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Effects of CeO2 nanoparticles on biological nitrogen removal in a SBBR and mechanism of toxicity


Bioresource Technology 191 (2015) 73–78

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Effects of CeO2 nanoparticles on biological nitrogen removal in a sequencing batch bio?lm reactor and mechanism of toxicity
Jun Hou, Guoxiang You, Yi Xu, Chao Wang, Peifang Wang ?, Lingzhan Miao, Yanhui Ao, Yi Li, Bowen Lv
Key Laboratory of Integrated Regulation and Resources Development on Shallow Lakes, Ministry of Education, Hohai University, Nanjing 210098, People’s Republic of China College of Environment, Hohai University, Nanjing 210098, People’s Republic of China

h i g h l i g h t s
 A sequencing batch bio?lm reactor

g r a p h i c a l a b s t r a c t

was exposed to CeO2 nanoparticles (NPs).  Low concentrations (1 mg/L) exerted no impact on the bio?lm performance.  Total nitrogen removal decreased at high dosage levels (10 and 50 mg/L).  The bio?lm adsorbed the NPs, increasing production of reactive oxygen species.  Key enzyme activities were inhibited and bacterial viability declined at high doses.

a r t i c l e

i n f o

a b s t r a c t
The effects of CeO2 nanoparticles (CeO2 NPs) exposure on biological nitrogen removal in a sequencing batch bio?lm reactor (SBBR) were investigated. At low concentration (1 mg/L), no signi?cant effect was observed on total nitrogen (TN) removal. However, at high concentrations (10 and 50 mg/L), the TN removal ef?ciency reduced from 74.09% to 64.26% and 55.17%, respectively. Scanning electron microscope imaging showed large amounts of CeO2 NPs adsorbed on the bio?lm, which increased the production of reactive oxygen species. The exposure at only 50 mg/L CeO2 NPs measurably affected the lactate dehydrogenase release. Confocal laser scanning microscopy showed that high concentrations of CeO2 NPs reduced bacterial viability. Moreover, after a short-term exposure, extracellular polymeric substances (EPS) were observed to increase, forming a compact matrix to protect the bacteria. The activities of nitrate reductase and ammonia monooxygenase were inhibited, but there was no signi?cant impact on the activity of nitrite oxidoreductase. ? 2015 Elsevier Ltd. All rights reserved.

Article history: Received 3 March 2015 Received in revised form 29 April 2015 Accepted 30 April 2015 Available online 7 May 2015 Keywords: CeO2 NPs Bio?lms Sequencing batch bio?lm reactor (SBBR) Nitrogen removal Toxicity mechanism

Abbreviations: AMO, ammonia monooxygenase; APHA, American Public Health Association; CLSM, confocal laser scanning microscopy; COD, chemical oxygen demand; DO, dissolved oxygen; EPS, extracellular polymeric substances; LB, loosely bound; LDH, lactate dehydrogenase; NOR, nitrite oxidoreductase; NPs, nanoparticles; NR, nitrate/nitrite reductase; ROS, reactive oxygen species; SBBR, sequencing batch bio?lm reactor; SBR, sequencing batch reactor; SEM, scanning electron microscopy; SND, simultaneous nitri?cation and denitri?cation; TB, tightly bound; TN, total nitrogen; TP, total phosphorous; WWTP, wastewater treatment plant. ? Corresponding author at: College of Environment, Hohai University, 1 Xikang Road, Nanjing 210098, People’s Republic of China. Tel./fax: +86 25 83787332. E-mail addresses: pfwang2005@hhu.edu.cn (P. Wang), mlz1988@126.com (L. Miao). http://dx.doi.org/10.1016/j.biortech.2015.04.123 0960-8524/? 2015 Elsevier Ltd. All rights reserved.

1. Introduction Nanomaterial research and development has experienced explosive growth in recent decades; today, nanoparticles (NPs) are used in medicine, manufacturing, biological studies, pigments, and photochemical catalysis (Bose and Wu, 2005), among others. One consequence of the wide use of nanomaterials is their unintentional or accidental release into the environment, which can lead to adverse effects (Hendren et al., 2011). Scientists have

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demonstrated that signi?cant amounts of NPs can be adsorptively removed from wastewater during treatment (Gottschalk et al., 2009; Kiser et al., 2009). For biological wastewater treatment processes with activated sludge, the adsorbed NPs could decrease microbial populations, disturb microbial diversity, and lead to a reduction in ef?ciency (Wang et al., 2012; Zheng et al., 2011a). In addition to activated sludge, bio?lms have become a principal treatment modality in wastewater remediation. Composed of a variety of microorganisms, bio?lms play a very important role in the puri?cation of water. Compared to the ?occulent structure of activated sludge, bio?lms have complex 3-D structures comprising pores, channels, and irregular protuberances (Stewart and Franklin, 2008). Furthermore, masses of extracellular polymeric substances (EPS), which are produced by microorganism metabolism or cell lysis, substantially enhance the ability of bio?lms to more effectively adsorb and aggregate NPs (Choi et al., 2010). Further studies have shown that EPS tend to bind with toxic materials and can thus improve the resistance of the microbial community to NPs (Hou et al., 2014; Sheng and Liu, 2011). Although several studies have focused on the effects of NPs on activated sludge, the impact of NPs on the process of wastewater treatment by a bio?lm remains unknown. Based on the sequencing batch reactor (SBR), SBBRs operate with different ?lters on which bio?lms can easily adhere and grow. The system provides aerobic, low-oxygen, and anaerobic environments in which to conduct nitri?cation/denitri?cation processes and assimilate/release phosphorus, to realize more effective nitrogen and phosphorus removal. Additionally, during the aerobic period, an SBBR system can achieve simultaneous nitri?cation and denitri?cation (SND), thus reducing operating costs (Yang et al., 2014). Accordingly, a bio?lm-based nitrogen-removal reactor is a system that fully exploits the metabolic activities of ammonia-oxidizing, nitrifying, and denitrifying bacteria in the bio?lm. For each of these bacteria, enzymes play important roles in effecting metabolic activities (Zheng et al., 2011b). However, limited knowledge is available concerning the in?uence of NPs on bio?lm bacteria or the key enzymes responsible for nitrogen removal. As a metal oxide of the lanthanide series, CeO2 NPs have become one of the most widely used nanomaterials, in applications as widely ranging as polishing solutions, oxygen sensor, and solid oxide fuel cells (Zhu et al., 2003). Limbach et al. (2008) found that a large number of CeO2 NPs were released into a model wastewater treatment plant (WWTP). Recent studies have demonstrated the visible cytotoxicity, oxidative stress, and genotoxicity effects of CeO2 NPs (Auffan et al., 2009; Yokel et al., 2009). Although some conclusions have been drawn from existing studies about the toxicity of CeO2 NPs toward single bacterial strains, scant attention has been paid to the effects of these nanomaterials on the process of biological nitrogen and phosphorus removal or the related toxicity-producing mechanisms in SBBRs. The aim of this study was to investigate how CeO2 NPs affect the nitrogen removal ef?ciency in an SBBR system, as well as the mechanisms by which harm is caused after short-term exposure, by answering the following questions: (1) How do different concentrations of CeO2 NPs affect the ? ? transformations of NH+ 4–N, NO2 –N, and NO3 –N, as well as the nitrogen removal ef?ciency, during an operating cycle? (2) What are the different responses of the bio?lm in terms of membrane integrity and bacterial viability in the presence and absence of CeO2 NPs, and how do the EPS respond when faced with toxicity? (3) What is the potential impact of CeO2 NPs on the key enzymes related to nitrogen removal in the bio?lm?

2. Methods 2.1. Synthetic wastewater The in?uent pH of the synthetic wastewater was kept at 7.4 by adding NaOH or HCl. The predetermined COD and TN and TP contents were 380, 26, and 6.8 mg/L, respectively. The carbon content was derived from glucose. The remaining components and concentrations were FeSO4?7H2O (35 lM), MnSO4?H2O (4 lM), ZnSO4?7H2O (14 lM), CaCl2 (200 lM), MgCl2?6H2O (85 lM), CuSO4?5H2O (1 lM), (NH4)6Mo7O24 (0.141 lM), NH4Cl (300 lM), NaNO3 (300 lM), and Na2HPO4 (570 lM) (Hou et al., 2014). 2.2. SBBR operation and bio?lm culturing Bio?lms were cultured in an anaerobic–low dissolved oxygen (DO) (0.1–0.45 mg/L) SBBR with a working volume of 3 L, ?lled with combined packing that made of polyester ?ber (Fig. A.1, Supplementary Material). In the ?rst stage, secondary activated sludge from the original water taken from a secondary sedimentation tank was inoculated with aeration, maintained at 21 ± 1 °C. After inoculation for 24 h, the aeration was stopped and sedimentation was allowed for 2 h. Then, the supernatant was discarded and the residue was poured into fresh synthetic wastewater to afford a ?nal activated sludge concentration of 3 mg/L. The DO level was maintained between 0.1 and 0.45 mg/L with detection by a portable hand-held DO meter (Hach sensION6, USA). During the bio?lm culturing period, the SBBR was operated for three cycles daily, including 6.5 h aeration, 1.5 h low oxygen and sedimentation, and instant intake and discharge. After culturing for a few days, the total nitrogen (TN), total phosphorus (TP), NH+ 4–N, ? NO? 2 –N, and NO3 –N contents, as well as the chemical oxygen demand (COD), were continuously monitored until the removal ef?ciencies remained unchanged. When the color of the bio?lm on the ?lter deepened and the bacteria densely covered the ?lter surface, it was judged to be mature; then, the exposure experiments were carried out. The characteristics of the bio?lm are listed in Supplementary Material Table A.1. 2.3. Nanoparticles Commercially produced CeO2 NPs were purchased from Sigma– Aldrich (St. Louis, MO). A scanning electron microscopy (SEM) image of the CeO2 NPs was obtained using a Hitachi S-4800 SEM to visually inspect their shape (Fig. A.2, Supplementary Material). To prepare a stock suspension, the CeO2 NPs (50 mg) were mixed with Milli-Q water (0.5 L, pH 6.9) and the suspension was ultrasonicated for 1 h (20 °C, 250 W, 40 kHz) (Keller et al., 2010) before the experiment. The particle size distribution and the zeta potential of the CeO2 NPs in the stock suspension was measured by a Malvern Zetasizer Nano ZS90 (Malvern Instruments, UK). The average size of the aggregates of CeO2 NPs was approximately 290 ± 16 nm, based on the number distribution with more than ?ve separate measurements per sample. The zeta potential of the CeO2 NPs in the Milli-Q water was ?20 ± 4.21 mV. 2.4. Short-term exposure experiments In this work, 1, 10, and 50 mg/L CeO2 NPs were chosen as the three test concentrations. The lowest level, 1 mg/L, was chosen as the environmentally relevant concentration of CeO2 NPs in WWTPs (Limbach et al., 2008). This experiment was designated as SBBR1. However, with CeO2 NPs listed as one of the most

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interesting metal oxide NPs by the Organization for Economic Co-operation and Development, they would be produced between 100 and 1000 t year?1, and more would be on their way (Piccinno et al., 2012). Considering the rapid development and growth in NPs use, 10 and 50 mg/L CeO2 NPs were also considered (SBBR2 and SBBR3, respectively). To conduct the experiments, 0, 30, 300, and 1500 mL of the CeO2 NPs stock solution (100 mg/L) were injected into the control SBBR, SBBR1, SBBR2, and SBBR3, respectively, and then the synthetic wastewater was added to afford a ?nal volume of 3 L. Aeration was started to maintain the DO at 0.1– 0.45 mg/L for 6.5 h, after an initial water sample was withdrawn. Thereafter, aliquots were extracted from each sample for analysis at designated times throughout the 8 h experiment. 2.5. Scanning electron microscopy SEM images were used to observe the different surface morphologies of the wastewater bio?lms with and without exposure to CeO2 NPs. At the end of the exposure period, the bio?lm samples on the ?lter were scraped out, washed three times with 0.1 M phosphate buffer (pH 7.4), and dehydrated using an ethanol gradient (50%, 70%, 80%, 90%, and 100%, 15 min per step). After drying in air, the bio?lm samples were coated with platinum and examined by SEM (Hitachi S-4800) at 5.0 kV. 2.6. Membrane integrity of bio?lm The bacterial cell membrane integrities in the bio?lms were determined by lactate dehydrogenase (LDH) release assays (Chen et al., 2012), which statistically compared the quantity of LDH released by bio?lms after exposure to different concentrations of CeO2 NPs. The LDH content was measured with a LDH kit (Jiancheng Bioengineering Co. Ltd., Nanjing, China) in accordance with the manufacturer’s instructions. Bio?lms exposed to different concentrations of CeO2 NPs were scraped out and centrifuged at 12000g for 5 min. Then, samples were prepared as instructed and measured at an absorption wavelength of 340 nm. 2.7. Viability assessment by confocal laser scanning microscopy (CLSM) The assessment of bacterial viability was conducted via a two-color ?uorescence assay of bacterial viability that depended on membrane integrity. The viable cells are stained by SYTO? 9 and ?uoresce green, while the damaged cells are stained by propidium iodide and ?uoresce red. The stained bio?lm structure was observed under a confocal laser-scanning microscope (Nikon A1, Japan). Further details are available in the Supplemental Material. 2.8. Other analytical methods and statistical analysis
? ? In the ef?uent, the concentrations of NH+ 4–N, NO2 –N, and NO3 – N were measured using AutoAnalyzer3 (BRAN+LUEBBE, Germany), while the TN, TP, and COD values were determined using APHA standard methods (American Public Health Association (APHA, 1998). The key enzymatic activities that play major roles in nitrogen removal were assessed with the relevant ammonia monooxygenase (AMO), nitrite oxidoreductase (NOR), nitrate/nitrite reductase (NR) enzymatic kits (Jiancheng Bioengineering Co. Ltd., Nanjing, China). The procedures for separating and quantifying EPS are in the Supplemental Materials. All tests were conducted in triplicate and the results are shown as the mean ± standard deviation. Statistical analyses were performed with SPSS 16.0. For values of p < 0.05, the statistical analysis was considered signi?cant.

3. Results and discussion 3.1. Effects of CeO2 NPs on biological nitrogen removal ef?ciency To evaluate the effects of CeO2 NPs on nitrogen removal, the decrease of TN in the ef?uent as a primary indicator was surveyed. Fig. 1 illustrates that the TN removal ef?ciency in SBBR1 was 74.09%, quite comparable with the control (75.50%), suggesting that 1 mg/L CeO2 NPs in the WWTP does not exert an obvious in?uence on TN removal. However, for the two SBBRs exposed to higher concentrations of CeO2 NPs (10 and 50 mg/L), the TN removal ef?ciencies decreased to 64.26% and 55.17%, respectively, which were statistically different compared with the control (p < 0.05). To elucidate the mechanisms of inhibition by the CeO2 NPs, the transfor? ? mations of NH+ 4–N, NO2 –N, and NO3 –N were investigated. ? As seen from Fig. 2, the ef?uent concentrations of NH+ 4–N, NO2 – ? N, and NO3 –N in the presence of 1 mg/L CeO2 NPs did not show much difference compared with the control. However, when the concentrations of CeO2 NPs were 10 and 50 mg/L, ammonia oxidation and denitri?cation were seriously inhibited during the expo? sure time. The ?nal NH+ 4–N and NO3 –N removal ef?ciencies were 73.22% and 55.25% in SBBR2 and were 45.21% and 46.09% in SBBR3, respectively, which were signi?cantly lower than those in the control (78.44% and 67.15%). Moreover, in the four SBBRs, no accumulation of NO? 2 –N was observed, although in SBBR3, the maximum concentration of NO? 2 –N in the ef?uent decreased significantly, from 1.05 to 0.51 mg/L, due to the deterioration of ammonia oxidation. ? ? The changes in the NH+ 4–N, NO2 –N, and NO3 –N contents during the process of biological nitrogen removal (Fig. 2) suggest that the reduction in nitrogen removal ef?ciency can be attributed to the inhibitory effects of the CeO2 NPs on the ammonia oxidation and denitri?cation phases, rather than the process of nitrite to nitrate. Similar conclusions were also drawn by Zheng et al. (2011a, 2011b), who studied the in?uences of different kinds of NPs on nitrogen removal in activated sludge systems. Furthermore, due to the complex and special structure of bio?lm, the denitrifying bacteria that live deeper in the anoxic bio?lm layer with carbon storage would stimulate the SND process (Yang et al., 2014). Thus, because denitri?cation takes place during the entire process

Fig. 1. Effects of CeO2 NPs on the nitrogen removal ef?ciency in four SBBRs. Error bars represent standard deviations of triplicate measurements. Asterisks indicate statistical differences (p < 0.05) from the control.

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? ? Fig. 2. Effects of different concentrations of CeO2 NPs on the contents of (a) NH+ 4–N (empty symbols) and NO2 –N (?lled symbols), and (b) NO3 –N. Error bars represent standard deviations of triplicate tests.

of nitrogen removal, it was less inhibited than the ammonia oxidation process by the higher concentrations of CeO2 NPs in this study, in contrast to the activated sludge system.

3.2. Adsorption of CeO2 NPs onto bio?lm and the possible effect on cytomembrane integrity Due to their broad, complex structural diversity with numerous binding sites, bio?lms have been widely used in WWTPs to adsorb and remove contaminants such as nitrogen, phosphorus, and metal ions. (Guibaud et al., 2009). Biosorption by EPS is major physical removal mechanism for pollutants, leading to the accumulation of these compounds in bio?lms (Kiser et al., 2010). In this study, when 10 and 50 mg/L CeO2 NPs were added into the SBBRs, 95% of the NPs were trapped in the EPS or bio?lm matrixes, consistent with previous studies (Kiser et al., 2010). The SEM and energy dispersive spectrometer image in Fig. A.3 (Supplementary Material) reveal a large number of aggregated NPs adhering onto the surface of the bio?lm after short-term exposure to 50 mg/L CeO2 NPs. However, it may be that once the NPs aggregate, their toxicity

may decline due to the increase in aggregates size (Choi and Hu, 2008). Several NPs, including those made of TiO2, CeO2, and ZnO, have been reported to cause damage to cell membranes and viability when attached to bacteria (Kim et al., 2010; Kocbek et al., 2010). For example, when the commonly used laboratory cell line BEAS-2B was exposed to different concentrations of CeO2 NPs, the rising concentrations of reactive oxygen species (ROS) and the advance of glutathione (GSH) activity were used to explain the mechanism of cell death (Horton and Khan, 2006). Therefore, experiments to observe LDH release and ROS production were conducted to assess the impact of such poisons on cell viability and growth (Gu et al., 2014). The extracellular LDH value is regarded as an indicator of membrane integrity (Hou et al., 2014), whereas the ROS production is used to measure oxidative stress (Applerot et al., 2012). Therefore, these two parameters were surveyed to assess the possible toxicity mechanisms of the CeO2 NPs toward bio?lm. As shown in Fig. 3, the intracellular ROS production in bio?lms was observed to increase with the rise in CeO2 NPs concentration, in accord with existing research (Fang et al., 2010) that described the stresses exerted by CeO2 NPs on Nitrosomonas europaea.

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Fig. 3. Relative ROS production and LDH release in bio?lms exposed to different concentrations of CeO2 NPs. Error bars represent standard deviations of triplicate measurements. Asterisks indicate statistical differences (p < 0.05) from the control.

However, a measurable difference in LDH was only seen with 50 mg/L CeO2 NPs, indicating that cell leakage occurred under these conditions (p < 0.05). This result agreed with previous research in which cells that were close to a large quantity of CeO2 NPs suffered signi?cant damage, such as large cavities and bubbles (Fang et al., 2010). Horton and Khan (2006) also considered that once NPs are taken up by living cells, they penetrate the cytoplasm and agglomerate around the cell nucleus, where they produce a harmful cellular response through contact with the bio-macromolecule directly. Further studies from Zhang et al. (2011) reported that low concentrations (1–100 nM) of CeO2 NPs could lead to a decrease lifespan of Caenorhabditis elegans. However, in our study, there was no sign of cell leakage in bio?lm under the exposure of 1 and 10 mg/L CeO2 NPs, which may attribute to the presence of EPS in the bio?lm in this study. In the coming text, the protective mechanisms of EPS in bio?lm were investigated. 3.3. Role of EPS during exposure Although the SEM images in Fig. A.3 show that CeO2 NPs can be adsorbed onto or penetrate the bio?lms, they do not necessarily mean that all the CeO2 NPs cross into the cells in the bio?lms (Fig. 3). The retention of the sorbed nanoparticles by the EPS could protect the cells from damage caused by the CeO2 NPs. The changes in the EPS during exposure were investigated to reveal the self-protection mechanism of a bio?lm facing toxicity (details are provided in the Supplementary Material). Mainly two kinds of EPS were observed to increase in the bio?lm during exposure to 10 and 50 mg/L CeO2 NPs; the EPS were mainly present as tightly bound (TB)-EPS, which are highly related to the packing ability (Sheng et al., 2010); while the loosely bound (LB)-EPS on the outmost layer of the bio?lm, were thought to contact and interact with the CeO2 NPs. These results indicated that the defense mechanism of the produced EPS is the formation of a dense matrix to protect the bacteria from the NPs (Puay et al., 2015), instead of enveloping the bio?lm by aggregation. In addition, particle aggregation together with retarded diffusive transportation due to the EPS can cause the bio?lms to be more resistant to CeO2 NPs and therefore reduce toxicity (Choi et al., 2010). 3.4. Viability of the bacteria before and after the exposure Maintaining the diversity of the bacteria species as well as a steady microbial community is important for achieving successful

biological nitrogen and phosphorus removal in municipal WWTPs. In this study, the CLSM technique was used to observe the live and dead cells in the bio?lm to explore the viability of bacteria exposed to different dosages of CeO2 NPs. As seen from Fig. A.4 (Supplementary Materials), there is a quite spectacular contrast between the density of live and dead cells in the absence and at high concentrations of CeO2 NPs. The image of the bio?lm exposed to 1 mg/L CeO2 NPs showed no noticeable difference from the control. However, in the presence of 10 and 50 mg/L CeO2 NPs, the number of dead cells apparently increases after exposure, which agrees with the results for ROS production and LDH release. Though the images show that the bacteria exposed to high concentrations of CeO2 NPs are damaged, the unique spatial structure of the bio?lm (as compared to a granulometric activated sludge system) enables the bacteria in the deeper layers to avoid the deadly impact of the NPs. Hou et al. (2014) concluded that, due to the protective barrier formed by the EPS in the bio?lm, ZnO NPs inhibited microbial activity only in the outer bio?lm layer, whereas the bacteria located deeper in the layer, such as denitrifying bacteria, became even more active. In contrast, the structure of the LB-EPS coating on the outside of sludge particles would permit the NPs to penetrate the deeper layers of the bio?lm, and thus, the protective capacity is limited under high concentrations (Hou et al., 2015; Ma et al., 2013). Thus, in this study, the reason for the inhibited denitri?cation in the SBBR may not have been caused by the denitrifying bacteria, in seeming contradiction to the inhibition of NPs in activated sludge systems (Zheng et al., 2011b). 3.5. Toxicity of CeO2 NPs on the activities of key denitrifying enzymes in bio?lm Obviously, successful biological nitrogen removal in WWTPs relies on a well-rounded system that includes ammonia oxidation, nitri?cation, and denitri?cation; these three processes are related to the activities of special enzymes correlated with nitrogen removal (Fig. A.5, Supplementary Materials). Researchers have determined that ammonia monooxygenase (AMO) plays a role in ammonia oxidation by ammonia-oxidizing bacteria, and then nitrite is catalytically transformed to nitrate by nitrite oxidoreductase (NOR) via nitrite-oxidizing bacteria. Finally, the nitrate reductase (NR) enzyme in the denitrifying bacteria achieves nitrogen removal (Zheng et al., 2011a). Given this enzymatic pathway, the impact of CeO2 NPs exposure on these three enzymes was examined to further explore the probable mechanism for the reduction of nitrogen removal ef?ciency.

Fig. 4. Effects of different concentrations of CeO2 NPs on the relative activities of AMO, NOR, and NR in bio?lms. Error bars represent standard deviations of triplicate measurements. Asterisks indicate statistical differences (p < 0.05) from the control.

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The activities of the three enzymes in the bio?lm in response to different concentrations of CeO2 NPs are shown in Fig. 4. Among the four SBBRs, the relative activity of NOR remained nearly constant. The AMO and NR activities in bio?lms exposed to 1 mg/L CeO2 NPs also showed no signi?cant differences from the controls. However, after exposure to 10 and 50 mg/L CeO2 NPs, the relative activities of AMO obviously decreased to 91.21% and 88.36% of the controls, and 67.49% and 76.03% for NR of the controls, respectively. The possible reason for these phenomena may be due to the highly increased content of ROS, which was reported to cause the denaturation of protein. Generally, the variations of these enzymes are consistent with the lack of measurable impact by the CeO2 NPs on nitrogen removal at a concentration of 1 mg/L, ? ? and the transformations of NH+ 4–N, NO2 –N, and NO3 –N in the corresponding SBBRs. 4. Conclusions The effects of CeO2 NPs on nitrogen removal by bio?lms were evaluated. Low CeO2 NPs concentrations did not affect the bio?lms. At higher levels, ammonia oxidation and denitri?cation decreased, reducing nitrogen removal ef?ciency. The EPS’ protective effects caused the inhibition of denitri?cation less than ammonia oxidation. CeO2 NPs were adsorbed as aggregates, and the resulting ROS production increase in?uenced cell growth. However, LDH release due to cell leakage occurred only for the highest exposure. Bacterial viability declined at higher dosages and two key enzyme activities related to nitrogen removal were inhibited. Acknowledgements We are grateful for the grants from Project supported by National Science Funds for Creative Research Groups of China (No. 51421006), National Natural Science Foundation of China (Nos. 51479047, 51479065, 51209069), National Science Funds for Distinguished Young Scholars (No. 51225901), Program for Changjiang Scholars and Innovative Research Team in University (No. IRT13061), the Key Program of National Natural Science Foundation of China (No. 41430751), Jiangsu Province Ordinary University Graduate Student Scienti?c Research Innovation Plan (No. KYZZ14_0157) and PAPD. 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.2015.04. 123. References
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