Effect of long-term low concentrations of TiO2 nanoparticles on dewaterability of activated sludge and the relevant mechanism: the role of nanoparticle aging

Nanoparticles can undergo aging phenomena in sewage treatment systems, which alter their physical and chemical properties. However, the effect of aged nanoparticles on the dewatering performance of activated sludge under long-term low concentrations is yet to be reported in sewage treatment systems. Here, we compared the chronic effects of pristine and aged TiO2 nanoparticles on the sludge dewatering index, which includes specific resistance to filtration (SRF) and bound water (BW) in a sequencing batch reactor (SBR) at μg/L concentration levels, and analyzed the relevant mechanisms. The results indicated that aging in the sludge supernatant altered the photosensitivity and water stability of nanoparticles, which was mainly due to the changes in the zeta potential and energy band of the particle and was ultimately attributed to the combined effect of particle surface inclusions such as organic matter and inorganic salt. At 10 μg/L, nanoparticles reduced the sludge dewaterability, which observed an improvement at 100 μg/L. This is because 10 μg/L promoted the secretion of extracellular polymeric substances (EPS), which regulated the structure of sludge flora and increased the abundance of secreted quorum sensing-acyl-homoserine lactones (QS-AHL) and EPS genera, while the corresponding exposure results for 100 μg/L were the opposite, owing to the damage and necrosis effects caused by exposure under long-term light, which reduced EPS production and increased sludge density. Interestingly, aging could alleviate the effects of two exposure concentrations on sludge dewatering, mainly because of the decrease in the photoactivity of the nanoparticles. The results of this study show that environmental aging could delay, but not reverse the results of exposure to specific concentrations of nanoparticles. However, the significantly different ecological effects of photosensitive nanoparticles with two environmentally relevant concentration should be refined and confirmed again in freshwater environments to provide a basis for subsequent scientific management and control of photosensitive nanoparticles.


Introduction
TiO 2 nanoparticles (TiO 2 NPs) are widely used in catalysts, sunscreens, cosmetics, coatings, plastics, medicine, and environmental management because of their superior physicochemical properties (Li et al. 2020a). The accelerated application of TiO 2 NPs increases their concentration in wastewater treatment plants (WWTPs). Recently, raw sewage was found to contain approximately 100-3000 μg/L of Ti, and its concentration in sludge was as high as 23.2 mg/kg (Gottschalk et al. 2009). Therefore, it is inevitable that TiO 2 NPs would interact with microbial aggregates, such as activated sludge or biofilms in WWTPs. The WWTPs can reportedly intercept more than 95% of the TiO 2 NPs, and most are adsorbed on the microbial aggregate surface or are infiltrated into the cells (Kiser et al. 2009). This might adversely affect the physical and chemical properties and functional activities of microbial aggregates. Most previous studies were focused on short-term or high-concentration acute exposure experiments. For example, TiO 2 NPs with high-exposure concentrations (1-1000 mg/L) exhibited acute toxicity toward microbial aggregates by inhibiting their activity , reducing their photosynthetic efficiency (Li et al. 2017a), screening and remodeling their microbial community structures (Garcia et al. 2012), and reducing the functional capacity of microbial aggregates, such as nitrogen and phosphorus removal (Li et al. 2014;Li et al. 2017b). However, reports on the long-term low-concentration exposure effect of TiO 2 NPs on the physical and chemical properties of microbial aggregates, such as the dewaterability of activated sludge in WWTPs, are limited. The urgent need to study this practical problem is not only the key in evaluating the response and adaptation mechanism of sewage treatment systems when challenged with the duress of nanoparticles, but also the basis for comprehensively evaluating the ecological risk of TiO 2 NPs in sewage. More realistically, in this study, it is possible to determine the response mechanism of activated sludge and other microbial aggregate dehydration indicators based on the engineering premise, along with mitigation methods or treatment measures when encountering nanoparticles.
The ecological effect of nanomaterials is closely related to their incubation time in environmental media and their corresponding surface characteristic conditions (Nowack et al. 2012). Furthermore, nanoparticles can reportedly undergo aging in environmental media, that is, the transformation of comprehensive characteristics (such as physicochemical properties) after experiencing complex environmental behaviors. Notably, the aging conditions of nanomaterials, such as TiO 2 NPs, and their aging experimental methods in different environmental media, such as soil/sediment or organic matter have been recognized by researchers (Fan et al. 2017;Lei et al. 2016;Wang et al. 2015). For example, Li et al. (2020bLi et al. ( , 2020c reported that TiO 2 NPs could incubate and age in municipal sewage and natural surface waters, respectively, which could restructure its surface properties, ultimately affecting its water stability and photosensitivity. This might significantly impact the aquatic ecological properties. The stability of a particle in water corresponds to the change in particle size; the term nanomaterials is only applicable when the material size is in the nanoscale range and its specific nanoeffects emerge. The surface activity and hydrophilicity of TiO 2 NPs reportedly change with the change in particle size (Wang et al. 1997). In addition, particle size is also a key factor for long-distance transmission or short-distance penetration, which is responsible for the ecological risk of nanomaterials . Previous studies only focused on the toxic effects of pristine nanoparticles (Dwivedi et al. 2015;Qian et al. 2017) but ignored the process effect (such as aging transformation) of nanoparticles in environmental media such as the sewage treatment systems (Dwivedi and Ma 2014). The changes in the characteristics of nanoparticles incubated in the exposure medium for long durations are hardly recognized, and the changes in biological behaviors and responses caused by this particular environmental process are unknown. The leading factors and continuance of the risks of nanoparticles in the exposed system are also unknown.
Furthermore, as a type of photoactive nanomaterial, the photosensitivity of TiO 2 NPs is expected to develop into an important ecological process in aquatic environments (Sun et al. 2014). This not only concerns ecologists about the phototoxicity of TiO 2 NPs but also arouses their thinking about the stability of photocatalytic activity of TiO 2 NPs after their surface structure is modified and functional groups regulated by long-term incubation in the relevant environmental media (Pan et al. 2011;Wang et al. 1997). Reportedly, the surface properties and photosensitivity of TiO 2 NPs could be altered by aging processes in sewage transportation and freshwater environments, mainly due to the encapsulation and passivation of organic compounds and inorganic ions in the corresponding water bodies (Li et al. 2020b(Li et al. , 2020c. However, when TiO 2 NPs collect in WWTPs and are detained there for a long duration, the changes in their surface characteristics and photosensitivity are unknown, which may affect the evaluation of the persistent adverse risk of TiO 2 NPs in wastewater treatment processes.
In view of the above analysis, we systematically elaborated the changes in sludge dewatering performance and EPS secretion in the sequencing batch reactor activated sludge process. The related mechanisms were analyzed in terms of the responses of bacterial cell death modes, quorum sensing and sludge density, and key microbial abundance in activated sludge after long-term exposure to low concentrations of TiO 2 NPs, particularly focusing on the impact of changes in size and photosensitivity of aged TiO 2 NPs to supplement the study on the persistent biological effects of nanoparticles, with relatively real particle states and environmentally relevant concentrations in WWTPs.

TiO 2 NP and reagents
Commercially bare anatase TiO 2 NPs (100 nm, 99.8% purity) were obtained from Shanghai Aladdin Biochemical Technology Co., Ltd. All chemicals and reagents from Aladdin (Shanghai, China) used in this study were of analytical grade, except for the standard products used to represent specific markings, such as proteins and polysaccharides.

Aging experiment of nanoparticles
Two suspensions (100 μg/L) of TiO 2 NPs were prepared by adding 200 μg of the TiO 2 NP each to 2 L (beaker) of Milli-Q water (pH=7 ± 0.1) and filtered aerobic tank suspension (0.45 μm filter membrane) collected from Tiebei WWTPs (Nanjing, China) accompanied by typical municipal water quality, which was followed by 1 h of ultrasonication (20°C /250 W/40 kHz). The hydrodynamic diameters and zeta potential profiles of the TiO 2 NPs in the two stock suspensions were determined on day 0 (dispersion state) using a Malvern Zetasizer Nano ZS90 equipment (Malvern Instruments, UK), and the photosensitivities of TiO 2 NP in the two suspensions were detected using UV-vis spectrophotometry. Thereafter, the two nanoparticle suspensions were stirred (200 rpm) for 300 days under simulated sunlight with an illumination period of 12:12 h (light/dark). The water evaporated in the beaker could be replenished any time with Milli-Q water or filtered aerobic tank sewage. After incubation, the particle size distribution, zeta potential, and photosensitivity of the samples were re-characterized, and the suspensions were centrifuged to discard the supernatant, and the pellet was gently washed with Milli-Q water several times to remove any residual microorganisms on the surface of the aged nanoparticles and finally freeze-dried. The surface properties of the dried TiO 2 NP powder were characterized to explore their remolding after incubation. High-resolution transmission electron microscopy (HRTEM) and X-ray diffraction (XRD) patterns were obtained to determine the morphology, microstructure, and crystalline pattern of TiO 2 NPs. Additionally, the band-gap of aged particles was calculated using UV-visible diffuse reflectance according to a previous study ).

Activated sludge domestication culture and exposure experiments
The activated sludge was collected from a secondary sedimentation tank at the Tiebei WWTP in Nanjing, China. The concentration of titanium (Ti) in sludge matrix is considerably low (0.5± 0.08μg Ti/g VSS); however, after 45 days of acclimation, Ti was virtually undetectable in the sequence batch reactor (SBR), indicating that the sludge was no longer preselected for titanium tolerance. After acclimatization for approximately 45 days, the sludge maintained steady operational performance with an average chemical oxygen demand (COD) removal efficiency of 96.36%. For the exposure experiment, considering the purpose of long-term low concentration and the actual concentration level of TiO 2 NPs in WWTPs (Gottschalk et al. 2009;Kiser et al. 2009), both 10 and 100 μg/L were chosen as the environmentally relevant concentrations of TiO 2 NPs in the WWTPs, with an exposure time of 300 days (chronic exposure). Considering that WWTPs mainly operate outdoors, a simulated solar irradiation with a radiation intensity of 3.6 mW/cm 2 UVA, 0.19 mW/ cm 2 UVB, and 75 μE/m 2 s photosynthetically active radiation, representing the environmentally realistic UV irradiation reaching the water surface on a summer day (Schug et al. 2014), was used in the acclimation and exposure experiment of sludge, with a light cycle of 12:12 h. For comparison, we studied the SBR without TiO 2 NP exposure, but with light irradiation.

Extracellular polymeric substance (EPS) extraction and analysis
Briefly, after the experiments, 25 mL of activated sludge samples were suspended in 50-mL centrifuge tubes, mixed with 0.05% (w/w) NaCl solution to form a 50 mL suspension, and then centrifuged at 6000 g for 10 min to remove the loose slime polymers attached to the surface of the activated sludge. The sludge pellets accumulated at the bottom of the centrifuge tubes were resuspended in a 0.05% (w/w) NaCl solution and then ultrasonicated for 2 min at 20 kHz. The homogeneous mixtures obtained from horizontal vibration in a thermostat incubator at 150 rpm for 15 min were immediately centrifuged at 8000 × g for 10 min. The supernatants were regarded as loosely attached EPSs. The residual pellets were resuspended to their initial volume with 0.05% (w/w) NaCl solution, followed by ultrasound for 2 min at 20 kHz, and then heated at 70°C for 30 min. Finally, the suspension was centrifuged at 11,000 × g for 30 min, and the supernatant was carefully collected as the tight binding EPS. All the EPS fractions were mixed and filtered using 0.45-μm acetate cellulose membranes and stored at −20°C before analysis. The main components of EPS, such as proteins (PRO) and polysaccharides (PS), were also quantified according to previous studies ).

Flow cytometry analysis
To verify whether the sludge cells underwent apoptosis and necrosis after long-term exposure to low concentrations, the physiological status of sludge cells was evaluated by multiparameter flow cytometry using an Annexin V-FITC/PI Apoptosis Kit (AV/PI; Invitrogen) (Dwyer et al. 2012;Eray et al. 2015). The externalization of phosphatidylserine (PPS) in apoptotic bacteria was detected using recombinant annexin V conjugated to green-fluorescent FITC dye and in dead bacteria using propidium iodide (PI) with red fluorescence (Dwyer et al. 2012;Eray et al. 2015). Annexin V is a Ca 2+dependent phospholipid-binding protein with a molecular weight of 35.8 kD. It can bind to PPS with high affinity during apoptosis (Pester et al. 2012). Annexin V-FITC staining can identify apoptosis at an early stage, along with the late stages of cell death resulting from either apoptotic or necrotic processes. The preparation of the sludge single-cell suspension, the staining process, and the machine test were all based on previous methods . Briefly, binding buffer (1×working) was used to prepare a single-cell suspension (100 μL) with a concentration of 1×10 6 -1×10 7 cells/mL and then incubated for 10 min after adding 2.5 μL annexin V-FITC in dark at 25°C, following which 5-μL PI was added and immediately washed with pre-cooled PBS, and analyzed after incubating for 1 min in dark at room temperature.

DNA extraction and high-throughput sequencing
At the end of the exposure, sludge samples (0.5 g) homogeneously obtained from the SBR were immediately stored at −20°C for DNA extraction. DNA was extracted using an E.Z.N.A. Soil DNA kit (OMEGA, D5625-01, USA) according to the manufacturer's instructions. The concentration of the extracted DNA was determined using 0.8% (w/v) agarose gel electrophoresis. The microbial communities with or without TiO 2 NP exposure were analyzed using an Illumina MiSeq platform according to standard protocols and was conducted by Guangdong Magigene Biotechnology Co., Ltd. China (Wang et al. 2018). Detailed information is available in SI.

Other analytical methods and statistical analysis
The specific resistance to filtration (SRF) and bound water (BW) contents were measured to characterize the dewatering capacity of the sludge (You et al. 2017). The intracellular nanoparticles accumulated inside the activated sludge were measured, and the intracellular oxidative damage (ROS) in sludge cells was tested using the methods described previously . Changes in the concentration of lactate dehydrogenase (LDH) outside the activated sludge cells were used to evaluate the permeability of bacterial cytoplasmic membrane. The density of activated sludge floc and quorum sensing (QS) signaling molecule responses via acylhomoserine lactones (AHLs) were determined. Details of the experiments and calculation methods are provided in the SI.
All tests were conducted in triplicate, and the values are presented as the mean ± standard deviation. One-way analysis of variance (ANOVA) was used to identify statistical significance (p < 0.05 significant and p < 0.01 highly significant) between the control and exposure groups or between different exposure groups.

Results and discussion
Characteristics of the pristine and aged TiO 2 NP As shown in Fig. 1a-d, after 300 days of sludge supernatant aging experiment, HRTEM (JEM-2100F, Japan) and XRD (Model D8, Advanced X-ray diffractometer, Germany) analyses revealed obvious changes in the surface morphology and elemental distribution of the TiO 2 NPs. The clear crystal texture on the surface of the nanoparticles become fuzzy and cohesive, mainly due to the encapsulation of organic matter and inorganic salts, such as NaCl, in the sludge supernatant. However, the crystallinity, grain size, and arrangement of the TiO 2 NPs influence its original photocatalytic activity and selectivity (Vance et al. 2015) and are unaffected by aging. In addition, both pristine and aged nanoparticles comprise internal monocrystalline structures with a dominant crystal surface index (101) (the number in parentheses represent the Miller index), which does not affect the toxic behavior of faceted TiO 2 NPs in terms of their distinctive crystallographic qualities (Liu et al. 2016).
Interestingly, the aging experiment in the sludge supernatant changed the light absorption capacity of the nanoparticles, which caused a redshift in the photosensitivity (Fig. 1e) and reduced the band-gap energy (Eg) of TiO 2 NP from 3.2 to 3.1 eV (Fig. 1f). This appears to differ from previous reports, where both surface water bodies and piping sewage aging experiments could increase the Egs of anatase nanoparticles and decrease the energy band of rutile nanoparticles (Li et al. 2020b(Li et al. , 2020c. This might be attributed to the differences in aging water quality and incubation time, for example, organic substances such as humic acid in the water can photosensitize the nanoparticles (Leads and Weinstein 2019), while the presence of salt crystals reduce the photocatalytic efficiency of TiO 2 NP by the "sheltering phenomenon of deposited salt" (Xu et al. 2019). Moreover, compared to previous studies, the delay in the aging time could alter the composition and thickness of deposited or wrapped such as organic matter and inorganic ions on the surface of the nanoparticles, where the surface microstructure and photoactivity of the nanoparticles are the result of the comprehensive regulation of the above aging behaviors (Halle et al. 2020).
In addition, Fig. S1 shows that aging not only increases the stability of the nanoparticle water environment but also increases the particle size range, which corresponds to the increase in the absolute value of the zeta potential on the surface of the aged nanoparticles. This is mainly due to the fact that the surface of the aged nanoparticles is wrapped by a large amount of negatively charged organic matter in the sludge supernatant, which increases the electrostatic repulsion and steric hindrance effects of nanoparticles (Gilbert et al. 2007).

Dewaterability and EPS secretion properties of activated sludge
Here, two indexes, SRF and BW, were used to evaluate sludge dewaterability under laboratory conditions. Figure 2A shows that 10-μg/L nanoparticles significantly (p<0.01) increased the SRF, whereas 100-μg/L TiO 2 NP significantly (p<0.01) reduced SRF compared with the control, and the BW contents observed a similar trend as that of the SRF (Fig. 2B), which indicated a severe decrease or increase in sludge dewaterability at two concentration levels (μg/L). Interestingly, aging could reduce the influence of nanoparticles on the dewatering performance of activated sludge; for example, compared with the pristine nanoparticles, the application effects of the aged nanoparticles significantly weakened, displaying an obvious increase in the SMP at 10-μg/L TiO 2 NP (p<0.05), while a 100-μg/L TiO 2 NP evidently (p<0.05) decreased the SRF. Furthermore, the change in the trend of BW was consistent with that of the SRF.
EPS, a gel-like matrix that accounts for 80% of the total sludge mass, with significant influences on sludge hydrophobicity, surface charge, microbial aggregates, flocculation, and adhesion (Liu and Fang 2003), and is reportedly the major limiting factor for sludge dewatering .
Here, Fig. 2C shows that the total amount of EPS, which is mainly composed of PRO and PS, maintained the same changing trend as that of both SRF and BW of the sludge; that is, whether aging or not, 10 μg/L promoted the secretion of EPS (compared with the control group, EPS increased by 34.0% and 10.9% in the pristine and aging exposed groups, respectively), while 100 μg/L inhibited the production of EPS (compared with the control group, EPS decreased by 34.1% and 17.7% in the pristine and aging exposed groups, respectively), implying that EPS and sludge dewatering performance are closely related. The increase or decrease in these EPS secretions appears to be related to changes in the abundance of EPS-secreting bacteria and quorum sensing signal flux in the activated sludge (Table 1 and Fig. 4A). Evidently, the damage and necrosis of sludge surface cells caused by exposure to high concentration (100 μg/L) result in an increase in sludge density (Fig. 4B), which might also be one of the reasons for the decrease in EPS secretion (the following will be a detailed discussion). Similarly, in previous reports, TiO 2 NPs not only affected the sludge EPS secretion but also regulated the EPS secretion signal transmission or gene expression of natural biofilm, which in turn affected sludge dewatering or the colonization and development of natural biofilms and is closely related to exposure concentrations, crystal form, Fig. 1 Physicochemical transformation of the crystalline phase of TiO 2 NP after 300 days of aging. a TEM image of pristine TiO 2 NP, b TEM image of aged TiO 2 NP, and c locally enlarged TEM image with d corresponding XRD patterns (squares indicated). Green arrows represent inclusions of aged NPs. eUV-vis diffuse reflections of TiO 2 NP and f corresponding band-gaps aging, and hydration conditions Li et al. 2020c). In addition, Li et al. (2019) reported that the composition and structure of EPS significantly influence the dewatering performance of sludge; for example, in the EPS, PRO was responsible for the change in SRF, while the change in BW was dominated by PS. Therefore, in this study, PRO and PS in EPS either increased (10 μg/L, p<0.05) or decreased significantly (100 μg/L, p<0.05) compared to the control, which explains the changing trends of SRF and BW of activated sludge in the corresponding exposure group.

Mode of microbial cells death in sludge and related causes
The cell death mode in the sludge has been reportedly related to EPS secretion and composition, which may explain the difference in sludge dewatering performance in different exposure groups . Figure 3A shows two ways of sludge cell death, apoptosis (cell membrane integrity) and necrosis (cell membrane leakage), which have been confirmed in previous reports (Chaloupka and Vinter 1996;Dwyer et al. 2012). Compared with the control group and exposure groups with 10-μg/L nanoparticles, 100-μg/L nanoparticles displayed severe inflammatory injury, leading to significant (p<0.01) LDH leakage in sludge cells (Fig. 3B), resulting in obvious cell necrosis (Fig. 3A). Aging could notably delay the cell necrosis caused by TiO 2 NP in the sludge, but it appears to increase the induction of apoptosis in sludge cells (Fig. 3A). This is mainly attributed to the reduced photo-oxidative degradation ability of TiO 2 NPs due to aging. In contrast, the aged nanoparticles were increasingly stable in the sewage environment and maintained a stable small particle size range in the sewage system (Fig. S1), finally promoting the accumulation of particles in sludge cells (Fig. 3C), leading to intracellular peroxidation and oxidative damage (Fig. 3D). Notably, the amount of intracellular ROS, mainly generated by intracellular accumulation of nanoparticles, appears to be only related to the amount of apoptosis, because the amount of intracellular ROS as an autoinducer (Li et al. 2020c), which only induces apoptosis in this study (Fadeel and Garcia-Bennett 2010;Rahman et al. 2002), and does not cause necrosis, as shown in Fig. 3A, Fig. 3B, and Fig. 3C. Thus, at lethal exposure concentrations (100 μg/L), the pristine nanoparticles advance toward sludge cell necrosis, while the aged nanoparticles readily induce the apoptosis of microbial cells, which have been verified in previous studies Li et al. 2020b). However, as for 10 μg/L, regardless of aging, this exposure level does not cause damage.
Previous studies have reported that cell death patterns in the sludge, such as apoptosis and necrosis, are notably related to EPS secretion and composition and affect the final sludge dewatering performance . Similarly, Fig. 2 and Fig. 3A show that regardless of the promoting effects of low concentration (10 μg/L) and inhibiting effects of high concentration (100 Fig. 2 Effects of TiO 2 NP with or without aging on the A SRF, B BW contents, and C EPS compositions. Single asterisk and double asterisks indicate the statistical difference (p <0.05) and high significance (p < 0.01) from that of the control, respectively. Note: 10 μg/L and 100 μg/L represent the exposure concentrations of aged nanoparticles. Error bars represent standard deviations (n = 3). μg/L) on EPS secretion, as compared with the control group using the ratios of PRO to PS (necrotic cells to apoptotic cells), it can be roughly discerned that apoptosis contributes to the secretion of polysaccharides in sludge, while necrosis contributes to protein secretion. This ultimately affects the response mechanism of sludge dewatering performance .

Changes of quorum sensing and sludge density and related causes
It has been reported that quorum sensing (QS) information of the microbial community in the activated sludge regulates changes in the EPS secretion and density of the sludge (Shrout and Nerenberg 2012), which significantly affects the sludge dewatering performance (Wu and Wu 2001;Zhang et al. 2017). Therefore, after exposure, the QS signal (AHL) response and density change of the activated sludge were also monitored. Figure 4A and Fig. 4B show that in all exposed groups, the change trends of QS-AHL secretion and sludge density were contradictory; for example, 10-μg/L TiO 2 NP improved the QS-AHL secretion and enhanced sludge organic trend by improving EPS production with a decrease in sludge density, while 100-μg/L TiO 2 NP inhibited the secretion of QS-AHL and enhanced the inorganic tendency of sludge by reducing EPS production with an increase in sludge density.
Notably, aging could attenuate the responsiveness of QS-AHL and sludge density caused by the two concentrations of pristine nanoparticles, but complete alleviation was difficult, with significant (p<0.05) levels still present relative to the control group (Fig. 4). This also suggests that regardless of aging, 10 μg/L of nanoparticles had promoting effects, and 100 μg/L inhibited EPS generation and the physiological behavior of the entire microbial community in activated sludge. The QS molecule was in reasonable agreement with the regulation of gene expression and the biosynthesis of EPS during mixed-species aerobic granule formation (Shrout and Nerenberg 2012;Wang et al. 2018). In addition, QS signals secreted and differentially altered by TiO 2 NP-exposed groups influenced the social and physiological environments (community structure and diversity) of the microbial population in sludge.
Considering the above analysis, this study used highthroughput sequencing technology to monitor the key genera secreting QS-AHL and genera secreting specific EPS. First, as shown in Table S2, regardless of aging, after exposure, 10-μg/L TiO 2 NPs promoted the richness and diversity of the activated sludge microbial community in terms of the OTUs and Shannon index, while 100 μg/L reduced the richness and microbial diversity in sludge based on the Chao and Shannon indices, compared with the corresponding indexes of the control group. This might indicate that the structure of the microbial community significantly differed or the specific microbial communities in activated sludge evolved after exposure to different concentrations of nanoparticles. In addition, the EPS production of certain bacteria is directly controlled by QS-AHL (Gilbert et al. 2009;Shrout and Nerenberg 2012). In this study, QS bacteria and few specific EPSsecreting genera in the activated sludge, which are directly or indirectly controlled by QS-AHL, are shown in Table 1. These QS bacterial abundances either increased or decreased after exposure to TiO 2 NPs at different concentrations; however, considering the increase (10 μg/L) or decrease (100 μg/L) of QS-AHL (Fig. 4A), the genera with a significant abundance (Table 1), such as Acinetobacter, Aeromonas, Comamonas, Nitrobacter, Nitrosomonas, Pseudomonas, and Sphingomonas, could play a key role in the secretion of AHLsignaling molecules. Moreover, the bacterial genera Bradyrhizobium, Burkholderia, and Sphingomonas are responsible for the significant increase in PS, one of the main components of EPS, while the bacterial genera Flavobacterium, Bosea, and Ralstonia are responsible for the significant increase of other components, such as proteins in EPS in the 10-μg/L nanoparticle-exposed groups. In contrast, for 100 μg/L, the abundance of the aforementioned specific EPS-secreting species substantially decreased, which is consistent with the trend of sludge density increasing at higher concentration (100 μg/L) (Fig. 4B). The above analysis suggests that nanoparticles can regulate the bacterial community structure and secretion of signal molecules. QS bacteria and genera secreting specific EPS inside the sludge play a key role in the QS-controlled genes related to sludge floc formation and density, where the bacteria emit and sense chemical signal molecules as a means to gauge population density and control EPS gene expression (Li et al. 2020a), thus affecting the dewatering performance of sludge.

Conclusions
We compared the physicochemical properties and water stability of pristine and aged TiO 2 NPs and studied the effects of aging on sludge dewatering performance after chronic Fig. 4 Effects of TiO 2 NP on A quorum sensing and B sludge density of activated sludge. Single asterisk and double asterisks indicate the statistical difference (p < 0.05) and high significance (p < 0.01) from the control, respectively. Error bars represent standard deviations (n = 3) exposure to TiO 2 NPs at two (10 and 100 μg/L) concentration levels and analyzed the related mechanisms. The main conclusions are as follows: (1) the aging of nanoparticles in sludge supernatant changed the surface properties of TiO 2 NP, such as zeta potential, functional groups, and energy bands, and increased the stability of the nanoparticles in the water environment, but did not change the crystallinity of TiO 2 NP; (2) regardless of aging, nanoparticles reduced sludge dewaterability at an exposure concentration of 10 μg/L and improved the sludge dewaterability at an exposure concentration of 100 μg/L, but aging could weaken the effect of two exposure concentrations on sludge dewatering; (3) exposure of nanoparticles could change the microbial community structure and the richness of both QS bacteria and genera secreting specific EPS because of the damaging effect of 100 μg/L and the hormonal effect of 10 μg/L for up to 300 days of exposure, leading to changes in the secretion and composition of EPS, ultimately affecting the density and dewatering performance of the sludge. The results of this study supplement the results of long-term low concentrations of nanoparticles on the physiological and biochemical effects of microbial aggregates in sewage treatment systems and suggest that the impact of environmental media aging should be included in evaluating the ecological effects of nanoparticles because the incubation of nanoparticles in the environment is likely to impact their own ecological effects.
Author contribution Chengyu Jiang designed the study, analyzed the data, and wrote the manuscript. Qingjin Chen performed the experiment and contributed to the manuscript writing.
Funding This study was financially supported by the Treatment Project of Black and Smelly River of Nanjing City (2017).
Data Availability Not applicable.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication Not applicable.
Competing interests The authors declare no competing interests. Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/s11356-021-16451-4.