Effects of ZnO Nanoparticles on Wastewater Biological Nitrogen and

Mar 7, 2011 - With the increasing utilization of nanomaterials, zinc oxide nanoparticles (ZnO NPs) have been reported to induce adverse effects on hum...
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Effects of ZnO Nanoparticles on Wastewater Biological Nitrogen and Phosphorus Removal Xiong Zheng, Rui Wu, and Yinguang Chen* State Key Laboratory of Pollution Control and Resources Reuse, School of Environmental Science and Engineering, Tongji University, 1239 Siping Road, Shanghai 200092, China

bS Supporting Information ABSTRACT: With the increasing utilization of nanomaterials, zinc oxide nanoparticles (ZnO NPs) have been reported to induce adverse effects on human health and aquatic organisms. However, the potential impacts of ZnO NPs on wastewater nitrogen and phosphorus removal with an activated sludge process are unknown. In this paper, short-term exposure experiments were conducted to determine whether ZnO NPs caused adverse impacts on biological nitrogen and phosphorus removal in the unacclimated anaerobic-low dissolved oxygen sequencing batch reactor. Compared with the absence of ZnO NPs, the presence of 10 and 50 mg/L of ZnO NPs decreased total nitrogen removal efficiencies from 81.5% to 75.6% and 70.8%, respectively. The corresponding effluent phosphorus concentrations increased from nondetectable to 10.3 and 16.5 mg/L, respectively, which were higher than the influent phosphorus (9.8 mg/L), suggesting that higher concentration of ZnO NPs induced the loss of normal phosphorus removal. It was found that the inhibition of nitrogen and phosphorus removal induced by higher concentrations of ZnO NPs was due to the release of zinc ions from ZnO NPs dissolution and increase of reactive oxygen species (ROS) production, which caused inhibitory effect on polyphosphate-accumulating organisms and decreased nitrate reductase, exopolyphosphatase, and polyphosphate kinase activities.

’ INTRODUCTION Nanoparticles (NPs) are widely applied in many commercial industrial and consumer products such as semiconductors, cosmetics, textiles, and pigments.1 However, researchers found that once released into the environment NPs might pose the potential risks to human health and microorganisms.2,3 Many studies were thus conducted to investigate the behavior of NPs in the environment and predict their environmental concentrations.4,5 The increasing utilization of nanoparticlescontaining products, however, was observed to result in the release of NPs into wastewater treatment plants (WWTPs).1,6 Limbach et al.7 found that large amounts of cerium oxide NPs released into a mode WWTP were adsorbed by activated sludge. The adsorption to activated sludge was the major mechanism for NPs removal in conventional activated sludge systems.8 Nevertheless, few studies have been conducted to determine if the adsorbed NPs could induce the adverse effect on activated sludge. Although silver NPs were reported to be toxic to the respiration of nitrifying bacteria,9 the potential influence of NPs on biological nitrogen and phosphorus removal is still unknown and needed to be explored. Large amounts of zinc oxide (ZnO) NPs have been used in semiconductors, plastic additives, pigments, and cosmetics, which is likely to cause the particles release into the environment.10 Recent study confirmed that ZnO NPs were r 2011 American Chemical Society

present in sewage sludge and effluents.1 Previous toxicological studies showed that 10 mg/L of ZnO NPs could induce the significant growth inhibition (up to 90%) of Bacillus subtilis, but only caused 22% of the growth inhibition of Escherichia coli.11 These studies indicated that the toxicity of ZnO NPs was dependent on the species of bacteria. Activated sludge in WWTPs involves various species of bacteria, and therefore it seems difficult to figure out the potential impact of ZnO NPs on activated sludge based on the toxicity of ZnO NPs to some pure bacteria. Furthermore, an important function of activated sludge is to achieve biological nitrogen and phosphorus removal and the performance of biological nutrient removal, especially phosphorus release and uptake, is related to the transformations of intracellular polyhydroxyalkanoates (PHA) and glycogen.12,13 However, the effect of ZnO NPs on transformations of PHA and glycogen in biological nutrient removal process has never been reported. ZnO NPs are slightly soluble and can dissolve to form zinc ions (Zn2þ) under aqueous conditions.14 The released Zn2þ was believed to account for the toxicity of ZnO NPs to Mycobacterium Received: January 8, 2011 Accepted: February 22, 2011 Revised: February 21, 2011 Published: March 07, 2011 2826

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Environmental Science & Technology smegmatis,10 Pseudokirchneriella subcapitata15 and marine phytoplankton.16 However, researchers also found that some species of bacteria such as Shewanella oneidensis MR-1 and E. coli were insensitive to the released Zn2þ even at high concentrations (>40 mg/L) of ZnO NPs.10 Therefore, determining the impact of the released Zn2þ on biological nitrogen and phosphorus removal is important to explain the potential effects of ZnO NPs on activated sludge. Moreover, oxidative stress induced by ZnO NPs was reported to cause the cell membrane damage and cytoplasmic leakage due to the lipid peroxidation.14,17 Intracellular reactive oxygen species (ROS) were found to be toxic to cytoplasmic lipids, proteins and other intermediates in cells.14 It is well-known that biological nitrogen and phosphorus removal consists of a series of biochemical processes, such as nitrification, denitrification, and phosphorus anaerobic release and aerobic or anoxic uptake.12,18 The performance of these processes is directly related to the activities of some key enzymes, such as ammonia monooxygenase (AMO), nitrite oxidoreductase (NOR), nitrate reductase (NR), nitrite reductase (NIR), exopolyphosphatase (PPX), and polyphosphate kinase (PPK).19-22 However, it is unknown how ZnO NPs affect these key enzymes activities in activated sludge. The aim of this study was to investigate the effects of ZnO NPs on biological nitrogen and phosphorus removal. The intracellular ROS production was measured to examine the oxidative stress in the presence of ZnO NPs. The impact of Zn2þ released from ZnO NPs on biological nutrient removal was also studied. Scanning electron microscope (SEM) and lactate dehydrogenase (LDH) release assays were used to determine the surface integrity of activated sludge. The activities of some key enzymes related to biological nitrogen and phosphorus removal were further measured to explore the potential effect of ZnO NPs on activated sludge. In this study, the anaerobic-low dissolved oxygen (DO) sequencing batch reactor (SBR) was used to culture activated sludge, because this biological wastewater treatment technology was proven to simultaneously achieve nitrogen and phosphorus removal and concurrently save the energy of oxygen supply.18

’ MATERIALS AND METHODS Nanoparticles. Commercially produced ZnO NPs were purchased from Sigma-Aldrich (St. Louis, MO). X-ray diffraction (XRD) analysis was conducted using a Rigaku D/Max-RB diffractometer equipped with a rotating anode and a Cu KR radiation source and shown in Figure S1 (Supporting Information (SI)). The specific surface area (SSA) of ZnO NPs was measured to be 39.1 ( 5.4 m2/g via a Micromeritics Tristar 3000 analyzer by nitrogen adsorption at 77 K using the BrunauerEmmett-Teller (BET) method. Before use the NPs stock suspension (100 mg/L) was prepared by dispersing 0.1 g of ZnO NPs in 1 L of Milli-Q water, followed by 1 h of ultrasonication (25 °C, 250 W, 40 kHz) according to the literature.23 The average diameter of the particles in the stock suspension was determined to be 89 nm by dynamic light scattering (DLS) using a Malvern Autosizer 4700 (Malvern Instruments, UK). Parent Sequencing Batch Reactor Operation. Activated sludge was cultured in the anaerobic-low DO (0.15-0.50 mg/L) parent SBR with a working volume of 4 L, which was operated to achieve biological nitrogen and phosphorus removal. The SBR was worked at 21 ( 1 °C with three cycles each day. Each cycle (8 h) consisted of 1.5 h anaerobic and 3 h low DO periods, followed

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by 1 h settling, 10 min decanting and 140 min idle periods. Three liters of synthetic wastewater were pumped into the reactor in the first 15 min of the anaerobic stage. The influent pH was adjusted to 7.5 by adding 4 M NaOH or 4 M HCl. In the low DO period, air was provided intermittently using an on/off control system with online DO detector to maintain the DO level between 0.15 and 0.50 mg/L. Sludge was wasted to keep the solids retention time (SRT) at approximately 22 days according to the previous publication.24 The reactor was constantly mixed with a magnetic stirrer except for the settling, decanting, and idle periods. After cultivation for 105 days, the stable removal efficiencies of nitrogen and phosphorus were observed in the parent SBR. Fluorescence in situ hybridization (FISH) analysis showed that the parent SBR was dominated by polyphosphate-accumulating organisms (PAO) (represented 43 ( 5% of the biomass) and glycogen-accumulating organisms (GAO) (represented 21 ( 4% of the biomass). Synthetic Wastewater. Three liters of synthetic wastewater contained 8 mL of concentrated feed, 2.2 mL of phosphorus stock solution, 0.38 g of ammonium chloride, 2 mL of trace element feed and 1.1 mL of acetic acid. The concentrated feed contained (g/L): 25.88 peptone, 4.24 yeast extract, 33.94 MgCl2 3 6H2O, 19.09 MgSO4 3 7H2O and 8.91 CaCl2 3 2H2O. The phosphorus stock solution contained (g/L): 29 KH2PO4 and 33 K2HPO4. The trace element feed contained (g/L): 1.50 FeCl3 3 6H2O, 0.03 CuSO4 3 5H2O, 0.12 MnCl2 3 4H2O, 0.06 Na2MoO4 3 2H2O, 0.12 ZnSO4 3 7H2O, 0.15 CoCl2 3 6H2O, 0.18 KI, 0.15 H3BO3 and 10 ethylenediamine tetraacetic acid. Nanoparticles Exposure to Activated Sludge. Three test concentrations (1, 10, and 50 mg/L) of ZnO NPs were examined in the short term exposure experiments. The environmentally relevant concentration of ZnO NPs in WWTPs was chosen to be 1 mg/L according to the literatures.1,6 Since the environmental release of NPs might increase due to the large-scale production,2 the potential effects of higher concentrations (10 and 50 mg/L) of ZnO NPs were also investigated in this study according to the references.10,11 To conduct the experiments, 1600 mL of mixture was withdrawn from the parent SBR at the end of low DO stage, centrifuged at 100g for 5 min, washed with NaCl solution (0.154 M) 3 times, and resuspended in 400 mL of deionized (DI) water. The 0, 1, 10, and 50 mg/L of ZnO NPs were prepared in 4 reactors by adding 0, 4, 40, and 200 mL of ZnO NPs stock suspension (100 mg/L), respectively. Then, 100 mL of resuspended sludge and 100 mL of synthetic wastewater (containing 0.11 mL of acetic acid, 0.038 g of ammonium chloride, 0.22 mL of phosphorus stock solution, 0.8 mL of concentrated feed, and 0.2 mL of trace element feed) were fed into each reactor. DI water was added to make the final volume of each reactor to be 400 mL, resulting in the initial concentrations of chemical oxygen demand (COD), ammonia-nitrogen (NH4þ-N) and soluble ortho-phosphorus (SOP) of approximately 300, 25, and 10 mg/L, respectively. The initial pH in each reactor was adjusted to 7.5 by adding 4 M NaOH or 4 M HCl. All reactors were bubbled with nitrogen gas for 10 min, sealed and anaerobically stirred for 1.5 h, and then each reactor was aerobically stirred under low DO (0.15-0.50 mg/L) condition for 3 h. Determination of Reactive Oxygen Species Production Induced by Nanoparticles. Intracellular ROS production was determined using an established fluorescence assay.25 Activated sludge was centrifuged at 100g for 5 min and washed with 0.1 M phosphate buffer (pH 7.4) for 3 times. The pellets were resuspended in 0.1 M phosphate buffer containing 50 μM of 2827

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Figure 1. Effects of ZnO NPs on the variations of NH4þ-N (white), NO3--N (black), SOP (white) and NO2--N (black) during one cycle in unacclimated activated sludge culture. Error bars represent standard deviations of triplicate tests.

dichlorodihydrofluorescein diacetate (H2DCF-DA, Molecular Probes, Invitrogen) and incubated at 21 ( 1 °C in dark. After 30 min of incubation, the phosphate buffer containing H2DCF-DA was removed by centrifugation. The pellets were resuspended in synthetic wastewater (pH 7.5) containing the test concentrations (0, 1, 10, and 50 mg/L) of ZnO NPs and plated into a 96-well plate. The generated fluorescein DCF was measured after 4.5 h using a microplate reader (BioTek, Winooski, VT) with 485 nm excitation and 520 nm emission filter. Measurement of Nanoparticles Dissolution in Synthetic Wastewater. The released Zn2þ in synthetic wastewater due to the dissolution of ZnO NPs was determined according to the literature.10 Briefly, 4 vials containing the synthetic wastewater (pH 7.5) and ZnO NPs (0, 1, 10, and 50 mg/L) were shaken at 21 ( 1 °C, and ZnO NPs were then removed by high speed centrifugation (12 000g) for 20 min. Finally, 0.5 mL of the supernatant was added to 4.5 mL of Milli-Q water (containing 2% ultrahigh purity HNO3). The Zn2þ concentration in the resulting solution at different time points was measured by inductively coupled plasma mass spectrometry (ICP-MS, Agilent Technologies, Santa Clara, CA). Scanning Electron Microscopy. SEM images were used to analyze the surface morphology of activated sludge after exposed to ZnO NPs. At the end of the short-term exposure experiments, the aliquots were centrifuged at 100g for 5 min. The pellets were then washed 3 times with 0.1 M phosphate buffer (pH 7.4), and fixed in 0.1 M phosphate buffer (pH 7.4) containing 2.5% glutaraldehyde at 4 °C for 4 h. After rinsing twice with 0.1 M phosphate buffer (pH 7.4), the pellets were dehydrated in ethanol serials (50%, 70%, 80%, 90%, and 100%, 15 min per step), and then dried in air. Finally, the images were obtained using the FEI Quanta 200 SEM at 20 kV. Lactate Dehydrogenase Release. The cell membrane integrity of activated sludge was measured by the LDH release assay. LDH activity was determined by using a cytotoxicity detection kit (Roche Applied Science) according to the manufacturer’s instructions. At the end of the short-term exposure experiments, the mixture was centrifuged at 12 000g for 5 min and then the supernatant was seeded on a 96-well plate, followed by the addition of 50 μL of substrate mix (Roche Applied Science). After 30 min of incubation at room temperature in dark, 50 μL of stop solution (Roche Applied Science) was added to each well

and the absorbance was recorded at 490 nm using a microplate reader (BioTek). Analytical Methods. The detailed procedures of FISH assay and measurements of AMO, NOR, NR, NIR, PPX and PPK activities are presented in the SI. The determinations of NH4þ-N, nitrite-nitrogen (NO2--N), nitrate-nitrogen (NO3--N), total nitrogen (TN), SOP, mixed liquor suspended solids (MLSS) and mixed liquor volatile suspended solids (MLVSS) were conducted in accordance with the Standard Methods.26 The analyses of PHA (including polyhydroxybutyrate (PHB), ployhydroxyvalerate (PHV), and polyhydroxy-2-methylvalerate (PH2MV)) and glycogen were reported in our previous publication.27 Statistical Analysis. All tests were performed in triplicate and the results were expressed as mean ( standard deviation. An analysis of variance (ANOVA) was used to test the significance of results and p < 0.05 was considered to be statistically significant.

’ RESULTS AND DISCUSSION Effects of ZnO NPs on Biological Nitrogen and Phosphorus Removal. Figure 1 presents the transformations of

nitrogen and phosphorus in the anaerobic and low DO stages at ZnO NPs concentrations of 1, 10, and 50 mg/L. It was found that the average TN and SOP removal efficiencies were respectively 80.8% and almost 100% at ZnO NPs concentration of 1 mg/L, which were almost the same as those observed in the control test (81.5% and almost 100%), suggesting that 1 mg/L of ZnO NPs showed no measurable effect on both nitrogen and phosphorus removal. However, when the concentrations of ZnO NPs were 10 and 50 mg/L, the effluent concentrations of NO3--N increased to 6.1 ( 0.4 and 7.3 ( 0.3 mg/L, respectively, and the corresponding TN removal efficiencies were 75.6% and 70.8%, which were lower than the TN removal efficiency in the control test (81.5%) (p < 0.05). This result indicated that the nitrogen removal was inhibited by higher concentrations of ZnO NPs. Nevertheless, all NH4þ-N was removed and no accumulation of NO2--N in effluent was observed at ZnO NPs concentrations of 10 and 50 mg/L (Figure 1). Moreover, when activated sludge was exposed to 10 and 50 mg/L of ZnO NPs, the anaerobic SOP releases were 73.2 ( 2.6 and 69.4 ( 3.1 mg/L, respectively, which were lower than in the control test (79.6 ( 2.8 mg/L) (p < 0.05). In the subsequent low DO stage, the 2828

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Figure 2. Effects of ZnO NPs on the intracellular ROS production (a) and LDH release (cell membrane damage marker) (b). Asterisks indicate statistical differences (p < 0.05) from the control test. Error bars represent standard deviations of triplicate tests.

Figure 3. SEM images of activated sludge exposed to different concentrations of ZnO NPs. Control (a), 1 mg/L (b), 10 mg/L (c), and 50 mg/L (d) of ZnO NPs.

effluent SOP concentrations increased to 10.3 ( 1.1 and 16.5 ( 1.6 mg/L, respectively, which were higher than the influent SOP (9.8 ( 0.3 mg/L), and thus there was no net SOP removal (Figure 1b). Apparently, higher concentrations (10 and 50 mg/L) of ZnO NPs were capable of inhibiting the SOP release, uptake and net removal. It is well-known that biological nitrogen removal depends on the successful ammonia oxidation and nitrate denitrification to nitrogen gas.18 The above studies showed that the decrease of biological nitrogen removal induced by higher concentrations of ZnO NPs was not caused by the inhibition of ammonia oxidation, but due to the inhibitory effect on denitrifying process (Figure 1a). Usually, the successful enhanced biological phosphorus removal means that the aerobic phosphorus uptake is greater than the anaerobic phosphorus release, leading to the net phosphorus removal.12 In the presence of 10 and 50 mg/L of ZnO NPs, the average aerobic SOP uptakes (72.9 and 62.9 mg/L, respectively) were lower than the corresponding anaerobic SOP releases (73.2 and 69.4 mg/L, respectively), which resulted in no net SOP removal. This observation indicated that the sudden appearance of higher concentrations of ZnO NPs could lead to the loss of normal phosphorus removal in a biological WWTP. In the coming text the mechanisms of the effect of ZnO NPs on biological nitrogen and phosphorus removal were investigated. Mechanisms of ZnO NPs Affecting Biological Nitrogen and Phosphorus Removal. It was reported in the literature that

ZnO NPs were able to cause oxidative stress and induce adverse effects on model organisms.14 High ROS production might lead to the damage of cell membrane or cytoplasmic proteins in human cells28 and E. coli.17 In this study, the intracellular ROS production was observed to increase with the increase of ZnO NPs concentration (Figure 2a). However, SEM analysis showed that there was no observed damage on the activated sludge surface (Figure 3). The result of LDH release assay in Figure 2b also confirmed that no measurable cell leakage was found at any concentration of ZnO NPs (p < 0.05). Also, recent studies have suggested that the toxicity of ZnO NPs to model organisms arises from the release of Zn2þ due to particles dissolution.14-16 In this study, kinetics study of ZnO NPs dissolution was conducted to determine the total concentration of the released Zn2þ in synthetic wastewater. The average concentrations of the released Zn2þ were, respectively, 0.12, 0.45, and 1.15 mg/L at ZnO NPs concentrations of 1, 10, and 50 mg/L (Figure 4a). Batch tests were conducted to examine the potential effects of these concentrations of Zn2þ on biological nitrogen and phosphorus removal. It was found that activated sludge exposed to 0.12 mg/L of Zn2þ showed similar effluent concentrations of NO3--N (4.4 ( 0.4 mg/L) and SOP (nondetectable) to those in the control test (4.6 ( 0.4 mg/L and nondetectable, respectively). However, higher concentrations (0.45 and 1.15 mg/L) of Zn2þ were inhibitory to biological nitrogen and phosphorus removal (Figure 4b). The average TN removal efficiencies decreased to respectively 78.8% and 75.2% at 2829

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Figure 4. Kinetics of ZnO NPs dissolution (a) and the effects of the released Zn2þ on the effluent SOP and NO3--N concentrations (b). Asterisks indicate statistical differences (p < 0.05) from the control test. Error bars represent standard deviations of triplicate tests.

Figure 5. Effects of ZnO NPs on the transformations of (a) PHA (white) and glycogen (black), as well as (b) PHB, PHV, and PH2MV during one cycle in unacclimated activated sludge culture. Error bars represent standard deviations of triplicate tests.

0.45 and 1.15 mg/L of Zn2þ, whereas the effluent concentrations of SOP increased to 5.8 ( 0.6 and 8.6 ( 0.8 mg/L, respectively. By comparing the effluent NO3--N and SOP concentrations induced by ZnO NPs with those induced by the corresponding Zn2þ, it can be seen that the released Zn2þ was mainly responsible for the inhibitory effects of ZnO NPs. High concentration of Zn2þ released from ZnO NPs was reported to induce the toxicity to many organisms.14,15 However, these studies mainly focused on the toxicity of ZnO NPs to cell growth. The current study showed that the released Zn2þ was capable of inhibiting the normal function of activated sludge and was responsible for the inhibitory effects of ZnO NPs on biological nitrogen and phosphorus removal. Also, it was found in this study that the denitrifying bacteria and PAO were sensitive to Zn2þ, but other bacteria, such as ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB) and GAO, seemed insensitive in the short term exposure experiments. In the literature, M. smegmatis and Cyanothece 51142 were found to be more sensitive to Zn2þ than S. oneidensis MR-1 and E. coli, which also suggested that the toxicity of Zn2þ to pure bacteria was dependent on the species of bacteria.10

It is well-known that biological phosphorus removal is related to the transformations of intermediate metabolites including intracellular PHA and glycogen.12,13 As shown in Figure 5a, the intracellular PHA was synthesized and glycogen was utilized in the anaerobic stage, whereas the opposed observations were made in the low DO stage in all reactors. However, compared with the control test, activated sludge exposed to 10 and 50 mg/L of ZnO NPs showed greater variations of total PHA and glycogen in both anaerobic and low DO stages (Figure 5a) (p < 0.05). It is reported that the transformations of PHA and glycogen in biological phosphorus removal are associated with PAO and GAO, and higher transformation of glycogen indicates that the metabolism of GAO might be activated.12,29 As seen in Figure 5b, four reactors had almost the same anaerobic synthesis of PHB and PH2MV, but the PHV synthesis increased with the increase of ZnO NPs concentration. Thus, higher PHA synthesis at ZnO NPs concentrations of 10 and 50 mg/L was due to the increase of PHV production. Some publications pointed out that the energy source for anaerobic acetic acid uptake by GAO came mainly from glycogen degradation, and when fed with acetic acid GAO tended to produce more PHV and total PHA than PAO due to 2830

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Figure 6. Schematic diagram of the anaerobic metabolic pathway for conversion of acetate and glycogen into PHA by GAO. The succinate-propionate pathway is shown by dashed line.

Table 1. Activities of the Key Enzymes Related to Biological Nitrogen and Phosphorus Removal in the Presence of ZnO NPsa AMOb

NRb

NIRb

PPXc

PPKd

0.021 ( 0.002

0.086 ( 0.004

0.046 ( 0.002

0.287 ( 0.005

0.022 ( 0.002

0.286 ( 0.005

1

0.023 ( 0.003

0.084 ( 0.003

0.048 ( 0.003

0.284 ( 0.003

0.020 ( 0.003

0.289 ( 0.003

10 50

0.019 ( 0.002 0.020 ( 0.003

0.088 ( 0.004 0.085 ( 0.002

0.037 ( 0.003 0.032 ( 0.004

0.286 ( 0.005 0.283 ( 0.004

0.014 ( 0.004 0.009 ( 0.003

0.167 ( 0.005 0.113 ( 0.004

control ZnO NPs (mg/L)

NORb

The data reported are the averages and their standard deviations in triplicate tests. b The unit is μmol nitrite/(min 3 mg protein). c The unit is μmol pnitrophenol/(min 3 mg protein). d The unit is μmol NADPH/(min 3 mg protein). a

the partial conversion of pyruvate to propionyl-CoA through the succinate-propionate pathway 12,29,30 (Figure 6). It seems that the presence of higher concentrations of ZnO NPs benefits the carbon source (acetic acid) consumption of GAO due to PAO being inhibited. Further investigation showed that ZnO NPs influenced the activities of enzymes relevant to nitrogen and phosphorus removal. AMO and NOR are two key enzymes in nitrification,20 and denitrification is relevant to NR and NIR,19 whereas phosphorus removal is directly related to the activity of PPX and PPK 12,22 (Figure S2, SI). As shown in Table 1, among four nitrogen metabolism enzymes (AMO, NOR, NR, and NIR), higher concentrations (10 and 50 mg/L) of ZnO NPs decreased the specific activity of NR (Table 1) (p < 0.05), but showed no measurable influence on the specific activities of AMO, NOR, and NIR (Table 1) (p > 0.05). These observations were in correspondence with higher effluent concentrations of NO3--N and lower TN removal efficiencies at ZnO NPs concentrations of 10 and 50 mg/L (Figure 1a). Furthermore, compared with the control test, activated sludge exposed to 10 and 50 mg/L of ZnO NPs showed lower specific activities of PPX and PPK (Table 1) (p < 0.05), which were also consistent with the observed lower phosphorus release and uptake (Figure 1b). These results suggested that higher concentrations of ZnO NPs could inhibit biological nitrogen and phosphorus removal by decreasing the activities of NR, PPX and PPK.

’ ASSOCIATED CONTENT

bS

Supporting Information. This file contains additional methods details, Table S1, and Figures S1 and S2. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Phone: þ86 21 65981263; fax: þ86 21 65986313; e-mail: [email protected].

’ ACKNOWLEDGMENT This work was financially supported by the Foundation of State Key Laboratory of Pollution Control and Resources Reuse (PCRRK09002). ’ REFERENCES (1) Gottschalk, F.; Sonderer, T.; Scholz, R. W.; Nowack, B. Modeled environmental concentrations of engineered nanomaterials (TiO2, ZnO, Ag, CNT, Fullerenes) for different regions. Environ. Sci. Technol. 2009, 43, 9216–9222. (2) Colvin, V. L. The potential environmental impact of engineered nanomaterials. Nat. Biotechnol. 2003, 21, 1166–1170. 2831

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