . The genomic DNA of the isolate was extracted and was used as a template for 16S rDNA amplification as described by Ruan et al 30 . The universal primers were 27F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492R (5′ACGGHTAC CTT GTT TAC GACTT-3′). The purified PCR product was sequenced by Bomad Technology (Beijing, China). The obtained sequence was deposited in GenBank. The strain was further identified by performing multiple sequence alignment using Clustal X sofware, and phylogenetic relationships were analyzed via the neighbor-joining (NJ) method with MEGA 6 software 31 .
Inoculum preparation. Before the degradation experiment, strain activation was performed. S. mizutaii LLE-5 was inoculated into 100 mL of LB medium and incubated at 30 °C on a rotary shaker at 150 rpm. After 12 h, the bacterial cells were harvested by centrifugation at 4000 g for 10 min. The precipitate was washed two times by phosphate buffered saline (PBS) solution and suspended for subsequent studies 32  where Y i refers to the predicted response, X i and X j are variables, b o is a constant, b i denotes the linear coefficient, b ii represents the quadratic coefficient, and b ij corresponds to the interaction coefficient.
Biodegradation tests. The SMX degradation test was performed under optimal conditions. S. mizutaii LLE-5 cells were inoculated into 100 mL of MSM supplemented with 50 mg/L of SMX. The residual of SMX and cell numbers of S. mizutaii LLE-5 were detected every day. The control was inoculated with killed cells of S. mizutaii LLE-5. Each test was conducted in triplicate 35 . The ability of S. mizutaii LLE-5 to degrade other structurally similar sulfonamides, including sulfadiazine, sulfaguanidine, sulfamisoxazole, and sulfadimidine, were evaluated. Analytical methods were the same as above described. The first-order kinetic model (Eq. 3) was created to elucidate sulfonamides degradation efficiency of S. mizutaii LLE-5 36 .
where C 0 is the initial concentration of sulfonamides at time zero, C t is the concentration of sulfonamides at time t, k is the degradation rate constant (d −1 ). The theoretical half-life (t 1/2 ) values of different sulfonamides were calculated by Eq. (4).
where ln 2 is the natural logarithm of 2 and k is degradation rate constant (d −1 ).

Identification of SMX biodegradation intermediates.
Intermediates generated during SMX degradation by S. mizutaii LLE-5 in MSM were analyzed by HPLC-MS (AB Sciex QTRAP 5500, USA). The extraction method was same as described above. The flow rate was 0.2 mL/min, the mobile phase A was 0.1% formic acid (V/V), and B was methanol. The targeted screening gradient elution program was: 0-2 min, 95% A; 2-25 min, 95%-5% A; 25-35 min, 5% A; and 35-40 min, 95% A. The sample injection volume was 20 μL, and the column temperature was 30 °C. The mass spectrometry conditions were: DuoSprayTM ion source, electrospray ionization (ESI), positive ion mode scanning, the ion source temperature was 550 ℃, the spray voltage was 5500 V, and curtain air (CUR) was 35 psi. The accumulation time was 0.25 s, and the collision voltage was (35 ± 15) eV. (2)