Short-term performance of enhanced biological phosphorus removal (EBPR) system exposed to erythromycin (ERY) and oxytetracycline (OTC)

Bioresource Technology 221 (2016) 15–25

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Short-term performance of enhanced biological phosphorus removal (EBPR) system exposed to erythromycin (ERY) and oxytetracycline (OTC) Zhetai Hu a, Peide Sun a,⇑, Zhirong Hu a,b, Jingyi Han a, Ruyi Wang a, Liang Jiao a, Pengfei Yang b a b

School of Environmental Science and Engineering, Zhejiang Gongshang University, Hangzhou 310012, China GL Environment Inc, Hamilton, Canada

h i g h l i g h t s  P-removal efficiency decreased to 34.6% under the effect of ERY (10 mg/L).  P-removal efficiency decreased to 0.0% under the effect of OTC (10 mg/L).  Aerobic stage in EBPR system was more seriously inhibited than anaerobic stage.  OTC showed more serious inhibitory effect on microorganism’s oxygen utilization.  Neither synergistic effect nor antagonistic effect was detected between ERY and OTC.

a r t i c l e

i n f o

Article history: Received 22 July 2016 Received in revised form 21 August 2016 Accepted 29 August 2016 Available online 7 September 2016 Keywords: Enhanced biological phosphorus removal ERY OTC Combined inhibition effect

a b s t r a c t The effects of Erythromycin (ERY) and oxytetracycline (OTC), including individual and combinative effect, on enhanced biological phosphorus removal (EBPR) system within a short-term (24 h) were evaluated in this study. Results showed that the P-removal efficiency decreased to 34.6% and 0.0% under the effect of ERY (10 mg/L) and OTC (10 mg/L) for 24 h. OTC concentration higher than 5 mg/L was sufficient to cause serious adverse impact on the EBPR performance. While the performance of EBPR system will be impacted by ERY above 10 mg/L. OTC, due to its special antibacterial action to the gram-negative bacteria which most PAOs belong to, has more serious negative effect on the EBPR performance than ERY does. Moreover, in the combined antibiotics test, neither synergistic nor antagonistic effect was detected between ERY and OTC. Finally, ERY (10 mg/L) and OTC (10 mg/L) could inhibit the microorganisms’ activity, while couldn’t induce serious microorganisms death within 24 h. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction Antibiotics, which are one of normally slather pharmaceuticals and personal care products (PPCPs), are increasingly discharged into the aquatic environment by wastewater treatment plants (WWTPs) effluents (Kümmerer, 2009, 2003). Moreover, most currently used antibiotics are difficult to be biodegraded due to its special physicochemical and biological characteristics (FattaKassinos et al., 2011; Halling-Sorensen et al., 1998; Xu et al., 2007; Li and Zhang, 2010), so it will be long-standing in WWTPs. Therefore, it is necessary to understand in-depth the effects of antibiotics on the performance of WWTPs. For all the processes of WWTPs, nutrient (carbon (C), nitrogen (N) and phosphorus (P)) removal is crucial to ensure good performance. The inhibitory effects of antibiotics on the process of N ⇑ Corresponding author. E-mail address: [email protected] (P. Sun). http://dx.doi.org/10.1016/j.biortech.2016.08.102 0960-8524/Ó 2016 Elsevier Ltd. All rights reserved.

and C removal have been reported abundantly (Katipoglu-Yazan et al., 2015; Collado et al., 2013; Alighardashi et al., 2009; Louvet et al., 2010; Meng et al., 2015). However, most researches ignored the effect of antibiotics on P removal process. Although Motlagh et al. (2015) found that 0.4 lg L 1 of ciprofloxacin sufficient to inhibit the process of phosphorus release (P-release) and phosphorus uptake (P-uptake) within one cycle, it’s insufficient to comprehensively understand the effect of antibiotics on EBPR system. Excessive phosphorus in aquatic environment induces eutrophication, which has become an important water quality problem worldwide (Zheng et al., 2013a,b). Enhanced biological phosphorus removal (EBPR) system has been widely accepted as the most economic and sustainable process for removing phosphorus from wastewater to control eutrophication problems (Lv et al., 2013). The performance of EBPR system operating in an anaerobic/aerobic (or anoxic) configuration depends on the enrichment of phosphate-accumulating organisms (PAOs) (Ye et al., 2010; Zheng et al., 2013a,b). Therefore, the high enrichment of PAOs

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plays an important role in achieving a high efficiency of biological phosphorus removal. Being regarded as another important microbial community in an EBPR system, glycogen accumulating organisms (GAOs) compete with PAOs for the carbon source in anaerobic stage, which should be studied as well. Erythromycin (ERY) and oxytetracycline (OTC) were selected as two representatives of antibiotics in this research. ERY, one of the most commonly used antibiotics in human medicine, belongs to macrolide antibiotics. It acts primarily through the inhibition of protein synthesis by combining with the ribosomal large subunit (50S) and restraining the translocation of peptidyl-tRNA (Alighardashi et al., 2009). ERY significantly inhibits the growth of aquatic photosynthetic organisms even at low concentrations (lg L 1) (Gonz alez-Pleiter et al., 2013; Liu et al., 2011). Meanwhile, microorganisms in activated sludge could also be inhibited by low concentration ERY in long term and at high concentration in short term (Alighardashi et al., 2009; Fan and He, 2011; Louvet et al., 2010). However, the effects of ERY on EBPR system have never been reported. On the other hand, OTC, one of the most widely used veterinary antibiotics, belongs to tetracyclines antibiotics (Álvarez et al., 2010; Li et al., 2008a). OTC could inhibit protein production by combining with the ribosomal small subunit (30S) and restraining the peptide growth. The concentration of OTC is up to 0.34 mg L 1 in surface water and even higher than 50.0 mg L 1 in the effluent of an OTC production facility in China (Li et al., 2008b). Some researches proposed that OTC (50–100 mg L 1) were able to obviously inhibit the process of N removal in short term (Yang et al., 2013; Noophan et al., 2012), while no research looked into the effect of OTC on EBPR system. Additionally the influent of WWTPs, especially those in China, contains a mixture of various antibiotics. Ozbayram et al. (2014) indicated that the combined effect of antibiotics was stronger than the effect of individual compound on acetoclastic methanogenic activity due to the synergistic effects among antibiotics. According to the past researches, the combined effect of antibiotics on C removal, volatile fatty acids (VFAs) and methane yield and microbial community shift have been reported (Aydin et al., 2015a,b; Ozbayram et al., 2014). However, the combined effects of antibiotics on EBPR system have never been reported. So, it is important and necessary to study into the combined and individual effects of antibiotics on EBPR system. The primary objective of the current research was to investigate the effects of ERY and OTC on EBPR performance in terms of variation of solution ortho-P (SOP), VFA, glycogen, poly-bhydroxyalkanoates (PHAs), total protein (TPN), extracellular polymeric substance (EPS), specific oxygen uptake rater (SOUR) and total bacterial population (TBP). In the research, firstly the individual effects of ERY and OTC were compared, and then their combined effects were also examined.

2. Materials and methods 2.1. Chemicals and sludge Chromatography-grade ERY (CAS NO. 114-07-8) and OTC (CAS NO. 6153-64-6) were purchased from Shanghai Aladdin biotechnology Ltd, China. Activated sludge was obtained from the Hangzhou Qige Municipal wastewater treatment plant, China. Anaerobic-anoxic-aerobic process was used in the domestic wastewater treatment plant. The main parameters of concentrated sludge were as following: mixed liquor suspended solids (MLSS) 7.8 ± 0.5 g/L, the ratio of mixed liquor volatile suspended solid (MLVSS) to MLSS was 57 ± 6%, and pH was 6.8–7.5.

2.2. Synthetic wastewater Synthetic wastewater containing 30 mg P/L phosphate, and 600 mg chemical oxygen demand (COD)/L (acetate and propionate in the ratio of 1/3 as the mixed carbon source) was prepared for experiment. The synthetic wastewater consisted of (per liter water): 0.256 g CH3COONa, 0.4 mL CH3CH2COOH, 0.2293 g NH4Cl, 0.0875 g KH2PO4, 0.147 g K2HPO4
3H2O, 0.09 g MgSO4, 0.0222 g CaCl2, 0.0015 g peptone, 0.0015 g yeast extract powder, 0.0072 g allylthiourea (ATU) and 0.6 mL trace elements solution. 1.8 mg/L ATU was added to inhibit nitrifiers. The trace elements solution prepared according to Smolders et al. (1994).

2.3. Batch experiments One 10 L lab-scale anaerobic-aerobic sequencing batch reactor (SBR) fed with 3.3 L synthetic wastewater was used to enrich PAOs. The batch experiments were operated at 21 ± 1 °C and for three cycles each day. Each cycle (8 h) consisted of following steps in order: 5 min for feeding synthetic wastewater, 120 min for anaerobic phase, 180 min for aerobic phase, 20 min for sedimentation, 5 min for extracting 2.5 L supernatant and 150 min for idling. The synthetic wastewater was constantly mixed with a magnetic stirrer in all steps except for the settling, decanting, and idling. The agitation speed during the operation was controlled at around 200 rpm. Dissolved oxygen (DO) level during the aerobic phase was maintained at 6–7 mg L 1. The pH was adjusted to 7.0–7.5 with 0.5 M NaOH and 0.5 M HCl during the operation. Sludge was wasted to keep the solids retention time (SRT) at approximately 7–9 days. After three months, stable phosphorous removal efficiencies were observed in the parent SBR. Batch experiments were carried out with the sludge from each reactor to examine the impact of ERY and OTC on VFA consumption, P-release, P-uptake, TPN, EPS, SOUR, and production and consumption of glycogen and PHAs. Eight parallel SBR reactors (termed as R1, E1, E2, E3, O1, O2, O3 and EO) with the working volume of 5 L were used in this study. R1 was used for blank test, where no antibiotics were added. ERY was added to keep the influent antibiotics concentration in the E1, E2 and E3 at 1 mg/L, 5 mg/L and 10 mg/L, respectively. Meanwhile, OTC was added to keep the influent antibiotics concentration in the O1, O2 and O3 at 1 mg/L, 5 mg/L and 10 mg/L, respectively. ERY and OTC were added together into EO to make the concentration of both ERY and OTC in influent at 5 mg/L respectively.

2.4. Analytical methods Mixed liquor samples were taken at the beginning of batch tests and at the end of anaerobic and aerobic stage. Then they were immediately filtered through 0.22 lm millipore filters before being analyzed. The MLSS, mixed liquor volatile suspended solid (MLVSS) and orthophosphate (PO34 -P) were analyzed according to standard methods (APHA, 1998). VFA were measured using gas chromatographs with a flame ionization detector. The column used was Elite FFAP (30 m * 0.32 mm). The set point of the oven and maximum temperature of the inlet were 100 °C and 240 °C, respectively. Helium gas was used as a carrier gas at a rate of 0.8 mL/min. GC–MS system (Agilent6890N/5975B inert GC–MSD system, USA) was used to determine the amount of poly-b-hydroxybutyrate (PHB), poly-bhydroxyvalerate (PHV) and poly-b-hydroxy-2-methylvalerate (PH2MV) (Oehmen et al., 2005). For the glycogen analysis, preweight samples of lyophilized sludge were added to 5 mL of 0.6 M HCl and digested at 100 °Cfor 6 h. After cooling, the samples

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were centrifuged and 0.5 mL of the supernatant liquid was taken out to be analyzed by the anthrone method (Zou et al., 2015). EPS were extracted using a modified heat extraction method as described previously (Li and Yang, 2007). Modified Lowry method, diphenylamine colorimetric method and anthrone method were applied to measure the proteins (PN), polysaccharides (PS) and humic acid (HA) contents in EPS, respectively (Liu and Fang, 2002). TBP were measured using the spread plate within agar culturemedium in a bacteria-free operating environment. The initial samples were diluted by 103, 104 and 105, respectively. 2.5. SOUR Oxygen uptake rater (OUR) was determined by the slope of DO versus time consumption line and the SOUR was calculated dividing OUR by MLVSS. The detail measures were according to Henriques and Love (2007). Mixed liquor samples (1 L) were taken from the test reactor by pre-aerated and placed in a biochemical oxygen demand (BOD) bottle (1 L) with 100–120 mg/L COD added in advance, to ensure that respiration was not inhibited during the experiment. DO readings were started at the beginning of the test and then recorded every 30 s with an oxygen electrode until the concentration of DO became lower than 1 mg/L. 3. Results and discussion 3.1. Effects of ERY and OTC on P removal and VFA consumption 3.1.1. Inhibitory effects of ERY on P removal and VFA consumption Inhibitory effects of ERY (concentrations in the range of 1– 10 mg/L) on the P removal and VFA consumption process were shown in Fig. 1 and Fig. S1. At the first cycle, P was regularly released at the anaerobic phase and taken up at the aerobic phase in all test reactors, indicating that good EBPR performance was achieved. While during the second and third cycles, P cannot be entirely removed by PAOs when the concentration of ERY reached 5 mg/L. It was notable that the phosphorus removal (P-removal) efficiency in E2 and E3 declined to 94.1% and 54.1% respectively at the second cycle, and further to 86.7% and 34.6% at the end of the 24-h inhibition. Decline in P-uptake rate was the main reason for the decreased P-removal efficiency. P-uptake rate declined from 12.0 mg P (gVSS h) 1 in R1 to 11.0, 10.3 and 7.2 mg P (gVSS h) 1 in E1, E2 and E3 respectively at the end of the third cycle. The process of P-release was also inhibited by ERY, declining from 14.4 mg P (gVSS h) 1 in R1 to 12.5, 12.6 and 9.8 mg P (gVSS h) 1 in E1, E2 and E3 respectively at the end of the third cycle. Moreover, the sludge P content was only 59.9, 57.8 and 45.1 mg P (gVSS) 1 in E1, E2 and E3 at the third cycle respectively, which was much lower than that in R1 (70.2 mg P (gVSS) 1) (Table 1), indicating that the ability of PAOs to store P has been suppressed by ERY. Detailed variation of SOP concentration under the effect of ERY was shown in Fig. S1. It is interesting that low ERY concentration (1 mg/L) can slightly inhibit the process of P-release and Puptake, but has no obvious influence on P-removal efficiency. VFA is the main carbon source during the anaerobic periods of EBPR system. It can be taken up by PAOs and then released in form of phosphate into the liquid phase through poly-P degradation (Puig et al., 2008). Above decision shows that the P-release process was partly inhibited by high concentration (10 mg/L) ERY, while the VFA was consumed regularly in anaerobic stage. Even after being influenced by the highest ERY concentration (10 mg/L) for three cycles, almost 97.0% of the VFA was consumed. Under the effect of 10 mg/L ERY, although PAOs was inhibited, other heterotrophic could also consume VFA (Cetecioglu et al., 2014).

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3.1.2. Inhibitory effects of OTC on the process of P removal and VFA consumption Fig. 2 and S2 present the effect of OTC (range in 1–10 mg/L) on P-release, P-uptake, P-removal efficiency and VFA consumption. At the first cycle, although the processes of P-release and P-uptake were inhibited by high concentration of OTC (10 mg/L), Premoval efficiency did not decline in any reactor. However, Premoval efficiency decreased to 38.6% and 28.9% in O2 and O3 at the second cycle, and further to 5.5% and 0.0% at the third cycle. Decline in P-removal efficiency was induced by the decrease of P-uptake rate. P-uptake rate declined from 12.0 mg P (gVSS h) 1 in R1 to 10.9, 5.5 and 4.6 mg P (gVSS h) 1 in O1, O2 and O3 at the third cycle, respectively. The process of P-release rate was also inhibited by OTC, declining from 14.4 mg P (gVSS h) 1 in R1 to 13.4, 8.1 and 7.3 mg P (gVSS h) 1 in O1, O2 and O3 at the third cycle, respectively. Moreover, the P content in sludge decreased from 70.2 mg P (gVSS) 1 in R1 to 69.7, 56.4 and 50.2 mg P (gVSS) 1 in O1, O2 and O3 at the second cycle, and then continued declining to 65.1, 45.6 and 38.1 mg P (gVSS) 1 in O1, O2 and O3 respectively at the third cycle (Table 1), suggesting that the ability of PAOs to stockpile P has been seriously inhibited by high concentration (above 5 mg/L) of OTC. Most VFA was consumed in anaerobic stage in all test reactors. Even in O3, almost 85.0% of the VFA was consumed in anaerobic stage at the third cycle. While in comparison with the control, VFA in test reactors was consumed more slowly. In the control test, VFA was completely used up within 30 min, while this time extended to almost 60 min and 120 min in O2 and O3. 3.1.3. Brief summary In general, high concentration of ERY (10 mg/L) and OTC (above 5 mg/L) seriously influenced the P-removal process within a short term, while different inhibitory performances were detected due to their disparate antibacterial characteristic. ERY concentration at 5 mg/L was insufficient to cause serious negative effect on the performance of P-removal process, while P consumption of PAOs was largely inhibited by 10 mg/L of ERY in short term. It could be concluded from the above result that the threshold of values of the ERY and OTC was 10 mg/L and 5 mg/L respectively in the EBPR system within 24 h inhibitory experiment. It could also be concluded from the inhibitory test of OTC that high concentration (above 5 mg/L) of OTC has more serious adverse effect on the P-removal process than the same concentration of ERY. OTC at 5 mg/L was sufficient to seriously destroy the P-removal process. Hence, microbial community in EBPR system was easier to be impacted by OTC than to ERY at the high concentration (above 5 mg/L). However, low concentration (1 mg/L) of OTC or ERY has no obvious impact on P-removal process in EBPR system. This situation possible due to that ERY with lower concentration (1 mg/L) could be biotransformed by the microbial community in EBPR system (Fernandez-Fontaina et al., 2015). Moreover, similar inhibitory effects were recognized between ERY and OTC. It is worth noting that the process of P-uptake was inhibited more serious than the P-release process. Compare to their inhibition on P-release and P-uptake among the three cycles, it could be concluded that the P-release process was suppressed prior to the P-uptake process. Many researchers draw similar conclusion in researching other inhibitory factors (Zheng et al., 2013a, b). Maybe the enzyme that control the P-uptake process was easier to be affected by the inhibition factors. 3.2. Inhibitory effects of ERY and OTC on glycogen and PHAs 3.2.1. Inhibitory effects of ERY on glycogen and PHAs Glycogen is a crucial energy substance in EBPR system that can be consumed in anaerobic stage and produced in aerobic stage.

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Fig. 1. Effects of ERY on the variations of P-removal process and VFA concentration during the first cycle (A (1), B (1)), second cycle (A (2), B (2)) and the third cycle (A (3), B (3)). Error bars represent standard deviations of triplicate tests.

Table 1 The concentration of TP in the each EBPR system (mg P (gVSS)

First cycle Second cycle Third cycle

1

).

R1

E1

E2

E3

O1

O2

O3

EO

70.2 71.3 70.7

71.1 65.2 59.9

70.5 62.8 57.8

65.9 55.2 45.1

70.9 69.7 65.1

65.1 56.4 45.6

63.8 50.2 38.1

65.6 55.3 41.7

PHAs, produced in anaerobic stage, are the foremost energy sources for PAOs to uptake orthophosphate and for microorganisms to grow in aerobic stage. The glycogen consumption was not influenced even under the effect of the highest ERY concentration (10 mg/L) (Fig. S3). However, less PHAs were consumed in E3 at the second and third cycles (Fig. S3), compared to the control. In comparison with the control, PHAs consumption in aerobic stage decreased by 8.7% and 23.4% in E2 and E3 at the third cycle. While

low concentration of ERY (1 mg/L) hardly changed PHAs consumption during the short-term experiment. Furthermore, PHAs production process also decreased by 12.1% in E3.

3.2.2. Inhibitory effects of OTC on glycogen and PHAs Fig. S4 show the variation of glycogen and PHAs under the effect of OTC. At the third cycle, glycogen was consumed regularly in

Z. Hu et al. / Bioresource Technology 221 (2016) 15–25

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Fig. 2. Effects of OTC on the variations of P-removal process and VFA concentration during the first cycle (A (1), B (1)), second cycle (A (2), B (2)) and the third cycle (A (3), B (3)). Error bars represent standard deviations of triplicate tests.

anaerobic stage in all test reactors, while glycogen in O3 could not completely recover (only by almost 90.0%) to the initial level. PHAs production and consumption processes were also influenced by high concentration of OTC (above 5 mg/L). Compared to the control, the PHAs consumption rate declined by 27.5% and 30.0% in O2 and O3 at the third cycle. Compared with glycogen production, PHAs consumption was inhibited more seriously in aerobic stage. Moreover, PHAs production in anaerobic stage decreased by almost 15.0% in O2 and O3 at the third cycle, compared to the control. 3.2.3. Brief summary In some aspects, such as glycogen and PHAs, the inhibitory performances of ERY and OTC were different. OTC has more serious negative impact on the production and consumption process of

glycogen and PHAs than ERY. ERY concentration at 1 and 5 mg/L has no obvious negative effect to the amount of glycogen and PHAs. However, OTC at 5 mg/L was sufficient to destroy the production and consumption process of glycogen and PHAs. Similar inhibitory performance was also recognized between these two antibiotics. The aerobic stage in EBPR system was easier to be impacted by antibiotics than the anaerobic stage. Table 2 shows that the PHArelease/VFAuptake ratio was not significantly decreased with the increase of antibiotics concentration, while the Glycogendegradation/VFAuptake ratio was increased due to the effect of antibiotics, compared to the control. Its explanation maybe that more VFA was consumed in the anaerobic stage by GAOs, which utilized more glycogen to provide energy for VFA uptake, and more PHAs was synthesized via succinate-propionate pathway (Filipe et al., 2001).

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Table 2 Effects of ERY and OTC on the anaerobic and aerobic metabolism. R1

E1

E2

E3

O1

O2

O3

EO

Anaerobic

Prelease/VFAuptake PHArelease/VFAuptake Glycogendegradation/VFAuptake

0.295 0.541 0.462

0.261 0.537 0.468

0.265 0.541 0.459

0.233 0.551 0.486

0.281 0.541 0.501

0.228 0.527 0.520

0.214 0.543 0.547

0.205 0.523 0.498

Aerobic

Puptake/PHAdegradation Glycogensynthesis/PHAdegradation

22.500 0.773

22.132 0.808

21.658 0.833

17.183 0.865

23.311 0.854

17.013 0.928

14.890 0.940

16.146 0.919

Fig. 3. Effects of ERY on the variations of EPS concentration, TPN (B) and SOUR (C) during the first cycle (A (1)), second cycle (A (2)) and the third (A (3)) cycle. Error bars represent standard deviations of triplicate tests.

3.3. Inhibitory effects of ERY and OTC on EPS and TPN 3.3.1. Inhibitory effects of ERY on EPS and TPN EPS is a complex high-molecular-weight mixture of polymers that produced by the microorganisms in EBPR at aerobic stage (Xu et al., 2013; Sheng et al., 2008). Both ERY and OTC affect pro-

tein synthesis in bacteria, so effects of these antibiotics on TPN was also studied in this research. TPN in EBPR system, as well as PN in EPS, was stable in all reactors at the first cycle (Fig. 3). However, at the second and third cycles, ERY showed adverse effect on TPN production. Compared to the control, TPN production declined by 6.2% in the second

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Fig. 4. Effects of OTC on the variations of EPS concentration, TPN (B) and SOUR (C) during the first cycle (A (1)), second cycle (A (2)) and the third (A (3)) cycle. Error bars represent standard deviations of triplicate tests.

and 10.5% in the third cycle. Moreover, PN in EPS decreased seriously according to the decline of TPN. At the third cycle, PN declined by 10.9% and 26.2% in E2 and E3, compared to the control. PS in EPS changed seriously throughout the whole experiment. At the first cycle, PS was 50.6 and 65.1 mg/L in E2 and E3, increasing by 11.1% and 42.9% when compared to the control. However, at the second and third cycles, PS was 40.1 and 37.1 mg/L in E2 and E3, declining by 20.8% and 43.0% when compared to the data of first cycle. Additionally, the content of HA in EPS was slight and stable. 3.3.2. Inhibitory effects of OTC on TPN and EPS Fig. 4 show that the TPN and PN in EPS were stable in all reactors at the first cycle. However, in comparison with those in control, PS in EPS increased by 55.8% and 66.7% in O2 and O3 respectively at the first cycle. However, TPN, PN and PS concentra-

tions dropped dramatically at the second and third cycles. At the third cycle, TPN decreased by 26.3% in O2 and 31.4% in O3 compared to the control. At the same time, PN declined by 33.2% and 36.9% and PS decreased by 22.9% and 25.3% when compared to the control. While HA content in EPS was slight and stable. 3.3.3. Brief summary TPN production was inhibited by high concentration of OTC (above 5 mg/L) and ERY (10 mg/L). It was widely known that the antibacterial action of ERY and OTC primarily through the inhibition of protein synthesis. However, OTC showed more serious negative impact. The microbial community in EBPR system was more fragile when it was exposed to OTC. Because more Gram-negative bacteria such as proteobacteria, which most microbial community in EBPR system belong to, was easy to be inhibited by OTC than ERY (Pala-Ozkok et al., 2014).

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Fig. 5. Effects of combined antibiotics on the variations of P-removal process and VFA concentration during the first cycle (A (1), B (1)), second cycle (A (2), B (2)) and the third cycle (A (3), B (3)). Error bars represent standard deviations of triplicate tests.

For the EPS, more PS was released to protect the microbial community from the impact of ERY and OTC at the first cycle. EPS acting as a barrier to permitted ERY and OTC into the cell membrane. This may be one reason for the stable performance in all the test reactors at the first cycle. However, at the second and third cycles, PN begin to decline possibly due to the decrease of TPN. It will be difficult for the microbial community in EBPR system to produce PN due to the existence of ERY and OTC. Hence, less PN will be transferred from intracellular to extracellular. More antibiotics into the cell membrane to inhibit the microorganisms’ activity due to the decline of EPS, and it possible induce the decrease of Premoval efficiency and the consumption of PHAs. Another interesting result was that, in the low concentration (1 mg/L) antibiotics test, PN largely increased at the second cycle, which was later than that in the high concentration (above 5 mg/L) test. One possible explanation was that, large antibiotics were adsorbed or biotransformation by microbial community at the first cycle due to its low

concentration, so no inhibitory performance was shown at the first cycle in O1. However, at the second and third cycles, microbial community cannot further adsorb or biotransformation antibiotics, therefore adverse effect emerged and more EPS was released to protect the microbial community. Furthermore, compare to the inhibitory effect of tetracycline, cefalexin and sulfamethoxazole to the production of EPS in anaerobic sludge system, ERY and OTC shown more serious adverse effect on the production of EPS in EBPR system (Hou et al., 2016; Lu et al., 2014; Zhang et al., 2015a,b). Two possible explanations should be given to this phenomenon. Firstly, the antibacterial characteristic among these antibiotics were different. ERY and OTC inhibit the bacterial growth through inhibition of protein synthesis, while cefalexin and sulfamethoxazole through other methods to inhibit the bacterial growth. Although the antibacterial action of tetracycline was also shown by inhibition of protein synthesis, the high adsorption rate by activated sludge possible reduce the

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Fig. 6. Effects of combined antibiotics on the variations of EPS concentration, TPN (B) and SOUR (C) during the first cycle (A (1)), second cycle (A (2)) and the third cycle (A (3)). Error bars represent standard deviations of triplicate tests.

Table 3 Compare the inhibitory performance between E2, O2 and EO.

E2 O2 EO

Prelease

Puptake

Premoval

VFA consumption

Glycogen consumption

Glycogen synthesis

PHAs synthesis

PHAs consumption

TPN

PN

PS

SOUR

12.6 8.1 7.3

10.3 5.5 7.7

86.7 5.5 3.7

100 84.7 87.4

1.7 1.7 1.7

2.0 1.8 1.7

2.6 2.2 2.2

2.4 1.9 1.8

97.8 83.7 19.1

9.4 33.2 38.4

18.7 25.3 22.9

92.1 73.9 68.9

Table 4 Variation of TBP in each reactor after exposure to antibiotics for 24 h compared to the control test (%). Error data represent standard deviations of triplicate tests.

TBP compare to the control test (%)

R1

E1

E2

E3

O1

O2

O3

EO

100

99.6 ± 0.2

98.8 ± 0.3

95.5 ± 0.3

99.1 ± 0.1

95.1 ± 0.4

94.4 ± 0.3

94.7 ± 0.2

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toxicity of tetracycline. Secondly, EBPR system which is high enrichment of one type of functional bacteria maybe more easy to be inhibited by inhibitory factors.

Furthermore, Table 3 compared the inhibitory performances among E2, O2 and EO, indicating that EO was inhibited more drastic than E2 and that was similar as O2. So, nor antagonistic effect was exists between ERY and OTC.

3.4. Inhibitory effects of ERY and OTC on SOUR OUR, as one of the classical indicator to measure the toxicity of inhibition factors to activated sludge, is based on respiration inhibition. SOUR was more scientific than OUR to reveal the inhibition effect. Fig. 3C summarizes the decreasing ratio of SOUR in the EBPR system when compare to the control after exposure to the ERY (range in 1–10 mg/L) for 24 h. It is shown that, compared to the control, the SOUR decreased by 4.9%, 7.9% and 23.5% in E1, E2 and E3 after exposure to ERY for 24 h, indicating that ERY could inhibit the oxygen utilization process of the microbial community in EBPR system. Fig. 4C shows that the SOUR in O1, O2 and O3 decreased by 9.5%, 36.1% and 39.4% compared to the control after exposure to OTC for 24 h. In summary, OTC and ERY could inhibit the process of oxygen utilization of the microbial community in the EBPR system. Compared to the other research, the toxicity of ERY at high concentration (10 mg/L) to oxygen utilization in EBPR is stronger than the toxicity of ZnO and TiO2 nanoparticles to that in conventional activated sludge system and the effect is milder at the low concentration (1 mg/L). This phenomenon may due to the different inhibition pathway among various of inhibit factors. OTC has more serious adverse effect on SOUR than ERY. As we know, most PAOs like Candidatus Accumulibacter phosphatis belong to proteobacteria, which is part of gram-negative microbial community. Most gram-negative microbial communities were more sensitive to OTC than ERY (Pala-Ozkok et al., 2014). This is the reason why OTC has more grievous adverse effect than ERY on the oxygen utilization of the microbial community in EBPR system. 3.5. Combined inhibition of ERY and OTC on the EBPR performance Figs. 5 and 6, S5 and S6 show the comparison between individual antibiotic and combined antibiotics at the same concentration on EBPR performance. 3.5.1. Combine inhibition effects on P-removal process, VFA, glycogen and PHAs Figs. 5 and 6 compare the inhibition effects between individual antibiotic and combined antibiotics on P-removal process (Prelease, P-uptake, P-removal efficiency), VFA, glycogen and PHAs at the first, second and third cycles. No synergistic effects were detected on the process of P-removal (P-release, P-uptake and Premoval efficiency), VFA and PHAs in EO. The P-removal efficiency decreased to 3.7% in EO at the third cycle, which was higher than that in O3 (0.0%) but lower than that in E3 (34.6%). Meanwhile, compared to the control, PHAs consumption rate in EO decreased by 28.8%, which was higher than that in E3 (11.5%) but lower than that in O3 (30.0%). However, similar adverse effect was detected on VFA consumption and glycogen variation in E3, O3 and EO. 3.5.2. Combine inhibition effects on TPN, EPS and SOUR The adverse effects on TPN, EPS and SOUR between individual antibiotic and combined antibiotics were also compared in this research (Figs. S5, S6). Compared to the control, TPN and SOUR declined by 19.1% and 33.1% after exposure to combined antibiotics for 24 h, which was as similar as that in O3 (declined by 18.4% and 31.4%). Meanwhile, PN and PS in EO declined by 39.4% and 22.9% at the third cycle. While HA content was stable through the whole experiment.

3.5.3. Brief summary According to the aforementioned information, neither synergistic effect nor antagonistic effect was detected between ERY and OTC. Although the adverse effect on TPN and SOUR in EO was slightly stronger than that in O3, it is insufficient to prove that synergistic effect exists between ERY and OTC. 3.6. Inhibition of antibiotics on the variation of TBP Table 4 presents the variation of TBP in each reactor after exposure to antibiotics for 24 h compare to the control test. It could be concluded from Table 4 that no serious bacterial death was detected even in the highest concentration antibiotics reactors. Hence, the inhibitory performance on each index mentioned above was possibly induced by the declined of microorganisms’ activity, not due to the microbial death. 10 mg/L of ERY and OTC could inhibit the activity of microorganisms, while did not induce microorganisms death within 24 h inhibitory test. 4. Conclution To summarize, the EBPR performance including P-release, Puptake, P-removal efficiency, PHAs consumption, TPN production, EPS and SOUR were significantly inhibited by ERY and OTC. Meanwhile, OTC has more serious adverse effect on the EBPR performance than ERY due to its special antibacterial action to the gram-negative bacterium which most PAOs belong to. Additionally, neither synergistic nor antagonistic effect exists between ERY and OTC. Finally, the inhibitory performance of each index such as P-removal efficiency, PHAs, TPN, EPS and SOUR possible induced by the decreased of microorganisms’ activity not due to the microbial death. Acknowledgement This research was financially supported by the National Natural Science Foundation of China (No. 21276236); Major Scientific and Technological Project of Zhejiang Province (No. 2014C03002). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2016.08. 102. References Alighardashi, A., Pandolfi, D., Potier, O., Pons, M.N., 2009. Acute sensitivity of activated sludge bacteria to erythromycin. J. Hazard. Mater. 172 (2–3), 685– 692. Álvarez, J.A., Otero, L., Lema, J.M., Omil, F., 2010. The effect and fate of antibiotics during the anaerobic digestion of pig manure. Bioresour. Technol. 101 (22), 8581–8586. APHA, 1998. Standard Methods for the Examination of Water and Wastewater. American Public Health Association, American Water Works Association, Water Pollution Control Federation, Washington, DC. Aydin, S., Cetecioglu, Z., Arikan, O., Ince, B., Ozbayram, E.G., Ince, O., 2015. Inhibitory effects of antibiotic combinations on syntrophic bacteria, homoacetogens and methanogens. Chemosphere 120 (4), 515–520. Aydin, S., Ince, B., Ince, O., 2015. Application of real-time PCR to determination of combined effect of antibiotics on bacteria, methanogenic archaea, archaea in anaerobic sequencing batch reactors. Water Res. 76 (3), 88–98.

Z. Hu et al. / Bioresource Technology 221 (2016) 15–25 Cetecioglu, Z., Ince, B., Ince, O., Orhan, D., 2014. Acute effect of erythromycin on metabolic transformations of volatile fatty acid mixture under anaerobic conditions. Chemosphere 124 (1), 129–135. Collado, N., Buttiglieri, G., Marti, E., Ferrando-Climent, L., Rodriguez-Mozaz, S., Barceló, D., Comas, J., Rodriguez-Roda, I., 2013. Effects on activated sludge bacterial community exposed to sulfamethoxazole. Chemosphere 93 (1), 99– 106. Fan, C., He, Z.J., 2011. Proliferation of antibiotic resistance genes in microbial consortia of sequencing batch reactors (SBRs) upon exposure to trace erythromycin or erythromycin -H2O. Water Res. 45 (10), 3098–3106. Fatta-Kassinos, D., Meric, S., Nikolaou, A., 2011. Pharmaceutical residues in environmental waters and wastewater: current state of knowledge and future research. Anal. Bioanal. Chem. 399 (1), 251–275. Fernandez-Fontaina, E., Gomes, I.B., Aga, D.S., Omil, F., Lema, J.M., Carballa, M., 2015. Biotransformation of pharmaceuticals under nitrification, nitratation and heterotrophic conditions. Sci. Total Environ. 541, 1439–1447. Filipe, C.D.M., Daigger, G.T., Grady, C.P.L., 2001. A metabolic model for acetate uptake under anaerobic conditions by glycogen accumulating organisms: stoichiometry, kinetics, and the effect of pH. Biotechnol. Bioeng. 76 (1), 17–31. Gonz alez-Pleiter, M., Gonzalo, S., Rodea-Palomares, I., Leganes, F., Rosal, R., Boltes, K., Marco, E., Fernández-Piñas, F, 2013. Toxicity of five antibiotics and their mixtures towards photosynthetic aquatic organisms: implications for environmental risk assessment. Water Res. 47 (6), 2050–2064. Halling-Sorensen, B., Nors Nielsen, S., Lanzky, P.F., Ingerslev, F., Holten Lutzhoft, H. C., Jorgensen, S.E., 1998. Occurrence, fate and effects of pharmaceutical substances in the environment – a review. Chemosphere 36 (2), 357–393. Henriques, I.D.S., Love, N.G., 2007. The role of extracellular polymeric substances in the toxicity response of activated sludge bacteria to chemical toxins. Water Res. 41 (18), 4177–4185. Hou, G.Y., Hao, X.Y., Zhang, R., Wang, J., Liu, R.T., Liu, C.G., 2016. Tetracycline removal and effect on the formation and degradation of extracellular polymeric substances and volatile fatty acids in the process of hydrogen fermentation. Bioresour. Technol. 212, 20–25. Katipoglu-Yazan, T., Merlin, C., Pons, M.N., Ubay-Cokgor, E., Orhon, D., 2015. Chronic impact of tetracycline on nitrification kinetics and the activity of enriched nitrifying microbial culture. Water Res. 72 (3), 227–238. Kümmerer, K., 2003. Significance of antibiotics in the environment. J. Antimicrob. Chemother. 52 (2), 317. Kümmerer, K., 2009. The presence of pharmaceuticals in the environment due to human use – present knowledge and future challenges. J. Environ. Manage. 90 (8), 2354–2366. Li, X.Y., Yang, S.F., 2007. Influence of loosely bound extracellular polymeric substances (EPS) on the flocculation, sedimentation and dewaterability of activated sludge. Water Res. 41 (5), 1022–1030. Li, B., Zhang, T., 2010. Biodegradation and adsorption of antibiotics in the activated sludge process. Environ. Sci. Technol. 44 (9), 3468–3473. Li, Z.J., Yao, Z., Zhang, J., Liang, Y., 2008. A review on fate and ecological toxicity of veterinary antibiotics in soil environments. Asian J. Ecotoxicol. 3 (1), 15–20. Li, K., Yediler, A., Yang, M., Schulte-Hostede, S., Wong, M.H., 2008. Ozonation of oxytetracycline and toxicological assessment of its oxidation by-products. Chemosphere 72 (3), 473–478. Liu, H., Fang, H.H.P., 2002. Extraction of extracellular polymeric substances (EPS) of sludges. J. Biotechnol. 95 (3), 249–256. Liu, B.Y., Nie, X.P., Liu, W.Q., Snoeijs, P., Guan, C., Tsui, M.T.K., 2011. Toxic effects of erythromycin, ciprofloxacin and sulfamethoxazole on photosynthetic apparatus in Selenastrum capricornutum. Ecotoxicol. Environ. Saf. 74 (4), 1027–1035. Louvet, J.N., Giammarino, C., Potier, O., Pons, M.N., 2010. Adverse effects of erythromycin on the structure and chemistry of activated sludge. Environ. Pollut. 158 (3), 688–693. Lu, X.Q., Zhen, G.Y., Liu, Y., Hojo, T., Estrada, A.L., Li, Y.Y., 2014. Long-term effect of the antibiotic cefalexin on methane production during waste activated sludge anaerobic digestion. Bioresour. Technol. 169 (5), 644–651.

25

Lv, X.M., Shao, M.F., Li, C.L., Li, J., Gao, X.L., Sun, F.Y., 2013. A comparative study of the bacterial community in denitrifying and traditional enhanced biological phosphorus removal processes. Microbes Environ. 29 (4), 261–268. Meng, L.W., Li, X.K., Wang, K., Ma, K.L., Zhang, J., 2015. Influence of the amoxicillin concentration on organics removal and microbial community structure in an anaerobic EGSB reactor treating with antibiotic wastewater. Chem. Eng. J. 274, 94–101. Motlagh, A.M., Bhattacharjee, A.S., Goel, R., 2015. Microbiological study of bacteriophage induction in the presence of chemical stress factors in enhanced biological phosphorus removal (EBPR). Water Res. 81, 1–14. Noophan, P., Narinhongtong, P., Wantawin, C., Munakata-Marr, J., 2012. Effects of oxytetracycline on Anammox activity. J. Environ. Sci. Health A 47 (6), 873–877. Oehmen, A., Keller-Lehmann, B., Zeng, R.J., Yuan, Z., Keller, J., 2005. Optimisation of poly-b-hydroxyalkanoate analysis using gas chromatography for enhanced biological phosphorus removal systems. J. Chromatogr. A 1070 (1–2), 131–136. Ozbayram, E.G., Arikan, O., Ince, B., Cetecioglu, Z., Aydin, S., Ince, O., 2014. Acute effects of various antibiotic combinations on acetoclastic methanogenic activity. Environ. Sci. Pollut. Res. 22 (8), 1–6. Pala-Ozkok, I., Rehman, A., Ubay-Cokgor, E., 2014. Pyrosequencing reveals the inhibitory impact of chronic exposure to erythromycin on activated sludge bacterial community structure. Biochem. Eng. J. 90 (90), 195–205. Puig, S., Coma, M., Monclús, H., van Loosdrecht, M.C.M., Colprim, J., Balaguer, M.D., 2008. Selection between alcohols and volatile fatty acids as external carbon sources for EBPR. Water Res. 42 (3), 557–566. Sheng, G.P., Zhang, M.L., Yu, H.Q., 2008. Characterization of adsorption properties of extracellular polymeric substances (EPS) extracted from sludge. Colloids Surf. B 62 (1), 83–90. Smolders, G., Van der Meij, J., Van Loosdrecht, M., Heijnen, J., 1994. Model of the anaerobic metabolism of the biological phosphorus removal process: stoichiometry and pH influence. Biotechnol. Bioeng. 44 (7), 461–470. Xu, W.H., Zhang, G., Li, X.D., Zou, S.C., Li, P., Hu, Z.H., Li, J., 2007. Occurrence and elimination of antibiotics at four sewage treatment plants in the Pearl River Delta (PRD), South China. Water Res. 41 (19), 4526–4534. Xu, J., Sheng, G.P., Ma, Y., Wang, L.F., Yu, H.Q., 2013. Roles of extracellular polymeric substances (EPS) in the migration and removal of sulfamethazine in activated sludge system. Water Res. 47 (14), 5298–5306. Yang, G.F., Zhang, Q.Q., Jin, R.C., 2013. Changes in the nitrogen removal performance and the properties of granular sludge in an Anammox system under oxytetracycline (OTC) stress. Bioresour. Technol. 129 (129C), 65–71. Ye, L., Pijuan, M., Yuan, Z.G., 2010. The effect of free nitrous acid on the anabolic and catabolic processes of glycogen accumulating organisms. Water Res. 44 (9), 2901–2909. Zhang, J., Dong, Q., Liu, Y., Zhou, X., Shi, H.C., 2015a. Response to shock load of engineered nanoparticles in an activated sludge treatment system: insight into microbial community succession. Chemosphere 144, 1837–1844. Zhang, Z.Z., Zhang, Q.Q., Guo, Q., Chen, Q.Q., Jiang, X.Y., Jin, R.C., 2015b. Anaerobic ammonium-oxidizing bacteria gain antibiotic resistance during long-term acclimatization. Bioresour. Technol. 192, 756–764. Zheng, X.L., Sun, P.D., Lou, J.Q., 2013a. The long-term effect of nitrite on the granulebased enhanced biological phosphorus removal system and the reversibility. Bioresour. Technol. 132, 333–341. Zheng, X.L., Sun, P.D., Lou, J.Q., Cai, J., Song, Y.Q., Yu, S.J., Lu, X.Y., 2013b. Inhibition of free ammonia to the granule-based enhanced biological phosphorus removal system and the recoverability. Bioresour. Technol. 148, 343–351. Zou, J.T., Li, Y.M., Zhang, L.I., Wang, R.Y., Sun, J., 2015. Understanding the impact of influent nitrogen concentration on granule size and microbial community in a granule-based enhanced biological phosphorus removal system. Bioresour. Technol. 177, 209–216. 177C.