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Long-Term Effects of Titanium Dioxide Nanoparticles on Nitrogen and Phosphorus Removal from Wastewater and Bacterial Community Shift in Activated Sludge Xiong Zheng, Yinguang Chen,* and Rui Wu 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: The expanding use of titanium dioxide nanoparticles (TiO2 NPs) in a wide range of fields raises concerns about their potential environmental impacts. However, investigations of the potential effects of TiO2 NPs on biological nitrogen and phosphorus removal and bacterial community in activated sludge are sparse. This study evaluated the influences of TiO2 NPs on biological nutrient removal in the anaerobic-low dissolved oxygen (0.150.50 mg/L) sequencing batch reactor. It was found that 1 and 50 mg/L TiO2 NPs had no acute effects on wastewater nitrogen and phosphorus removal after shortterm exposure (1 day). However, 50 mg/L TiO2 NPs (higher than its environmentally relevant concentration) was observed to significantly decrease total nitrogen (TN) removal efficiency from 80.3% to 24.4% after long-term exposure (70 days), whereas biological phosphorus removal was unaffected. Denaturing gradient gel electrophoresis profiles showed that 50 mg/ L TiO2 NPs obviously reduced the diversity of microbial community in activated sludge, and fluorescence in situ hybridization analysis indicated that the abundance of nitrifying bacteria, especially ammonia-oxidizing bacteria, was highly decreased after long-term exposure to 50 mg/L TiO2 NPs, which was the main reason for the serious deterioration of ammonia oxidation. Further study revealed that 50 mg/L TiO2 NPs inhibited the activities of ammonia monooxygenase and nitrite oxidoreductase after long-term exposure, but had no significant impacts on the activities of exopolyphosphatase and polyphosphate kinase, and the transformations of intracellular polyhydroxyalkanoates and glycogen, which were consistent with the observed influences of TiO2 NPs on biological nitrogen and phosphorus removal.
’ INTRODUCTION Nanomaterials are increasingly used to produce large amounts of industrial and consumer products due to their novel physical and chemical properties.1 Particularly, titanium dioxide nanoparticles (TiO2 NPs) are widely applied in catalysts, sunscreens, cosmetics and wastewater treatment processes.2 These extensive applications of TiO2 NPs inevitably lead to their environmental release. Recently, TiO2 NPs have been found in soils, surface waters, wastewaters and sewage sludge,3,4 which raises concerns about their potential environmental impacts. Although the current predicted environmental concentration of TiO2 NPs is at μg/L level,4,5 their release to the environment may continuously increase due to their large-scale production. As a result, many studies have been conducted to investigate the toxicity of TiO2 NPs to model organisms such as human cells,6 zebrafish,7 marine phytoplankton8 and microorganisms9 at mg/L level. For instance, 0.14 mg/L Ag NPs was found to cause 50% inhibition to respiration of nitrifying bacteria,10 but no inhibitory effect on respiration of ammonia-oxidizing bacteria was observed at Cu r 2011 American Chemical Society
NPs concentration of 10 mg/L.11 Previous works mainly focus on the influence of NPs on bacterial respiration. Recent study indicates that ZnO NPs can lead to the acute effect on activated sludge,12 but for other NPs little information is available on their effects on microorganisms in activated sludge. Although it is found that some raw sewage contained 0.1 to nearly 3 mg Ti/L,13 the potential impacts of TiO2 NPs on nitrogen and phosphorus removal from wastewater are still unknown. Current knowledge of the toxicity of TiO2 NPs largely comes from studies of model organisms. Simon-Deckers et al. observed that TiO2 NPs had no effect on cell viability of Cupriavidus metallidurans up to 500 mg/L after 24 h of exposure,14 and the growth of Bacillus subtilis was also unaffected by 500 mg/L TiO2 NPs for 6 h.9 Nevertheless, these data were obtained from the Received: March 15, 2011 Accepted: July 22, 2011 Revised: June 29, 2011 Published: July 22, 2011 7284
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Environmental Science & Technology short-term exposure experiments (usually 124 h), and thus only showed the acute effects of TiO2 NPs on cell viability and growth of model bacteria. Prolonged exposure (3 months) to TiO2 NPs was found to induce the adverse effects on human keratinocyte cells, such as the decreased mitochondrial activity and loss of normal cell morphology, although the equal concentration of TiO2 NPs was not toxic to keratinocytes after shortterm exposure.6 It seems that only considering the acute effect of NPs is insufficient to assess their potential environmental risks. To comprehensively evaluate the potential impacts of TiO2 NPs on biological nitrogen and phosphorus removal, both short-term and long-term effects of TiO2 NPs should be investigated. It is well-known that wastewater nitrogen and phosphorus removal with an activated sludge process can be achieved by complex microbial populations responsible for nitrification, denitrification, and phosphorus anaerobic release and aerobic or anoxic uptake.15,16 Therefore, the diversity of microbial populations and the stable bacterial community structure play important roles in achieving high efficiency of biological nitrogen and phosphorus removal. However, to date, whether the presence of TiO2 NPs can affect bacterial community in activated sludge after long-term exposure is still unknown. The aims of this study were (1) to evaluate the effects of TiO2 NPs on nitrogen and phosphorus removal from wastewater, (2) to reveal bacterial community in activated sludge via polymerase chain reaction-denaturing gradient gel electrophoresis (PCRDGGE) analysis after long-term exposure to TiO2 NPs, and (3) to explore the influences of TiO2 NPs on the transformations of intracellular polyhydroxyalkanoates (PHA) and glycogen, and the activities of some key enzymes associated with biological nutrient removal, such as ammonia monooxygenase (AMO), nitrite oxidoreductase (NOR), nitrate reductase (NAR), nitrite reductase (NIR), exopolyphosphatase (PPX), and polyphosphate kinase (PPK). This work used an anaerobic-low dissolved oxygen (DO: 0.150.50 mg/L) wastewater treatment process to achieve biological nutrient removal, because this technology was expected to save energy of oxygen supply and concurrently achieve high biological nutrient removal.16
’ MATERIALS AND METHODS Preparation of Nanoparticle Suspension. Commercially available TiO2 NPs (Sigma) were used in this study, which were confirmed to be pure anatase by X-ray diffraction (XRD) analysis via a Rigaku D/Max-RB diffractometer equipped with a rotating anode and a Cu KR radiation source (Figure S1, Supporting Information (SI)). Specific surface area of TiO2 NPs was measured to be 106 ( 8 m2/g via a Micromeritics Tristar 3000 analyzer by nitrogen adsorption at 77 K using the Brunauer EmmettTeller method.17 To produce 100 mg/L NP stock suspension, 100 mg of TiO2 NPs was dispersed into 1 L of MilliQ water by sonication for 1 h (25 °C, 250 W, 40 kHz) according to the literature.18 Although the particle size of TiO2 NPs reported by Sigma-Aldrich was less than 25 nm, the primary size of particles in stock suspension was determined to be in the range of 7090 nm by dynamic light scattering (DLS) using a Malvern Autosizer 4700 (Malvern Instruments, UK). Set-Up and Long-Term Operation of Sequencing Batch Reactors (SBRs). In this study, 1 mg/L was chosen as the environmentally relevant concentration of TiO2 NPs, and the potential effect of 50 mg/L TiO2 NPs was also investigated, because the environmental release of TiO2 NPs might continuously increase
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due to their large-scale production.4,13 To conduct the experiment, three SBRs were prepared to contain 1 L of synthetic wastewater and 1 L of inoculated sludge obtained from a parent SBR which had been operated over 100 days and achieved the stable biological nutrient removal (approximately 80% and 99% of nitrogen and phosphorus removal). Afterward, SBR 1 and SBR 2 were fed with 40 and 2000 mL of TiO2 NPs stock suspension (100 mg/L), respectively, and SBR 3 was operated as the control (i.e., without TiO2 NPs addition). Finally, deionized (DI) water was added to make the working volume of each SBR to be 4 L. Each SBR was set up in triplicate, covered with aluminum foil to avoid the possible light-induced effects, maintained at 21 ( 1 °C, and worked with three 8 h cycles per day. Each cycle consisted of 1.5 h anaerobic and 3 h low DO periods, followed by 1 h settling, 10 min decanting and 140 min idle periods. The operational time was recorded since TiO2 NPs were added. Because the concentrations of TiO2 NPs in SBR 1 and SBR 2 might slowly decrease due to the discharge of effluent and sludge, a certain amount of TiO2 NPs were supplemented every 2 days to recover to the initial concentrations after determining the total concentration of TiO2 in each reactor. In the first 15 min of each cycle, the SBRs were fed with 3 L of synthetic wastewater (containing 1.1 mL of acetic acid, 2.9 mL of “N-water”, 1.4 mL of “P-water”, 10 mL of “concentrated solution” and 2 mL of “trace-element solution”) to result in the initial concentrations of chemical oxygen demand (COD), ammonianitrogen (NH4+-N) and soluble ortho-phosphorus (SOP) of approximately 300, 25, and 10 mg/L, respectively. The compositions of “N-water”, “P-water”, “concentrated solution” and “trace-element solution” are detailed in the SI. 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. Before the end of low DO stage, sludge was wasted to keep the solids retention time (SRT) at approximately 22 days. After the settling period 3 L of supernatant was discharged. All SBRs were constantly mixed with a magnetic stirrer except for the settling, decanting and idle periods. The effluent concentrations of NH4+-N, nitrite-nitrogen (NO2-N), nitrate-nitrogen (NO3-N) and SOP in all SBRs were frequently determined until the nitrogen and phosphorus removal reached relatively stable (approximately 70 days). Investigations of Nitrogen and Phosphorus Transformations during One Cycle after Short-Term and Long-Term Exposure. The experiments were conducted on day 1 (i.e., shortterm exposure) and day 70 (i.e., long-term exposure), respectively, to evaluate the short-term and long-term effects of TiO2 NPs on transformations of nitrogen and phosphorus. Activated sludge in SBR 1, SBR 2 and SBR3 was washed 3 times with 0.9% NaCl solution before being resuspended in 4 L of synthetic wastewater, which made the same influent nitrogen and phosphorus in each SBR. All other operational conditions were the same as those described in the Set-Up and Long-Term Operation of Sequencing Batch Reactors (SBRs) section. The changes of NH4+-N, NO2-N, NO3-N, SOP, PHA, glycogen and the activities of AMO, NOR, NAR, NIR, PPX, and PPK were measured during the anaerobic and low DO stages. PCR-DGGE Analysis of Bacterial Community in Activated Sludge. Bacterial genomic DNA of activated sludge was first extracted according to our previous publication.19 Briefly, 2 mL of mixture was centrifuged, washed three times with STET buffer (8% Sucrose, 5% Triton X-100, 50 mM EDTA and 50 mM Tris, pH 8.0), and resuspended in 360 μL of STET buffer. After adding 7285
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Figure 1. Effects of exposure time on the effluent concentrations of (A) NH4+-N (empty symbols) and NO3-N (full symbols), and (B) NO2-N (empty symbols) and SOP (full symbols) at different concentrations of TiO2 NPs. All the standard deviations of triplicate measurements are less than 21%.
40 μL of lysozyme (50 mg/mL) followed by 10 min of incubation at 37 °C, 20 μL of 10% sodium dodecyl sulfate (SDS) and 2 μL of proteinase K (20 mg/mL) were added to the mixture which was then incubated at 37 °C for 60 min. After addition of 50 μL of 5 M NaCl and 50 μL of 10% cetyltrimethylammonium bromide (CTAB) and subsequent incubation at 65 °C for 10 min, 0.5 mL of Tris-saturated phenol, 0.5 mL of phenol-chloroform-isoamyl alcohol (25:24:1) and 0.5 mL of chloroformisoamyl alcohol (24:1) were used to extract DNA, respectively. Then, 0.5 mL of 3 M sodium acetate (pH 5.2) and 1 mL of ethanol were used to precipitate DNA for 1 h at 4 °C, followed by 10 min of centrifugation at 12 000g. The pellets were washed with 500 μL of 70% ethanol, and finally resuspended in 50 μL of elution buffer. The extracted DNA was checked by 1% agarose electrophoresis using ethidium bromide (EB) as staining dye, and then used as the template DNA for PCR amplification. The 16S rDNA variable V3 region of extracted DNA was amplified with primers 341f with a GC-clamp (50 -CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGCCTACGGGAGGCAGCAG-30 ) and 534r (50 -ATTACCGCGGCTGCTGG-30 ) according to the literature.20 PCR amplification was carried out in a total volume of 25 μL containing 10 ng of template DNA, 1 Ex Taq reaction buffer, 1 U Ex Taq polymerase, 1.5 mM MgCl2, 0.2 mM dNTPs and 0.5 μM primers (TaKaRa, Japan) using an Eppendorf Mastercycler Gradient thermocycler (Eppendorf, Germany). The amplification program consisted of an initial denaturation step at 94 °C for 5 min, 30 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s and extension at 72 °C for 30 s, followed by a final extension at 72 °C for 10 min. PCR products were electrophoresed on 8% polyacrylamide gel in 1 TAE buffer with gradients ranged from 30% to 60% denaturant (100% denaturant: 7 M urea and 40% (v/v) deionized formamide) at a constant voltage of 80 V for 15 h at 60 °C using a DCode Universal Mutation Detection System (BioRad). The gel was stained with EB for 15 min and viewed with a BioRad Gel Documentation system (BioRad). Prominent bands were then excised from the gel, and after cleanup treatment the recovered DNA was reamplified (initial denaturation at 94 °C for 5 min, 30 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s and extension at 72 °C for 30 s, followed by a final extension at 72 °C for 10 min), purified, cloned to
pMD19-T vector (TaKaRa, Japan) and sequenced via an ABI PRISM 3730 automated DNA sequencer (Applied Biosystems, USA). The sequences from this study have been submitted to the GenBank database under accession numbers JF449961 to JF449971, and the closest matching sequences were searched using the BLAST program. Analytical Methods. The determinations of NH4+-N, NO2N, NO3-N, TN (total nitrogen), SOP, mixed liquor suspended solids (MLSS) and mixed liquor volatile suspended solids (MLVSS) were conducted in accordance with the Standard Methods.21 PHA (including polyhydroxybutyrate (PHB), ployhydroxyvalerate (PHV) and polyhydroxy-2-methylvalerate (PH2MV)) and glycogen were measured according to our previous publication.19 The analyses of AMO, NOR, NAR, NIR, PPX, PPK, TiO2 NPs, TiO2 NPs dissolution, lactate dehydrogenase (LDH) release, fluorescence in situ hybridization (FISH) and scanning electron microscopy (SEM) are detailed in the SI. Statistical Analysis. All tests were performed in triplicate, and 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 TiO2 NPs on Biological Nitrogen and Phosphorus Removal from Wastewater. As seen from Figure 1,
the effluent concentrations of NH4+-N, NO2-N, NO3-N and SOP in the presence of 1 mg/L TiO2 NPs (SBR 1) were relatively stable with increasing exposure time and similar to those in the absence of TiO2 NPs (the control SBR) over a period of 70 days. The average TN removal efficiencies of SBR 1 and the control SBR were 79.4% and 80.3%, respectively, which were not statistically different, and both SBRs displayed >99% phosphorus removal (Figure 1B). These results indicated that the current environmentally relevant concentration of TiO2 NPs (1 mg/L) had no adverse effects on nitrogen and phosphorus removal. However, when activated sludge was exposed to 50 mg/L TiO2 NPs in SBR 2, the effluent NH4+-N significantly increased from nondetectable to approximately 17.5 mg/L with increasing exposure time to 16 days (Figure 1A). After 70 days of exposure, the effluent NO2-N and NO3-N in SBR 2 were
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Figure 2. Variations of (A) NH4+-N, (B) NO2-N, (C) NO3-N and (D) SOP during one cycle after short-term (empty symbols) and long-term (full symbols) exposure to different concentrations of TiO2 NPs. All the standard deviations of triplicate measurements are less than 10%.
around 0.68 and 0.73 mg/L, respectively. The TN removal efficiency of SBR 2 was 24.4%, which was remarkably lower than that in the control (80.3%). Nevertheless, almost all phosphorus was removed in SBR 2 (Figure 1B), suggesting that 50 mg/L TiO2 NPs did not influence wastewater phosphorus removal in long-term exposure time. The influences of TiO2 NPs on the transformations of NH4+N, NO2-N, NO3-N, and SOP under the anaerobic and low DO conditions were further investigated after short-term and long-term exposure, respectively. As seen in Figure 2, after shortterm exposure (day 1) the variations of NH4+-N, NO2-N, NO3-N and SOP showed no significant differences among TiO2 NPs concentrations of 0, 1, and 50 mg/L (p > 0.05), indicating that the presence of 1 and 50 mg/L TiO2 NPs had no acute effect on nitrogen and phosphorus removal. Similarly, it was reported in the literature that 100 mg/L TiO2 NPs did not cause acute toxicity to human keratinocyte cells.6 Other toxicological studies also pointed out that 500 mg/L TiO2 NPs was not toxic to the viability and growth of C. metallidurans and B. subtilis after 6 h of exposure.9,14 After long-term exposure (day 70), compared with the absence of TiO2 NPs, the presence of 1 mg/L TiO2 NPs also did not influence the variations of NH4+-N, NO2-N, NO3-N and SOP in both anaerobic and low DO stages (Figure 2). However, it was found that 50 mg/L TiO2 NPs caused the serious inhibition to ammonia oxidation in the low DO stage after long-term exposure (Figure 2A). The average removal efficiency of NH4+-N was
Figure 3. DGGE profile of bacterial communities in two SBRs after long-term exposure. L1 and L2 represent activated sludge in SBR 2 (with 50 mg/L TiO2 NPs addition) and the control SBR (without TiO2 NPs addition) (L2), respectively. Detailed information on the bands 111 is presented in Table 1.
observed to be 30.8%, which was significantly lower than that in the control SBR (>99%). The corresponding effluent NO3-N highly decreased from 5.5 to 0.8 mg/L due to the deterioration of ammonia oxidation. Nevertheless, the transformations of anaerobic 7287
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Table 1. DGGE Bands and Their Closely Related Sequence band ID
closely related sequence from GenBank
accession no.
identity (%)
1
uncultured Bacteroidetes bacterium clone QEDN4BF06
CU927760.1
99
2
Comamonas sp. MQ
HQ176414.1
100
3
uncultured Nitrosomonas sp. clone Z11
GU247146.1
98
4
Stenotrophomonas sp. enrichment culture clone ECC31
GU056297.1
100
5
uncultured Candidatus Accumulibacter sp. clone EMB clone_7
HM046420.1
98
6
uncultured Thiothrix sp. clone H2SRC116
FM165207.1
98
7
uncultured Actinobacterium clone CB01C05
EF471482.1
99
8 9
uncultured Candidatus Accumulibacter sp. clone EMB clone_4 Aeromonas sp. MDC2508
HM046417.1 HQ436040.1
98 100
10
uncultured Rhodocyclaceae bacterium clone R11
AM268362.1
100
11
uncultured Nitrospira sp. clone 6
HQ424533.1
99
phosphorus release, low DO phosphorus uptake and phosphorus removal were unaffected by the presence of 50 mg/L TiO2 NPs (Figure 2D). Bacterial Community Shift in Activated Sludge after Long-Term Exposure to TiO2 NPs. In the literature, the inhibitory effects of some nanomaterials such as ZnO NPs were attributed to the release of metal ions.12 However, despite the presence of 50 mg/L of TiO2 NPs, no titanium ions were detected in this study. Consistently, Kiser et al. have also reported that Ti in wastewater is expected to occur solely in solid phases, not in ionic forms, because TiO2 has very low solubility.13 It is well-known that maintaining the diversity of bacterial populations and the stable microbial community is important to achieve successful biological nitrogen and phosphorus removal in wastewater treatment plants.15,20 Therefore, the reason for the serious deterioration of ammonia removal caused by 50 mg/L TiO2 NPs was explored by determining the microbial community via PCRDGGE analysis. It can be seen from Figure 3 that activated sludge in the control SBR showed high bacterial diversity. According to the detailed information on the bands in DGGE profile (Table 1), the control SBR contained the typical ammoniaoxidizing bacteria (AOB) (band 3, related to Nitrosomonas sp.) and nitrite-oxidizing bacteria (NOB) (band 11, related to Nitrospira sp.), and these microorganisms were mainly responsible for the oxidation of ammonia to nitrate.22 Meanwhile, some bacteria were found to be affiliated to Candidatus Accumulibacter phosphatis (bands 5 and 8), Actinobacteria (band 7), and Aeromonas sp. (band 9), which were usually reported to carry out wastewater phosphorus removal.2325 As shown in Figure 3, however, 50 mg/L TiO2 NPs obviously reduced the microbial diversity in activated sludge and caused the shift in bacterial community after long-term exposure. Recently, some researchers observed that TiO2 NPs were able to alter the bacterial composition in soil and reduce microbial populations after 60 days of exposure.26 It should be noted that Nitrosomonas sp. (band 3) was found to be washed out of activated sludge due to the presence of 50 mg/L TiO2 NPs. In previous study, TiO2 NPs were reported to be toxic to the pure culture of N. europaea,27 which might explain the disappearance of Nitrosomonas sp. in activated sludge after long-term exposure to 50 mg/L TiO2 NPs. Interestingly, Stenotrophomonas sp. (band 4) was observed in the presence of 50 mg/L TiO2 NPs, and this microorganism was pointed out to enable to tolerate high levels of metal contaminants and carry out nitrate denitrification.28,29 Additionally, Candidatus Accumulibacter phosphatis (bands 5
Figure 4. Effects of 50 mg/L TiO2 NPs on the relative activities of AMO, NOR, NAR, NIR, PPX and PPK after long-term exposure. Asterisks indicate statistical differences (p < 0.05) from the control SBR. Error bars represent standard deviations of triplicate measurements.
and 8) and Rhodocyclaceae (band 10) were found to be the dominant polyphosphate-accumulating organism (PAO) in the presence of 50 mg/L TiO2 NPs. Furthermore, FISH analysis was carried out to investigate the quantitative changes of nitrifying bacteria (AOB and NOB) and PAO in activated sludge. The abundances of AOB and NOB in the control SBR accounted for 8% and 6% of total biomass, respectively, whereas those in the presence of 50 mg/L TiO2 NPs were only 1% and 3% of total biomass, respectively, after longterm exposure (Figure S2, SI). Compared to other heterotrophs, autotrophic AOB are always expected to grow very slowly and extremely susceptible to a wide variety of inhibitors.30 This study showed that 50 mg/L TiO2 NPs significantly decreased the abundance of nitrifying bacteria, especially the fraction of AOB, after long-term exposure, which might be the main reason for the serious inhibition of ammonia oxidation, thereby reducing the efficiency of nitrogen removal. Meanwhile, FISH results indicated that the abundance of PAO (Candidatus Accumulibacter sp.) accounted for 49% of total biomass after long-term exposure to 50 mg/L TiO2 NPs, which was similar to that in the control SBR (accounted for 46% of total biomass) (Figure S2, SI). Effects of TiO2 NPs on Activities of Key Enzymes and Metabolic Intermediates Related to Biological Nitrogen and Phosphorus Removal after Long-Term Exposure. Biological nitrogen and phosphorus removal from wastewater depend on 7288
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Environmental Science & Technology the successful nitrification, denitrification, and phosphorus release and uptake, and these processes are relevant to the activities of some key enzymes associated with nitrogen and phosphorus removal (Figure S3, SI). Usually, autotrophic AOB uses AMO to catalyze ammonia oxidation, and the subsequent oxidation of nitrite to nitrate is carried out with NOR in NOB.31 Denitrification is mainly catalyzed by NAR and NIR,32 whereas phosphorus transformation is relevant to PPX and PPK.33 As shown in Figure 4, 50 mg/L TiO2 NPs significantly inhibited the activities of AMO and NOR after long-term exposure (p < 0.05), which might be one reason for the serious deterioration of ammonia oxidation. However, it was found that the activities of PPX and PPK were unaffected by long-term exposure to 50 mg/L TiO2 NPs, which was in accordance with the similar phosphorus removal efficiencies in the presence and absence of TiO2 NPs. In biological phosphorus removal systems, it has been reported that hydrolysis of polyphosphate causes SOP release in the anaerobic stage, which is accompanied with PHA synthesis and glycogen consumption.16 In the subsequent low DO stage, the produced PHA is consumed to supply the energy for phosphorus uptake, and glycogen is concurrently replenished. Therefore, wastewater phosphorus removal is relevant to the anaerobic and low DO transformations of intracellular PHA and glycogen. After long-term exposure the anaerobic PHA synthesis and glycogen degradation in the presence of 50 mg/L TiO2 NPs were 3.20 and 2.21 mmol-C/g-MLVSS, respectively, which were similar to those in the control SBR (3.16 and 2.25 mmol-C/gMLVSS). In the low DO stage, the corresponding PHA consumption and glycogen replenishment were 3.19 and 2.18 mmolC/g-MLVSS, respectively, whereas those in the control SBR were 3.13 and 2.29 mmol-C/g-MLVSS, respectively. These results indicated that long-term exposure to 50 mg/L TiO2 NPs did not affect the transformations of intracellular PHA and glycogen in both anaerobic and low DO stages, which was also consistent with no observed influence on biological phosphorus removal. Effect of TiO2 NPs on Surface Integrity of Activated Sludge after Long-Term Exposure. Previous studies have shown that TiO2 NPs may induce oxidative damage to the cell membrane of bacteria9 and human cells.34 However, it was found in this study that the surface structure of activated sludge was not damaged by long-term exposure to 50 mg/L TiO2 NPs via SEM analysis, and the same observation was made at TiO2 NPs concentration of 1 mg/L (Figure S4, SI). Moreover, the LDH assay also showed that no measurable cytoplasmic leakage occurred after long-term exposure to TiO2 NPs (Figure S5, SI), which confirmed the surface integrity of activated sludge. It is well-known that activated sludge contains large amounts of extracellular polymeric substances (EPS), which are typically reported to keep microorganisms together.35 In this study, EPS secreted by activated sludge might protect the surface integrity of activated sludge from being affected by long-term exposure to TiO2 NPs. According to the above investigations, despite no acute influence on nitrogen and phosphorus removal from wastewater, 50 mg/L TiO2 NPs caused the significant inhibition to biological nitrogen removal after long-term exposure due to the serious deterioration of ammonia oxidation. The main reasons for this inhibitory effect were found to be the highly decreased abundance of AOB and the serious inhibition of AMO and NOR activities. However, it was observed that the activities of PPX and PPK related to phosphorus removal and
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the transformations of intracellular PHA and glycogen were unaffected, which was consistent with no measurable impact of TiO2 NPs on biological phosphorus removal. To the best of our knowledge, this is the first study to examine the potential effects of TiO2 NPs on biological nitrogen and phosphorus removal from wastewater and bacterial community shift in activated sludge.
’ ASSOCIATED CONTENT
bS
Supporting Information. Additional analytical methods, Table S1, and Figures S1S5. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Phone: +86 21 65981263; fax: +86 21 65986313; e-mail:
[email protected].
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