Biological conversion of sulfamethoxazole in an autotrophic denitrification system

Biological conversion of sulfamethoxazole in an autotrophic denitrification system

Water Research 185 (2020) 116156 Contents lists available at ScienceDirect Water Research journal homepage: www.elsevier.com/locate/watres Biologic...

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Water Research 185 (2020) 116156

Contents lists available at ScienceDirect

Water Research journal homepage: www.elsevier.com/locate/watres

Biological conversion of sulfamethoxazole in an autotrophic denitrification system Liang Zhang a, Faqian Sun b, Dan Wu a, Wangwang Yan a, Yan Zhou a, c, * a

Advanced Environmental Biotechnology Centre, Nanyang Environment and Water Research Institute, Nanyang Technological University, 637141, Singapore b College of Geography and Environmental Science, Zhejiang Normal University, Jinhua, 321004, China c School of Civil and Environmental Engineering, Nanyang Technological University, 639798, Singapore

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 March 2020 Received in revised form 8 June 2020 Accepted 4 July 2020 Available online 11 August 2020

Sulfamethoxazole (SMX) is a common antibiotic prescribed for treating infections, which is frequently detected in the effluent of conventional wastewater treatment plants (WWTPs). Its degradation and conversion in a laboratory-scale sulfur-based autotrophic denitrification reactor were for the first time investigated through long-term reactor operation and short-term batch experiments. Co-metabolism of SMX and nitrate by autotrophic denitrifiers was observed in this study. The specific SMX removal rate was 3.7 ± 1.4 mg/g SS-d, which was higher than those reported in conventional wastewater treatment processes. The removal of SMX by the enriched denitrifying sludge was mainly attributed to biodegradation. Four transformation products (three known with structures and one with unknown structure) were identified, of which the structures of the two transformation products (TPs) were altered in the isoxazole ring. Additionally, the presence of SMX significantly shaped the microbial community structures, leading to the dominant denitrifier shifting from Sulfuritalea to Sulfurimonas to maintain the stability of system. Collectively, the sulfur-based autotrophic denitrification process could effectively remove SMX in addition to efficient nitrate removal, and further polish the effluent from conventional WWTPs. © 2020 Elsevier Ltd. All rights reserved.

Keywords: SMX removal Biodegradation Sulfur oxidation Nitrate removal Autotrophic denitrifier

1. Introduction Sulfamethoxazole (SMX) is one of the widely used sulfonamide antibiotics, which is mainly used for diminishing inflammation and promoting livestock growth (Zuccato et al., 2010). SMX is frequently detected in the aquatic environments owing to its high usage and persistence, and enters the environment mainly via the discharge of the effluent from wastewater treatment plants (WWTPs) (Loos et al., 2013). The SMX concentration is at the level of ng/L to mg/L in the effluent of WWTPs (Loos et al., 2013; Zhou et al., 2013). When treating wastewater from pharmaceutical industries, the SMX concentration in the effluent could even reach a mg/L level (Cetecioglu, 2014). The ubiquitous presence of SMX may have directly toxic effects on aquatic organisms (Shimizu et al., 2013;

* Corresponding author. Advanced Environmental Biotechnology Centre, Nanyang Environment and Water Research Institute, Nanyang Technological University, 637141, Singapore. E-mail address: [email protected] (Y. Zhou). https://doi.org/10.1016/j.watres.2020.116156 0043-1354/© 2020 Elsevier Ltd. All rights reserved.

Johnson et al., 2015). The persistent exposure of microorganisms to SMX may also promote the development of antibiotic resistance genes, which may lead to long-term threats to human health and animals (Baran et al., 2011). As such, improving SMX elimination in WWTPs is deemed necessary to minimize its discharge into the environment. Meanwhile, nitrate (NO 3 ) commonly exists in the effluent of WWTPs, which causes eutrophication, deterioration of water resources, and poses potential hazards to human health (Ward et al., 2005). Heterotrophic denitrification process is typically employed for nitrate removal. However, the high organic consumption and a large amount of sludge production limit its application for treating wastewater with low C/N ratios (Sun et al., 2010), such as nitrate polluted groundwater, and secondary effluent from WWTPs. Autotrophic denitrification is a promising alternative to heterotrophic denitrification as the former does not consume organics, and has low sludge production and operational cost (Sahinkaya and Dursun, 2012). Elemental sulfur-based autotrophic denitrification is one of such biotechnologies, in which autotrophic denitrifiers reduce nitrate to nitrogen gas using elemental sulfur as the electron

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donor while generating sulfate (Equation (1)) (Zhang et al., 2015; Liu et al., 2016; Qiu et al., 2020). Many previous studies have demonstrated the feasibility of sulfur-based autotrophic denitrification process for producing drinking water from nitratecontaminated water (Sierra-Alvarez et al., 2007; Shao et al., 2010; Sahinkaya et al., 2012) and treating low C/N wastewater (Sahinkaya et al., 2014; Qiu et al., 2020). þ S0 þ 0:91NO 3 þ 0:69H2 O þ 0:07NH4 þ þ 0:36CO2 /0:07C5 H7 O2 N þ SO2 4 þ 0:45N2 þ 1:16H

(1)

In addition to autotrophic denitrifiers, low abundance of heterotrophic denitrifiers and sulfate-reducing bacteria (SRB) commonly co-exist with autotrophic denitrifiers in the sulfur-based denitrification system (Zhang et al., 2015; Qiu et al., 2020). Heterotrophic denitrifiers, such as Achromobacter and Pseudomonas are able to degrade SMX (Jiang et al., 2014; Reis et al., 2014). Jia et al. (2017) and Qiu et al. (2019) observed SMX could be removed under sulfate-reducing conditions. In this light, we could expect that the sulfur-based denitrification system could remove SMX. However, it still remains unknown if autotrophic denitrifiers could be involved in SMX removal in the system. In addition, it is not clear how the antibiotics would interact with the denitrifying system in terms of denitrification performance and microbial community responses. Therefore, this study aims to investigate the feasibility of a sulfur-based autotrophic denitrification system for simultaneous removal of nitrate and SMX. A laboratory-scale sequencing batch reactor (SBR) was established and continuously operated for 170 days to: a) investigate the long-term performance of SBR in terms of nitrate and SMX removal; b) determine the role of autotrophic denitrification on SMX conversion; c) identify the transformation products of SMX; d) elucidate the effects of SMX on functional microbial community.

2. Materials and methods 2.1. Experimental setup and operation A laboratory-scale sequencing batch reactor (SBR) with a working volume of 1.90 L (110 mm diameter x 200 mm height) was set up. The SBR was seeded with approximately 3.3 g/L activated sludge collected from a local water reclamation plant which is operated with conventional activated sludge process. The SBR was fed with synthetic wastewater prepared as per Qiu et al., 2020. Sublimed sulfur (20e40 mm in particle size) was daily supplemented through the top opening of the reactor according to the daily consumption rate of elemental sulfur. The SBR was operated with a cycle time of 10 min of feeding, 110 min of stirring, 35 min of settling and 5 min of decanting. In each cycle, 50% of the supernatant in the SBR were replaced by new synthetic wastewater, resulting in a hydraulic retention time of 5.3 h. The SBR was operated under constant room temperature (~25  C). The SBR was continuously operated for 170 days, which was divided into two stages (I and II). In Stage I (days 0e46), the SBR was fed with synthetic wastewater without SMX, providing approximately 45 ± 5 mg N/L of nitrate. In Stage II (days 47e170), in addition to the synthetic wastewater, SMX (Sigma-Aldrich) stock solution was spiked to the influent tank to achieve an environmentally relevant concentration of 22.4 ± 4.3 mg/L (Wei et al., 2019). The influent tank, tubing, and reactor were covered by aluminum foil to avoid photodegradation. During the whole operational period, the influent and effluent water samples were regularly collected to monitor the performance of the SBR.

2.2. Batch adsorption experiments To study if SMX could be adsorbed onto sulfur fine particles, equilibrium isotherm experiments were performed in duplicates in brown glass bottles with 50 mL of tap water and 2 g of elemental sulfur. These bottles were continuously agitated in a shaker (100 rpm) for 24h. Seven concentration levels of SMX (0, 10, 25, 50, 100, 500, and 1000 mg/L) were tested. At the end of the experiment (48h), the bottles were settled for 4 h to allow the settling of solid materials before the supernatant was collected for further analysis. Bottles without sulfur were used as blank to monitor the loss of SMX during the experiment. 2.3. Batch experiments - effects of nitrate or sulfur on sulfamethoxazole removal To study if autotrophic denitrification contributed to SMX removal in the SBR, three sets of batch tests were conducted with the enriched denitrifying sludge collected from the SBR in the presence or absence of nitrate. Each test was carried out in duplicates. The sludge was first washed with deoxygenated deionized water for three times and evenly distributed into six 500-mL glass bottles with caps. The concentrations of sludge and SMX in each bottle were approximately 0.2 g MLVSS/L and 20 mg/L, respectively. The initial pH was adjusted to 7.5 ± 0.1 via adding HCl/NaOH solution. All the tests were conducted at room temperature (~25  C). Two of the six bottles were used as the control group to determine the contribution of sorption and other bacteria (except denitrifiers) on SMX removal, in which only 0.5 g/L sulfur was fed. Approximately 30 mg N/L of nitrate and 0.5 g/L sulfur were added into the other four bottles. 10 mM sodium azide (NaN3) (Prieto et al., 2011) were spiked into two of the four bottles to investigate the contribution of sorption processes on SMX removal via inhibiting the microbial activity. The batch tests lasted for 12 h, during which water samples were collected at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h via a sampling point on the caps fitted with a hose. Concentrations of SMX, nitrate, nitrite, sulfate and sulfide as well as pH were measured. To further evaluate the contribution of other non-autotrophicdenitrifiers to SMX removal, a separate batch test was conducted in duplicates using the enriched denitrifying sludge in the absence of sulfur. To avoid the interference of residual sulfur from the parent reactor, the sludge taken out from the SBR was washed ten times using the deoxygenated deionized water to remove sulfur particles and then incubated for one week only with nitrate, micronutrients and NaHCO3 to consume all the remaining sulfur. Before performing the batch tests, the starved sludge was then washed again with deoxygenated deionized water and evenly distributed into two 250-mL glass bottles. The experiment was initiated with the addition of approximately 40 mg N/L of nitrate and 20 mg/L of SMX. In addition, a batch test in duplicates with 0.5 g/L of sulfur initially was conducted as a reference. The other test conditions were the same as those in above mentioned batch tests. The batch tests were conducted for 4 h, during which water samples were collected at 0 h, 2 h, and 4 h. 2.4. Co-metabolism of sulfamethoxazole and nitrate To explore the SMX degradation mechanism, two groups of batch tests were conducted in duplicates in the SBR. In the first group, nitrate was fed into the reactor as one pulse with different initial nitrate concentrations (0, 30, 60, and 120 mg N/L). The initial SMX concentration in all the tests was approximately 20 mg/L. The tests were performed for 120 min, during which the water samples were collected at 0 min, 5 min, 10 min, 20 min, 30 min, 40 min,

L. Zhang et al. / Water Research 185 (2020) 116156

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60 min, 100 min, and 120 min. In the second group, nitrate was added into the SBR as multiple pulses. The initial SMX concentration in the test was approximately 45 mg/L. The tests were conducted for 210 min, during which the water samples were collected at 0 min, 30 min, 60 min, 180min, and 210 min. Nitrate was added into the SBR at the starting of the batch tests and each sampling point. Nitrate concentration was 100 mg N/ L at 60 min and 35 mg N/L at the rest of the sampling time.

weighted UniFrac distance matrix in QIIME to study the beta diversity of bacterial communities (Lozupone et al., 2011). The statistical differences between the different groups in the PCoA plot were assessed using Analysis of Similarities (ANOSIM) with BrayCurtis distance, where the p value below 0.05 indicating a statistically significant difference.

2.5. Analytical methods

3.1. Performance of the SBR

Nitrate, nitrite, sulfate, and sulfide in water samples were measured after filtration (Millipore, 0.45 mm). Nitrate, nitrite, total dissolved sulfide (H2S, HS and S2), mixed liquor suspended solids (MLSS) and mixed liquor volatile suspended solids (MLVSS) were measured following the Standard Methods (APHA, 2005). Sulfate concentration was determined with an ion chromatograph (DIONEX ICS2100, CA, USA) with a conductivity detector and a Dionex IonPac AS11-HC analytical column. pH and temperature were measured using portable meters (Multi-Parameter Meter, HQ40D, Hach). Elemental sulfur content in sludge samples was measured with a high-performance liquid chromatograph (HPLC, Agilent 1260 Infinity, USA) equipped with a Luna C18 column (250  4.6 mm, 5 mm, 100 Å) and a UV detector at 254 nm according to the protocol described by McGuire and Hamers (2000). The SMX after filtration (PTFE filter, 0.22 mm) was measured by a triple Quadruple mass spectrometry equipped with an Agilent Jet Stream Electrospray ionization (AJS-ESI) (LCQQQ 6460, Agilent) and an [email protected] Omega polar LC C18 column (100  2.1 mm, 1.6 mm) under mobile phase A (MiliQ-water modified with 0.1% acetic acid) and mobile phase B (acetonitrile modified with 0.1% acetic acid) at a flow rate of 0.3 mL/min. Before being analyzed, the water samples were first purified using an online solid-phase extraction (OSPE) system (Agilent 1290 Infinity Flexible Cube module). The detailed procedures about the OSPE system and operation can be found in Anumol and Snyder (2015). SMX-13C6 (Sigma-Aldrich) was used as the internal standard to accurately quantify the SMX concentration. The SMX calibration ranges from 5 ng/L to 1250 ng/L. The SMX adsorbed by the enriched denitrifying sludge in the SBR was extracted according to the protocols described by Jia et al. (2017). The SMX transformation products (TPs) were analyzed using the above stated LCQQQ and mobile phases with an injection volume of 5 mL at a flow rate of 0.2 mL/min. The MS system was operated in MS2 mode in both positive and negative ionization modes to scan mass spectra with a range from m/z 30 to m/z 600. The SMX TPs without reference standards were further identified using an Agilent 6550 iFunnel LC-QTOF/MS (Agilent, USA), in which the QTOF/MS was operated with an electrospray ion source using Agilent Jet Stream Technology in positive and negative ionization modes. The column, mobile phase and flow rate (0.2 mL/min) were the same with those used in LCQQQ operation.

The laboratory-scale SBR was continuously operated for 170 days (Fig. 1). In Stage I without SMX addition (days 0e46), the influent nitrate was efficiently removed with an initial concentration of 44 ± 6 mg N/L, and no nitrite was detected in the effluent. Correspondingly, the nitrate reduction rate was 8.3 mg/L-h on average, which is comparable to other similar studies (Sahinkaya et al., 2014, 2015; Qiu et al., 2020). Sulfur was used as the electron donors in nitrate reduction and oxidized into sulfate, generating 123 ± 29 mg S/L. In Stage II (days 47e170), 22.4 ± 4.3 mg/L of SMX were spiked into the feed. The performance of SBR was not influenced in terms of nitrate removal. 47 ± 5 mg N/L of the influent nitrate were efficiently removed without nitrite accumulation. Meanwhile, 129 ± 16 mg S/L of sulfate were produced. 7 ± 2 mg S/L of sulfide were detected in the SBR effluent (Fig. 1b). The presence of sulfide may be attributed to a small extent of sulfur disproportionation to sulfate and sulfide (Sahinkaya et al., 2015) or the low activity of SRB (Qiu et al., 2020). In addition, the sulfur-based autotrophic denitrification process consumes alkalinity (Equation (1)), resulting in the lower pH values in the effluent than in the influent (Fig. S1). Collectively, the results indicate that the presence of SMX at an environmentally relevant concentration had a negligible effect on the autotrophic denitrification performance, which has never been previously reported.

2.6. DNA extraction, PCR, Illumina Miseq sequencing and analysis The sludge samples in triplicates were collected on day 46, 78, 112, and 170 during the operation of SBR to study the effect of SMX presence on microbial community. The total genomic DNA of the sludge samples was extracted with the fast DNA extraction Kit for soil (MP Biomedicals, Singapore) according to the manufacturer’s instructions. The extracted DNA was then stored at 20  C before being subjected to sequencing. A 515F-806R primer pair was employed to amplify the 16s rRNA to target the V4 regions of both the bacteria and archaea domains. The Illumina Miseq sequencing service was provided by Major bioTech. Co., Ltd. (Shanghai, China). Principal coordinates analysis (PCoA) was conducted based on the

3. Results and discussion

3.2. Sulfamethoxazole removal in the SBR The removal efficiency of SMX was 21.2 ± 6.3% during the entire operational period, corresponding with a specific SMX removal rate of 3.7 ± 1.4 mg/g SS-d (or 4.8 ± 1.9 mg/g VSS-d) (Fig. 2a). The removal rate is higher than those reported in conventional activated sludge (CAS) processes and methanogenic processes (see Table 1). For instance, Alvarino et al. (2014) and Kang et al. (2018b) found that the specific SMX removal rate in CAS processes was 1.4e2.2 mg/g VSS-d. Alvarino et al. (2014) observed that the specific SMX removal rate was only approximately 0.22 mg/g VSS-d in an up-flow anaerobic sludge blanket (UASB) reactor. It is also comparable to those reported in membrane bioreactors (MBR) (0.019e47.1 mg/g SS-d) (Clara et al., 2005; Hai et al., 2011; Xiao et al., 2017). However, the specific SMX removal rate obtained in this study was lower than that in sulfate reduction process. Jia et al. (2017) and Qiu et al. (2019) observed that the specific SMX removal rate was 11.2e15.2 mg/g SS-d in sulfate-reducing upflow sludge bed (SRUSB) reactors. The octanol-water distribution coefficient (log Kow) of SMX is 0.86, suggesting that SMX is relatively hydrophilic and is difficult to be sorbed (Qiu et al., 2019). In this light, the different SMX removal rates in different bioprocesses may be due to the discrepancy in the different microbial communities. In the sulfur-based autotrophic denitrification system, the predominant microbial communities are autotrophic denitrifiers (see Section 3.6), which is apparently different from those in sulfate-reducing reactors (e.g. Desulfomicrobium and Desulfobulbus etc.) (Jiang et al., 2013; Zhang et al., 2016a), aerobic (e.g. Pseudomonas and Nitrosospira etc.) (Katipoglu-Yazan et al., 2016) and anaerobic biosystems for treating

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Fig. 1. Long-term performance of the SBR in the absence or presence of SMX: (a) nitrate removal and (b) net sulfate production and effluent sulfide concentration.

Fig. 2. (a) Removal of SMX in the SBR during the long-term operational period, and (b) the contribution of sorption and biodegradation to the removed SMX in the long-term operation of SBR.

Table 1 Comparison of SMX removal performance. Influent concentration (mg/L)

SMX removal efficiency (%)

Specific removal rate (mg/g Reference SS-d)

Synthetic wastewater 5.3

22.4 ± 4.3

21.2 ± 6.3

3.7 ± 1.4/4.8 ± 1.9b

This study

Synthetic Synthetic Synthetic Synthetic

10 2 10 50e100

44 73e84 89 34e51

2.2b 1.2e1.5b 0.2b 11.2e15.2

750 0.15

66 61

47.1 0.014

Alvarino et al. (2014) Kang et al. (2018b) Alvarino et al. (2014) (Jia et al., 2017; Qiu et al., 2019) Hai et al. (2011) Clara et al. (2005)

2

68

0.78

Xiao et al. (2017)

Parameters

Wastewater

Sulfur-based autotrophic denitrification CAS CAS UASB reactora SRUSB reactor Aerobic or anoxic MBR Aerobic MBR Anaerobic MBR a b

wastewater wastewater wastewater wastewater

HRT (h)

24 12 24 5e6

Synthetic wastewater 24 Real domestic 12 wastewater Synthetic wastewater 6

The UASB reactor was used for methanogenic process. The unit is mg/g VSS-d.

SMX-contained waste water. In general, biodegradation, sorption, photodegradation, hydrolysis and volatilization are the possible removal pathways of organic micropollutants in activated sludge systems (Grandclement et al., 2017). In this study, the reactor was fully covered by aluminium foil, indicating that photodegradation barely occurred. The physical-chemical properties of SMX show that hydrolysis and volatilization could be ruled out (Table S1). In this light, microbial degradation and sorption could be the main pathway of SMX

removal. Elemental sulfur used in this study was in fine particles form, which may adsorb SMX to some extent. However, the adsorption experiment concluded that almost no SMX could be adsorbed by the elemental sulfur particles (Fig. S2). Meanwhile, the amount of SMX accumulated in the sludge during the long-term operation of SBR was negligible (<1%) (Fig. 2b), indicating that the removed SMX was mainly via biodegradation process. Similarly, Qiu et al. (2019a) observed that approximately 76% of the removed SMX were attributed to microbial biodegradation in sulfate

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reduction process. SMX removal in aerobic granules and CAS was also mainly ascribed to biodegradation (Kang et al., 2018b). 3.3. Sulfamethoxazole transformation products Owing to the low initial SMX concentration and removal efficiency, identifying transformation products (TPs) of SMX directly with the SBR effluent was challenging. A batch test with a higher SMX concentration (~350 mg/L) was therefore performed (methods described in SI). Approximately 40% of the spiked SMX were removed within 96 h (Fig. S3). Four TPs were detected during the batch test via LC-MS/MS and confirmed with LC-QTOF/MS. The potential SMX TPs and chemical structures except for an unknown TP (TP287) are listed in Table 2. The LC-MS/MS total ion chromatogram is presented in Fig. S4. The detected metabolites (Table 2 and Fig. 3) showed that the isoxazole ring was vulnerable which could be easily attacked. The result is in agreement with that reported under anaerobic sulfatereducing (Jia et al., 2017) and iron-reducing conditions (Mohatt et al., 2011). An unstable intermediate (SMXþ) could be first formed under reductive conditions in which the ring cleavage initiated in the NeO bond via hydrogenation (Fig. 3) (Jia et al., 2017). The unstable SMXþ was then transformed into the proposed stable end-products (TP1 (m/z 254) and TP2 (m/z 256)). Hydroxy-N-(5-methyl-3-isoxazole) benzene-1-sulfonamide (m/z 254) as one of the metabolites of SMX was also observed during the biodegradation by Rhodococcus rhodochrous. Sun et al. (2019) reported that 4-nitro-SMX could be abiotically generated from the reaction between SMX and nitrite during nitrification process. However, this metabolite was not detected at all during the entire batch test even at 4th h when approximately 70 mg N/L of nitrite were accumulated (data not shown). It may be ascribed to the different environments (anoxic in this study vs. aerobic). In addition, N4-hydroxyl-sulfamethoxazole (OH-SMX, m/z 270) was identified to be one of the metabolites in this study. Zhang et al. (2016b) also found that a strain of Alcaligenes faecalis could biotransform SMX into N4-hydroxy-sulfamethoxazole. In addition, more research needs to be done in the future to obtain more detailed degradation pathways of SMX, and to investigate the fate and toxicity of SMX TPs in the autotrophic denitrifying system. 3.4. Role of autotrophic denitrifiers on sulfamethoxazole removal In the presence of NaN3, nitrate and SMX concentration

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maintained stable during the 12-h tests (Fig. S5a), indicating that SMX was barely adsorbed by the enriched denitrifying sludge (Fig. 4a). Similarly, Wang and Wang (2018b) observed that SMX removal by adsorption in aerobic sludge was minor. The result could partially support the finding that the amount of accumulated SMX in the SBR was extremely low. In the absence of nitrate, only 9.4 ± 0.7% of the spiked SMX were removed during the batch tests. Conversely, in the presence of nitrate, the SMX removal efficiency was 27.6 ± 7.2%, which was nearly three-fold higher than that in the absence of nitrate, indicating that the denitrifiers played an important role in SMX degradation. Yang et al. (2018) found that heterotrophic denitrifiers could degrade SMX. Although no exogenous carbon was provided, the organic carbon from the endogenous respiration of bacteria may support the activity of heterotrophic denitrifiers in the SBR (Qiu et al., 2020), and heterotrophic denitrifiers were also detected in the SBR (indicated by the following microbial community analysis). We could postulate that heterotrophic denitrifers could contribute to the SMX removal in the SBR. To further evaluate the contribution of autotrophic denitrifiers to SMX removal, batch tests were conducted in the absence of sulfur. In the absence of sulfur, nitrate concentration maintained stable in the 4-h batch test, but it was completely removed in the presence of sulfur (Fig. S5b). Meanwhile, the SMX removal in the presence of sulfur (22.2 ± 0.9%) was significantly higher than that in the absence of sulfur (13.6 ± 3.4%) (Fig. 4b), revealing that autotrophic denitrifiers played an important role in SMX biodegradation. 3.5. Co-metabolic degradation of sulfamethoxazole To understand the SMX degradation mechanism, two groups of batch tests were conducted. In the first group, nitrate was completely removed within 120 min with the initial concentration ranged from 0 to 120 mg N/L (Fig. S6). Correspondingly, SMX removal increased with elevated initial nitrate concentration. In the absence of nitrate, only 7.7 ± 3.3% of the initial SMX were removed (Fig. 5a). The SMX removal efficiency increased to 26.9 ± 4.5% when the initial nitrate concentration increased to 120 mg N/L (Fig. 5a). The present results further confirm the crucial role of autotrophic denitrifiers in removing SMX. It should be noted that, in the batch test with 120 mg N/L of nitrate initially, SMX reduction only occurred within the first 60 min, and a small extent of degradation was observed in the following 60 min (Fig. 5a). Nevertheless, sufficient nitrate was still present (43 ± 6 mg N/L) at 60 min. Thus, we

Table 2 Analytical information of the transformation products of SMX biodegradation in the sulfur-based autotrophic denitrification system provided by the LC-TOF/MS. Denotation

RT (min)

Observed [M þ Hþ] (m/z)

Calculated [M þ Hþ] (m/z)

Error (ppm)

Molecular formula

SMX

7.254

254.0601

254.0594

2.7553

C10H11O3N3S

TP1

4.460

254.0595

254.0594

0.3936

C10H11O3N3S

TP2

3.574

256.0751

256.075

0.3936

C10H13O3N3S

TP3

6.647

270.0543

270.0543

0

C10H11O4N3S

Proposed structure

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Fig. 3. Transformation products and the possible degradation pathway of sulfamethoxazole in the enriched autotrophic denitrifying sludge.

Fig. 4. (a) SMX removal by the enriched denitrifying sludge in the presence of NaN3 or nitrate or in the absence of nitrate, (b) SMX removal by the enriched denitrifying sludge in the presence or absence of sulfur.

Fig. 5. (a) Effect of different initial nitrate concentrations (0, 30, 60, 120 mg N/L) on SMX removal in the batch tests; (b) effect of multi-pulsed nitrate addition on SMX removal.

speculate that competitive enzyme inhibition may occur. In other words, the pulse addition of nitrate at the beginning of batch test may induce the formation of certain enzymes that could convert SMX. With sufficient nitrate, the activated enzymes may be preferentially used by nitrate reduction. In this light, the multi-pulse feeding may mitigate the impact of competitive inhibition and could re-activate the enzyme activity for SMX removal. SMX removal with multi-pulse nitrate feed was investigated in

the second group experiment (Fig. 5b). Apparently, SMX removal pattern was different from that in the first group. A gradual decline in the SMX concentration from 46 mg/L to 35 mg/L was observed with the multi-feed strategy. Zhang et al. (2019) and Tang et al. (2017) observed a similar phenomenon in which they found that intermittent feeding of organics could enhance pharmaceutical removal in a sand filter and moving bed biofilm reactors (MBBRs) used for polishing the effluent of WWTPs. Interestingly, such co-

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metabolism would not take place if nitrate is continuously fed into the system without net nitrate accumulation (data not shown). At this stage, it still highlights the need towards further studies on shedding light on such competitive inhibition between SMX and nitrate removal in sulfur-based autotrophic denitrification systems. 3.6. Effect of sulfamethoxazole on microbial community analysis The sludge samples were taken during the different phases of the SBR operational period to analyse the effect of SMX on microbial community structures. The rarefaction analysis according to OTUs based on 97% similarity shows that the sequencing depth sufficiently represented the diversity of microbial community in the sludge samples (Fig. S7). Shannon index indicated that the presence of MSX reduced the microbial diversity during the longterm exposure (Fig. 6a). PCoA analysis based on unweighted UniFrac distances revealed that the microbial community presented a clear evolution trajectory response to the presence of SMX (R ¼ 0.1, p ¼ 0.001) (Fig. 6b), indicating that SMX apparently imposed a selective pressure on microbial compositions in the sulfur-based autotrophic denitrification reactor. Thiobacillus, Sulfurimonas, Sulfuritalea, Longilinea, Sulfuricurvum, Thauera, and Sulfurospirillum were the main denitrifying related genera (Fig. 7) (Zhang et al., 2009, 2015; Qiu et al., 2020). In the absence of SMX, the relative abundance of these denitrifying related genera were 27.8%, 8.3%, 22.2%, 0.5%, 0.1%, 0.04% and 0.03%, respectively. When the SBR received approximately 20 mg/L of SMX, the abundance of these genera exhibited different tendencies. The relative abundance of Thiobacillus and Sulfurimonas gradually increased to 36.0% and 23.5% on day 170, respectively, indicating that the two genera could tolerate the toxicity of SMX. Similarly, Yu et al. (2019) found that the abundance of Thiobacillus increased under ampicillin stress. Wolff et al. (2018) also observed that the relative abundance of Thiobacillus and Sulfurimonas significantly and positively correlated with antibiotics (e.g. diatrizoate, venlafaxine and tramadol). In this light, we could expect that these two denitrifiers may play an important role in SMX degradation. However, the mechanism towards SMX degradation by the genera still remains unknown, which merits further investigation. The relative abundance of Thauera maintained at 0.01%e0.04% throughout the entire operational period. The denitrifying genus Thauera is capable of both autotrophic and heterotrophic denitrification (Zhang et al., 2015). Yang et al. (2018) also reported that

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Thauera could play a vital role in SMX biodegradation in mangrove sediment under nitrate-reducing conditions. In contrast, the relative abundance of the rest of identified denitrifying related genera declined to below 0.01% except for Sulfuritalea, whose abundance reduced to 0.7%, indicating that these denitrifying genera could not endure the SMX biotoxicity. Accordingly, SMX stress reduced the diversity of nitrate-reducing related microbial communities, but the total abundance of denitrifiers maintained stable (59.0% on day 46 vs. 60.5% on day 170). The stable performance of SBR even under the SMX stress could be ascribed to the abundant denitrifiers. Additionally, Geobacter (0.7%e1.5%) and Azoarcus (0.01%e0.05%) were also detected over the entire operational period. They are associated with decomposing aromatic hydrocarbons, indicating they could degrade SMX (Weelink et al., 2010; Li et al., 2019). The similar abundance of Geobacter and Azoarcus regardless of the presence of SMX suggests that the SMX may not be the sole factor influencing the growth and activity of these potential SMX degraders. It should be pointed out that the genus Azoarcus could reduce nitrate to nitrogen gas using organics as the electron donors (Weelink et al., 2010), which could partially support the findings that the enriched denitrifying sludge could remove SMX without sulfur (Fig. 4b). Hydrogenophaga and Pseudomonas were present as the rare genera (not in the top 35 genera shown in Fig. 7) in the SBR, which have been reported to possess the capacity of degrading SMX (Yang et al., 2016, 2018; Kang et al., 2018a). 3.7. Implications This study for the first time demonstrated that the enriched autotrophic denitrifying sludge could degrade SMX in the presence of primary substrate (nitrate). The SMX removal rate was higher than or comparable with those observed in conventional wastewater treatment processes (Hai et al., 2011; Alvarino et al., 2014; Prasertkulsak et al., 2016). Multi-pulsed operational strategies could enhance SMX removal via enzyme induction, which merits further investigation. Other optimization methods, e.g. organics addition, should also be considered. Since some of heterotrophic denitrifiers (e.g. Pseudomonas and Achromobacter) have been found to have the capacity of degrading SMX (Jiang et al., 2014; Yang et al., 2018), supplementing sulfur-based autotrophic denitrification systems with organics could stimulate the growth of heterotrophic denitrifiers and then improve the removal of antibiotics via

Fig. 6. (a) a-Diversity index of the microbial communities on different dates (day 46, 78, 112 and 170) and (b) Principal coordinates analysis (PCoA) plot showing the unweighted UniFrac distances between the sludge samples on different dates. The indicated groups (ellipse encapsulations) are significantly different from each other (p < 0.05, Adonis).

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Fig. 7. The heatmap of the top 35 genera in each sludge sample was constructed, showing the relative abundance and distribution of the representative 16S rRNA sequences at genus level.

mixotrophic denitrification (Wang and Wang, 2018a). In fact, adding organics into sulfur-based autotrophic denitrification systems is a common practice as heterotrophic denitrification can reduce a part of nitrate, reducing sulfate production, which could alleviate secondary sulfate pollution (Zhang et al., 2015; Qiu et al., 2020). Additionally, there are many types of antibiotics existing in domestic wastewater effluent. It is meaningful to assess the feasibility of using sulfur-based autotrophic denitrification systems to eliminate other antibiotics, such as structurally diverse isoxazolecontaining antibiotics. 4. Conclusions This study revealed the feasibility of SMX conversion in a long-

term sulfur-based autotrophic denitrification reactor. The SMX removal rate was 3.7 ± 1.4 mg/g SS-d, which is higher than those reported in many conventional bioprocesses. The SMX was removed mainly via biodegradation process. SMX removal could be co-metabolically degraded with nitrate removal. Four transformation products (three TPs with known structures and one TP with unknown structure) were identified, among which two of the TPs were produced via altering the isoxazole ring of SMX. In addition to other bacteria, autotrophic denitrifiers played an important role in SMX biodegradation. The presence of SMX at an environmentally relevant concentration (20 mg/L) did not influence nitrate removal performance, but it significantly shaped the microbial communities, resulting in the predominant autotrophic denitrifiers shifting from Sulfuritalea to Sulfurimonas to maintain

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the system’s stability. Taken together, the sulfur-based denitrification process can be a promising technology used as a polishing step in WWTPs to remove nitrate and SMX simultaneously. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The authors acknowledge the financial support of Advanced Environmental Biotechnology Centre, Nanyang Environment and Water Research Institute (NEWRI), Nanyang Technological University. The authors would like to thank Elvy Riani Wanjaya from NEWRI for her assistance in sulfamethoxazole and transformation products analysis. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.watres.2020.116156. References Alvarino, T., Suarez, S., Lema, J., Omil, F., 2014. Understanding the removal mechanisms of PPCPs and the influence of main technological parameters in anaerobic UASB and aerobic CAS reactors. J. Hazard Mater. 278, 506e513. Anumol, T., Snyder, S.A., 2015. Rapid analysis of trace organic compounds in water by automated online solid-phase extraction coupled to liquid chromatographyetandem mass spectrometry. Talanta 132, 77e86. Apha, 2005. Standard Methods for the Examination of Water & Wastewater, twenty-first ed. American Public Health Association (APHA)/American Water Works Association (AWWA)/Water Environment Federation (WEF), Washington, DC, USA.  ska, J., Sobczak, A., 2011. Effects of the presence of Baran, W., Adamek, E., Ziemian sulfonamides in the environment and their influence on human health. J. Hazard Mater. 196, 1e15. Cetecioglu, Z., 2014. Aerobic inhibition assessment for anaerobic treatment effluent of antibiotic production wastewater. Environ. Sci. Pollut. Res. 21 (4), 2856e2864. Clara, M., Strenn, B., Gans, O., Martinez, E., Kreuzinger, N., Kroiss, H., 2005. Removal of selected pharmaceuticals, fragrances and endocrine disrupting compounds in a membrane bioreactor and conventional wastewater treatment plants. Water Res. 39 (19), 4797e4807. Grandclement, C., Seyssiecq, I., Piram, A., Wong-Wah-Chung, P., Vanot, G., Tiliacos, N., Roche, N., Doumenq, P., 2017. From the conventional biological wastewater treatment to hybrid processes, the evaluation of organic micropollutant removal: a review. Water Res. 111, 297e317. Hai, F.I., Li, X., Price, W.E., Nghiem, L.D., 2011. Removal of carbamazepine and sulfamethoxazole by MBR under anoxic and aerobic conditions. Bioresour. Technol. 102 (22), 10386e10390. Jia, Y., Khanal, S.K., Zhang, H., Chen, G.-H., Lu, H., 2017. Sulfamethoxazole degradation in anaerobic sulfate-reducing bacteria sludge system. Water Res. 119, 12e20. Jiang, B., Li, A., Cui, D., Cai, R., Ma, F., Wang, Y., 2014. Biodegradation and metabolic pathway of sulfamethoxazole by Pseudomonas psychrophila HA-4, a newly isolated cold-adapted sulfamethoxazole-degrading bacterium. Appl. Microbiol. Biotechnol. 98 (10), 4671e4681. Jiang, F., Zhang, L., Peng, G.-L., Liang, S.-Y., Qian, J., Wei, L., Chen, G.-H., 2013. A novel approach to realize SANI process in freshwater sewage treatmenteUse of wet flue gas desulfurization waste streams as sulfur source. Water Res. 47 (15), 5773e5782. Johnson, A.C., Keller, V., Dumont, E., Sumpter, J.P., 2015. Assessing the concentrations and risks of toxicity from the antibiotics ciprofloxacin, sulfamethoxazole, trimethoprim and erythromycin in European rivers. Sci. Total Environ. 511, 747e755. Kang, A.J., Brown, A.K., Wong, C.S., Huang, Z., Yuan, Q., 2018a. Variation in bacterial community structure of aerobic granular and suspended activated sludge in the presence of the antibiotic sulfamethoxazole. Bioresour. Technol. 261, 322e328. Kang, A.J., Brown, A.K., Wong, C.S., Yuan, Q., 2018b. Removal of antibiotic sulfamethoxazole by anoxic/anaerobic/oxic granular and suspended activated sludge processes. Bioresour. Technol. 251, 151e157. Katipoglu-Yazan, T., Merlin, C., Pons, M.-N., Ubay-Cokgor, E., Orhon, D., 2016. Chronic impact of sulfamethoxazole on the metabolic activity and composition of enriched nitrifying microbial culture. Water Res. 100, 546e555.

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