Long-Term Effects of Copper Nanoparticles on Wastewater Biological

State Key Laboratory of Pollution Control and Resources Reuse, School of Environmental Science and Engineering, Tongji University, 1239 Siping Road, S...
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Long-Term Effects of Copper Nanoparticles on Wastewater Biological Nutrient Removal and N2O Generation in the Activated Sludge Process Yinguang Chen,*,† Dongbo Wang,† Xiaoyu Zhu,† Xiong Zheng,† and Leiyu Feng† †

State Key Laboratory of Pollution Control and Resources Reuse, School of Environmental Science and Engineering, Tongji University, 1239 Siping Road, Shanghai 200092, China S Supporting Information *

ABSTRACT: The increasing use of copper nanoparticles (Cu NPs) raises concerns about their potential toxic effects on the environment. However, their influences on wastewater biological nutrient removal (BNR) and nitrous oxide (N2O) generation in the activated sludge process have never been documented. In this study the long-term effects of Cu NPs (0.1−10 mg/L) on BNR and N2O generation were investigated. The total nitrogen (TN) removal was enhanced and N2O generation was reduced at any Cu NPs levels investigated, but both ammonia and phosphorus removals were not affected. The mechanism studies showed although most of the Cu NPs were absorbed to activated sludge, the activated sludge surface was not damaged, and the released copper ion from Cu NPs dissolution was the main reason for TN removal improvement and N2O reduction. It was also found that the transformation of polyhydroxyalkanoates and the activities of ammonia monooxygenase, nitrite oxidoreductase, exopolyphosphatase, and polyphosphate kinase were not affected by Cu NPs, whereas the decreased metabolism of glycogen and the increased activities of denitrification enzymes were observed. Further investigation revealed that Cu NPs increased the number of denitrifiers (especially N2O reducing denitrifiers) but decreased nitrite accumulation. All these observations were in correspondence with the enhancement of TN removal and reduction of N2O generation.

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INTRODUCTION Copper nanoparticles (Cu NPs) are widely used in various applications such as air and liquid filtration, wood preservation, bioactive coatings, textiles, skin products, and coatings on integrated circuits due to their novel physical and chemical properties.1,2 The extensive applications of Cu NPs inevitably lead to their environmental releases, and their potential toxicities to the environment have attracted increasing concerns. Recently, many studies have been carried out to examine the toxicities of Cu NPs to aquatic model organisms, such as bacteria, protozoa, crustaceans, algae, and zebrafish, and their cytotoxic effects on these species have been reported.1,3,4 As the last barriers prior to their aquatic environmental risks, wastewater treatment plants (WWTPs) have been reported to remove most of the Cu NPs via aggregation, settling, precipitation, biosorption, or other biomass mediated processes.5 An important function of activated sludge in WWTPs is to remove nitrogen and phosphorus from wastewater via a series of biochemical processes, such as nitrification, denitrification, and phosphorus anaerobic release and aerobic/anoxic uptake.6,7 The intercepted Cu NPs in activated sludge may also bring risks to these biochemical processes. It was reported that 10 mg/L Cu NPs in wastewater had no significant toxic effect on coliform and ammonia oxidizing bacteria after 1 d exposure. 5 Also, some other publications © 2012 American Chemical Society

indicated that different nanoparticles and exposed time showed different effects on biological nutrient removal (BNR). For example, the sudden appearance of 50 mg/L ZnO NPs led to the inhibition of both nitrogen and phosphorus removal in a BNR process, but 50 mg/L TiO2 NPs had no obvious influence on wastewater nitrogen and phosphorus removal by short-term (1 d) exposure.8,9 After long-term (70 d) exposure, however, 50 mg/L TiO2 NPs were observed to significantly decrease the total nitrogen (TN) removal.9 It is well-known that WWTPs are long-term operated, and BNR is related to many functional bacteria (such as nitrifiers, denitrifiers, polyphosphate accumulating organisms (PAO)), the short-term toxic investigation of Cu NPs on the reported model bacteria cannot represent their influences on BNR, and the limited short- and long-term toxic studies of other nanoparticles on BNR also cannot reflect the effects of Cu NPs on wastewater nitrogen and phosphorus removal. Another important issue occurred in the BNR process is that a potent greenhouse gas, nitrous oxide (N2O), can be produced. N2O is a very important greenhouse gas, as it has a 300-fold stronger effect than carbon dioxide.10 The Received: Revised: Accepted: Published: 12452

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was selected for this study. To obtain more information about Cu NPs impacts on BNR and N2O generation, Cu NPs concentrations of 0.1, 1, and 5 mg/L were also performed. The effects of Cu NPs on BNR and N2O generation were conducted in an anaerobic − low DO aerobic (DO: 0.15−0.5 mg/L) BNR process. Five identical sequencing batch reactors (SBRs) with a working volume of 4 L each were operated. The seed sludge was withdrawn from the secondary sedimentation tank of a municipal WWTP in Shanghai, China, and was inoculated into the five SBRs simultaneously. All SBRs were maintained at 21 ± 1 °C in a temperature controlled room and operated with three cycles per day. Each cycle consisted of 2 h anaerobic and 3 h low DO periods, followed by 1 h settling, 10 min decanting, and 110 min idle periods. These five SBRs were, respectively, received wastewaters containing 0 (i.e., the control SBR), 0.1, 1, 5, and 10 mg/L Cu NPs, which were prepared by adding relevant volumes of Cu NPs stock suspension to wastewater. After settling phase 2 L of the supernatant was discharged from each SBR, and was respectively replaced with 2 L of 0, 0.1, 1, 5, and 10 mg/L Cu NPs wastewaters during the initial 10 min of the anaerobic period. In the low DO period, air was provided intermittently using an on/off control system with an online DO detector to maintain the DO level between 0.15 and 0.5 mg/L. The hydraulic retention time in these five SBRs was 16 h, while the sludge retention time was maintained at approximately 20 d. All reactors were constantly mixed with a magnetic stirrer except for the settling, decanting, and idle periods. The effluent concentrations of NH4+-N, nitritenitrogen (NO2−-N), nitrate-nitrogen (NO3−-N), SOP, and the amounts of N2O generation in all SBRs were determined twice per week during the acclimation period. It took 92 d before these measured data reached relatively stable, then the cycle studies were performed, and the data were reported. Effects of Cu NPs on Biomass Growth during One Cycle. This study was carried out to calculate the cycle biomass growth among five reactors. Five sludge mixtures (0.4 L each) were respectively taken from the above five SBRs at the end of aerobiosis but before settling. The mixtures were centrifuged (5000 rpm for 5 min) and washed three times with tap water to remove the residual ammonia, nitrite, nitrate, SOP, and SCOD and then were resuspended in tap water with a final volume of 0.2 L each before being transferred to five reactors. Allyl-N thiourea (a nitrification inhibitor) was added at a concentration of 2 mg/L to each reactor to inhibit the nitrification according to the literature.18 All reactors were then operated according to the method described in the section “Effects of Cu NPs on Biological Nutrient Removal and N2O Generation”. Due to the absence of nitrification in all reactors, the ammonia uptake can be used to calculate the biomass growth via the biomass composition (C5H7O2N).19 Determination of Cu NPs Dissolution in Wastewater. The determination of released Cu2+ from Cu NPs dissolution was conducted according to the method documented in the literature.20,21 The Cu NPs wastewater was maintained in an air-batch shaker (150 rpm) at 21 ± 1 °C. At different times, the samples were withdrawn and centrifuged at 12000 rpm for 30 min, and 0.5 mL of the supernatant was added to 4.5 mL of Milli-Q water (containing 2% ultrahigh purity HNO3). The Cu2+ concentration was then measured. Long-Term Effects of Released Cu2+ from Cu NPs on BNR and N2O Generation. The long-term experiments were conducted with the same method described in the section “Effects of Cu NPs on Biological Nutrient Removal and N2O

generation of N2O in BNR, which is directly related to the activities of some key enzymes, such as NO reductase and N2O reductase, has been reported to be affected by the operational parameters and environmental conditions, such as wastewater feed mode,11 dissolved oxygen (DO) concentration,12 nitrite oxidizing bacteria supplement,13 Cu2+ concentration,14 and wastewater composition.15 Cu NPs in wastewater might bring a potential impact on N2O generation during wastewater BNR. Up to now, the influence of Cu NPs on N2O generation in biological nitrogen and phosphorus removal process, however, has never been documented. The aim of this study was to investigate the effects of Cu NPs on wastewater BNR and N2O generation in the anaerobic-low DO aerobic process, which was proven to save oxygen supply and achieve high BNR concurrently.16 First, the mass balance of Cu NPs in wastewater treatment process was evaluated, and most of the influent Cu NPs was observed to be transformed into activated sludge. Then, the effects of Cu NPs ranging from 0.1 to 10 mg/L on biological nitrogen and phosphorus removal as well as N2O generation were investigated. Finally, the mechanisms of Cu NPs affecting biological nutrient removal and N2O generation were explored from the aspects of the integrity of activated sludge surface assayed by scanning electron microscopy (SEM) and lactate dehydrogenase (LDH) release, the transformations of metabolic intermediates, the activities of key enzymes, and the abundances of major microorganisms by quantitative real-time polymerase chain reaction (PCR) analysis.

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MATERIALS AND METHODS Preparation of Cu NPs Suspension. Commercially produced Cu NPs were purchased from Sigma-Aldrich (St. Louis, MO). X-ray diffraction analysis was conducted using a Rigaku D/Max-RB diffractometer equipped with a rotating anode and a Cu KR radiation source, and the results were shown in Figure S1 (Supporting Information). The specific surface area of Cu NPs was determined to be 14.5 ± 0.6 m2/g via a Micromeritics Tristar 3000 analyzer by nitrogen adsorption at 77 K using the Brunauer−Emmett−Teller method. In this study, 100 mg/L Cu NPs stock solution was produced by adding 0.1 g Cu NPs to 1.0 L Milli-Q water, followed by 1 h of ultrasonication (25 °C, 250 W, 40 kHz) according to the literature.17 The average diameter of the particles in the stock suspension was measured to be 220 ± 25 nm by dynamic light scattering using a Malvern Autosizer 4700 (Malvern Instruments, UK). Wastewater. The wastewater was obtained from the primary sedimentation tank outlet of a WWTP in Shanghai, China. Its characteristics are as follows: total chemical oxygen demand (TCOD) 176−209 mg COD/L, soluble chemical oxygen demand (SCOD) 140−170 mg COD/L, NH4+-N 19− 30 mg/L, TN 28−38 mg/L, soluble orthophosphate (SOP) 2.8−4.8 mg/L, total phosphate (TP) 3.0−5.5 mg/L, pH 7.4− 7.6. The municipal wastewater was supplemented by NH4Cl, KH2PO4, and CH3COONa to get an average initial NH4+-N, SOP, and SCOD of approximately 30, 12, and 350 mg/L, respectively. Long-Term Effects of Cu NPs on Biological Nutrient Removal and N2O Generation. To date, no data are available on the concentration of Cu NPs in actual wastewaters, but some scientists suggest that it is unlikely that Cu NPs concentration entering the WWTPs will be higher than 10 mg/ L.5 Accordingly, a maximum Cu NPs concentration of 10 mg/L 12453

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Table 1. Effects of Cu NPs on TN and TP Removals and N2O Generation during One Cyclea Cu NPs concentration (mg/L) item

0

0.1

1

5

10

TP removal efficiency (%) TN removal efficiency (%) N2O generation (mg N2O−N/mg N removed)b

98.8 ± 1.2 60.6 ± 1.9 0.441 ± 0.011

98.9 ± 1.1 64.1 ± 3.0 0.404 ± 0.021

98.5 ± 1.5 68.9 ± 2.8 0.360 ± 0.035

98.5 ± 1.4 72.8 ± 2.8 0.174 ± 0.017

98.0 ± 1.9 70.6 ± 1.7 0.269 ± 0.031

a

Results are the averages and their standard deviations of triplicate measurements. bThe data are the sum of N2O generated in the liquid and gas phases.

generation, however, was decreased with the increase of Cu NPs in the range of 0−5 mg/L. Although the generated N2O at Cu NPs of 10 mg/L was greater than that at 5 mg/L (0.269 against 0.174 mg N2O−N/mg N removed), it was still much lower than the control (0.269 versus 0.441 mg N2O−N/mg N removed). In the literature, metallic nanoparticles (e.g., TiO2 NPs and ZnO NPs) exposure was found to bring adverse impacts on a biochemical process such as wastewater BNR and waste activated sludge anaerobic digestion.8,9,21 However, it was observed in this study that long-term exposure of Cu NPs did not cause inhibitory effects on biological nitrogen and phosphorus removal but resulted in positive impacts on TN removal and N2O reduction. In the following text the reasons for long-term exposure of Cu NPs showing no influence on TP removal but enhancing TN removal and reducing N2O generation were investigated. Influence of Cu NPs on the Surface Integrity of Activated Sludge. It was reported that the morphological damage caused by nanoparticles resulted in functional harm when the toxic effect of various inhalable metal nanoparticles on human alveolar epithelial cells was studied.25 Previous studies also showed that nanoparticles could cause oxidative stress and thereby might induce oxidative damage to cell membrane.9,26 In this study SEM analysis was first conducted to evaluate whether the surface structure of activated sludge was damaged by the long-term exposure of Cu NPs. As seen in Figure 1, large amounts of cocci-shaped cells were observed in all activated sludge samples of the control and different Cu NPs exposed systems. It seems that the surfaces of cocci-shaped cells were not damaged by Cu NPs exposure at any concentrations investigated. Further LDH release (cell membrane damage marker) assay showed that no measurable cytoplasmic leakage

Generation” except that CuSO4 was used to replace Cu NPs, and the Cu2+ concentration was 0.002, 0.012, 0.094, and 0.176 mg/L, respectively, according to the results of kinetics studies of Cu NPs dissolution performed above. After operation for 85 d, nitrogen and phosphorus removals as well as N2O generation in all reactors reached relatively stable, and then the data were reported. Analytical Methods. The analyses of COD, NH4+-N, NO3−-N, NO2−-N, TN, SOP, TP, and volatile suspended solid (VSS) were conducted in accordance with standard methods.22 The measurements of sludge glycogen, poly-3-hydroxybutyrate (PHB), poly-3-hydroxyvalerate (PHV), and poly-3-hydroxy-2methylvalerate (PH2MV) were the same as described in our previous publication.23 The total polyhydroxyalkanoates (PHA) was calculated as the sum of measured PHB, PHV, and PH2MV. Copper ion was detected by ICP-MS (Agilent Technologies 7700 series). N2O concentration in gas and liquid phases was measured by the microsensors (Unisense, Denmark), and the N2O emission rate was calculated according to the reported method.24 The measurements of ammonia monooxygenase (AMO), nitrite oxidoreductase (NOR), nitrate reductase (NR), nitrite reductase (NIR), NO reductase, N2O reductase, exopolyphosphatase (PPX), and polyphosphate kinase (PPK) activities were reported in our previous publications.8,14 The SEM analysis, LDH release assay, and quantitative real-time PCR are detailed in the Supporting Information. Statistical Analysis. All experiments were performed in triplicate. An analysis of variance was used to evaluate the significance of results, and p < 0.05 was considered to be statistically significant.

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RESULTS AND DISCUSSION Effects of Cu NPs on Wastewater BNR and N2O Generation. In this study the Cu2+ concentration in the influent was nondetectable. According to the effluent copper level the removal of influent Cu NPs can be estimated. The results showed that more than 93% of the influent Cu NPs was intercepted in the reactors no matter what the influent Cu NPs concentration was (Table S1, Supporting Information), which clearly indicated that most of the influent Cu NPs was transformed into activated sludge during wastewater treatment. Thus, it was necessary to investigate their influences on the performance of BNR. As seen in Table 1, the average TP removal efficiencies at any Cu NPs concentration investigated were around 98%, which indicated that the influences of Cu NPs on phosphorus removal and the activities of PAO were negligible. However, the TN removal was significantly affected by Cu NPs concentration (p < 0.05). With the increase of Cu NPs from 0 to 5 mg/L, the average TN removal efficiency was gradually improved from 60.6% to 72.8%, and further increase of Cu NPs to 10 mg/L caused a slight decrease of TN removal to 70.6%. The N2O

Figure 1. SEM images of activated sludge exposed to different concentrations of Cu NPs. 0.1 mg/L (A), 1 mg/L (B), 5 mg/L (C), and 10 mg/L (D) of Cu NPs. 12454

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Figure 2. The kinetics of Cu NPs dissolution (a) and the impacts of released Cu2+ on the removals of TN and TP and the generation of N2O (b). Error bars represent standard deviations of triplicate determinations.

Figure 3. Effects of Cu NPs on the variations of (a) NH4+-N (black) and NO3−-N (white) and (b) TP (black) and NO2−-N (white) during one cycle after long-term exposure to different Cu NPs concentrations. Error bars represent standard deviations of triplicate measurements.

TN removal efficiency and N2O generation were respectively 63.2% and 0.402 mg/g, 69.4% and 0.346 mg/g, 71.2% and 0.152 mg/g, and 70.2% and 0.168 mg/g, while those were 64.1% and 0.404 mg/g, 68.9% and 0.360 mg/g, 72.8% and 0.174 mg/g, and 70.6% and 0.269 mg/g at Cu NPs of 0.1, 1, 5, and 10 mg/L (see Table 1). It can be easily seen that the released Cu2+ was mainly responsible for the positive effects of Cu NPs on TN removal and N2O reduction. It is known that copper containing nitrite reductase (CuNIR) is one of the enzymes catalyzing the biochemical transformation from NO2− to NO,29 and N2O reductase is the enzyme catalyzing the final step of bacterial denitrification (i.e., reducing N2O to N2).30 In general, CuNIR folds a homotrimeric structure with two distinct Cu-binding sites,29 and N2O reductase is a catalytic site called CuZ in its crystal structure, which comprises four copper ions.31 It was reported that the lack of Cu2+ caused an inhibitory effect on the denitrifying process, resulting in the accumulation of N2O.32 Accordingly, a pertinent release of Cu2+ from Cu NPs dissolution might activate CuNIR and N2O reductase, which thereby benefited the removal of TN and the reduction of N2O generation. In the literature, some studies also found that the influences of ZnO NPs on bacteria respiration and function were mostly due to the released zinc ion.8,21 Influences of Cu NPs on the Transformations of Nitrogen, Phosphorus, and Metabolic Intermediates during One Cycle. From Figure 3 it can be observed that the anaerobic and aerobic transformations of TP were very

took place after long-term exposure to 0.1, 1, 5, or 10 mg/L of Cu NPs (Figure S4, Supporting Information). The cell surface integrity of activated sludge in Cu NPs exposed systems might be attributed to the protective role of sludge extracellular polymeric substances.27 Influence of Cu2+ Released from Cu NPs on BNR and N2O Generation. Some studies suggested that one reason for the influence of Cu NPs on microorganisms was due to the release of Cu2+ from Cu NPs dissolution.28 The kinetics determination of Cu NPs dissolution in this study displayed that the average concentrations of the released Cu2+ were, respectively, 0.002, 0.012, 0.094, and 0.176 mg/L at Cu NPs concentrations of 0.1, 1, 5, and 10 mg/L (Figure 2a). The potential effects of the released Cu2+ on TN and TP removals and N2O generation are shown in Figure 2b. The TP removal was not affected significantly by the released Cu2+, but the TN removal and N2O generation were influenced. The improved TN removal and decreased N2O generation were observed with the increase of Cu2+ from 0.002 to 0.094 mg/L. The highest TN removal efficiency (71.2 ± 5.8%) and the lowest N2O generation (0.152 ± 0.03 mg/mg N removed) appeared at 0.094 mg/L of Cu2+. However, the TN removal efficiency and the N2O generation was, respectively, slightly decreased to 70.2 ± 3.7% and increased to 0.168 ± 0.04 mg/mg N removed when the Cu2+ concentration was further increased to 0.176 mg/L. At the released Cu2+ concentration of 0.002, 0.012, 0.094, and 0.176 mg/L the data in Figure 2b showed that the average 12455

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Figure 4. Effects of Cu NPs on the transformations of (a) PHA and (b) glycogen during one cycle after long-term exposure to different Cu NPs concentrations. Error bars represent standard deviations of triplicate measurements.

Table 2. Activities of the Key Enzymes Related to BNR at Different Cu NPs Concentrationsa Cu NPs concentration (mg/L) item AMOb NORb PPXc PPKd NARe NIRe NO reductase (N1)e N2O reductase (N2)e N1/N2

0 0.021 0.086 0.021 0.285 0.451 0.288 0.224 0.046 4.870

± ± ± ± ± ± ± ± ±

0.1 0.003 0.004 0.002 0.012 0.005 0.011 0.009 0.002 0.007

0.020 0.084 0.022 0.286 0.463 0.316 0.251 0.055 4.564

± ± ± ± ± ± ± ± ±

1 0.002 0.003 0.003 0.015 0.003 0.013 0.010 0.005 0.009

0.019 0.085 0.019 0.289 0.485 0.402 0.269 0.060 4.483

± ± ± ± ± ± ± ± ±

5 0.002 0.005 0.002 0.012 0.004 0.021 0.012 0.005 0.010

0.023 0.088 0.020 0.287 0.462 0.550 0.275 0.086 3.198

± ± ± ± ± ± ± ± ±

10 0.003 0.005 0.003 0.014 0.003 0.025 0.015 0.006 0.007

0.022 0.086 0.023 0.286 0.453 0.520 0.282 0.065 4.338

± ± ± ± ± ± ± ± ±

0.002 0.004 0.002 0.015 0.005 0.023 0.013 0.003 0.005

The data are the averages and their standard deviations of triplicate measurements. bThe unit is μmol NO2−-N/(min·mg protein). cThe unit is μmol pnitrophenol/(min·mg protein). dThe unit is μmol NADPH/ (min·mg protein). eThe unit is mg N/ (min·g VSS).

a

similar among five SBRs, which was in correspondence with almost the same TP removal in all reactors. Nevertheless, there were some differences in the variations of NO2−-N and NO3−N in the low DO phase. For example, at the end of the low DO stage, the NO2−-N and NO3−-N were, respectively, 6.83 ± 0.36 and 5.55 ± 0.06, 6.01 ± 0.30 and 4.94 ± 0.05, 4.78 ± 0.35 and 4.63 ± 0.05, 3.98 ± 0.24 and 4.17 ± 0.04, and 4.48 ± 0.21 and 4.40 ± 0.04 mg/L at Cu NPs of 0, 0.1, 1, 5, and 10 mg/L. Obviously, NOx−-N (especially NO2−-N) concentrations at the end of the low DO stage were decreased with influent Cu NPs increase. Also, the data in Figure 3 revealed that there were no obvious nitrification and denitrification that occurred in the anaerobic stage, whereas in the low DO stage both nitrification and denitrification occurred. From the variations of NH4+-N in the low DO stage it can be seen that the influence of Cu NPs on nitrification was insignificant (p > 0.05). Thus, the influence of Cu NPs on TN removal and N2O generation mainly occurred in the denitrification reaction of the low DO stage. It has been reported that many microorganisms (including PAO and glycogen accumulating organisms (GAO)) in activated sludge systems are capable of denitrification.33,34 Also, both PAO and GAO have the ability to synthesize PHA from wastewater−carbon source under anaerobic condition, and some PAO and GAO have been found to be able to use the intracellular PHA as the electron donor for denitrification.33 In this study most (>98%) of the influent biological oxygen demand in all reactors was consumed, and PHA synthesis and glycogen degradation were observed during the anaerobic period. In the followed low DO stage PHA was oxidized for

phosphorus uptake, denitrification, cell growth, and glycogen replenishment. Thus, in the low DO stage if less PHA were consumed for cell growth or phosphorus removal or glycogen synthesis, more PHA would be left for denitrification, and the TN removal efficiency would be increased. As seen from Figures 3b and 4a there were no significant differences in the anaerobic and aerobic transformations of phosphorus and PHA among five SBRs (p > 0.05). In addition, the cell growth was not found to be significantly affected by Cu NPs (Table S2, Supporting Information). The transformations of glycogen in the low-DO stage of the five SBRs, however, were different. The average glycogen synthesis in the low DO stage was 1.32 mmol-C/g VSS in the control SBR, which was, respectively, decreased to 1.23, 1.13, 0.99, and 0.95 mmol-C/g VSS with the increase of Cu NPs to 0.1, 1, 5, and 10 mg/L. Accordingly, less PHA was consumed for glycogen replenishment in the presence of Cu NPs, and more PHA was used for denitrification during the low DO stage, which might be one reason for the Cu NPs exposed reactors showing higher TN removal than the control. In addition, it has been reported that the transformation of glycogen is mainly associated with the activities of GAO, and N2O is the main denitrification product of GAO.34,35 Usually, a lower transformation of glycogen is associated with a lower metabolism of GAO.6,7 In the current study the exposure of Cu NPs decreased the transformation of glycogen, indicating that the activity of GAO was suppressed, and thus the generation of N2O was reduced. Effect of Cu NPs on Key Enzymes and Microbes. The performance of biological nitrogen and phosphorus removal is 12456

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directly related to the activities of some key enzymes. AMO and NOR are two key enzymes in nitrification, and denitrification is relevant to NAR, NIR, NO reductase, and N2O reductase, whereas phosphorus removal is directly related to the activity of PPX and PPK (Figure S5, Supporting Information). As shown in Table 2, the influences of Cu NPs on the activities of AMO, NOR, PPX, and PPK were insignificant (see Table S3 for detailed statistical analysis, Supporting Information), which agreed with the observations of both nitrification and phosphorus removal being unaffected by Cu NPs (Table 1 and Figure 3). However, the presence of Cu NPs significantly increased the activity of NAR, which reduced NO3−-N to NO2−-N, and the activity of NIR, bioconverting NO2−-N to NO, was also improved by Cu NPs (Table S3). Therefore, the accumulations of NO3−-N and NO2−-N in the reactors of Cu NPs were lower than those in the control reactor (Figures 3a and 3b), and higher TN removals were observed (Table 1). Some researchers reported that the levels of nitrite would significantly influence N2O accumulation in the biological nitrogen removal process, because the denitrifying reaction of N2O to N2 was readily inhibited by the toxicity of nitrite or HNO2 formed from nitrite.36 Furthermore, nitrite accumulation was also recognized as the reason for denitrification by the autotrophic nitrifier, the end product of which was N2O.37 As seen in Figure 3b, nitrite accumulation in all Cu NPs exposed reactors was obviously lower than that in the control reactor. Hence, the reduction of nitrite accumulation in the Cu NPs exposed systems was another important reason for lower N2O generation. Additionally, the level of N2O accumulation was reported to be relevant to the ratio of NO reductase activity/N2O reductase activity (N1/N2).38,39 The data in Table 2 showed that the Cu NPs exposed SBRs had a lower ratio of N1/N2 than the control, which consisted with the lower N2O generation. The biological reduction of nitrite ion to gaseous nitrogen monoxide (NO2− → NO) is catalyzed by two different enzymes called haem cd1-containing nitrite reductase (cd1NIR) and CuNIR. NirS and nirK are their respective product genes.29 Moreover, the nosZ gene plays a crucial role in encoding N2O reductase which can catalyze the reduction of N2O to N2.30 The densities of the copies of three genes (nirS and nirK, and nosZ) have been found to be correlated with the abundance of denitrifying bacteria,40 and the nosZ gene copy has been used to represent the abundance of N2O reducing bacteria.41 Therefore, quantitative real-time PCR assay targeting these three genes and the nosZ gene can be used to measure the abundance of denitrifying bacteria and the bacteria capable of reducing N2O to N2, respectively. In this study the average densities of nirS, nirK, and nosZ gene copies in the control reactor were respectively 6.01 × 106, 1.24 × 106, and 7.54 × 106 copies/g VSS, which were 8.98 × 106, 1.59 × 106, and 1.21 × 107 copies/g VSS at Cu NPs of 5 mg/L (Table S4, Supporting Information), indicating that the numbers of denitrifiers and N2O reducing cells were increased by long-term exposure to Cu NPs. Consequently, it can be easily understood that the presence of Cu NPs improved TN removal and reduced N2O generation.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86-21-65981263. Fax: +86-21-65986313. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was financially supported by the project of National Hi-Tech Research and Development Program (863) (2011AA060903), the Foundation of State Key Laboratory of Pollution Control and Resource Reuse (PCRRK09002), Shanghai Postdoctoral Scientific Program (12R21415700), and China Postdoctoral Science Foundation (2012M510888).

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Additional analytical methods, Tables S1−S4, and Figures S1− S5. This material is available free of charge via the Internet at http://pubs.acs.org. 12457

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Environmental Science & Technology

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