Influence of wastewater type in the effects caused by titanium dioxide nanoparticles in the removal of macronutrients by activated sludge

The imminent arrival of nanoparticles (NPs) to the wastewater treatment plants (WWTP) brings concern about their effects, which can be related to the wastewater composition. In this work, the effects of titanium dioxide (TiO2) NPs in the removal of carbon, nitrogen, and phosphorus by activated sludge bioreactors during the treatment of synthetic, raw, and filtered wastewaters were evaluated. Floc size, compaction of sludge, and morphological interactions between sludge and NPs were also determined. The main effect of TiO2 NPs was the inhibition of up to 22% in the removal of ammonia nitrogen for all types of wastewaters. This effect is strong dependent on combined factors of TiO2 NPs concentration and content of organic matter and ammonia in wastewater. The removal of dissolved organic carbon was affected by TiO2 NPs in lower level (up to 6%) than nitrogen removal for all types of wastewaters. Conversely to adverse effects, the removals of orthophosphate in the presence of TiO2 NPs were improved by 34%, 16%, and 55% for synthetic, raw, and filtered wastewater, respectively. Compaction of the sludge was also enhanced as the concentrations of NPs increased without alterations in the floc size for all types of wastewaters. Based on TEM and STEM imaging, the main interaction between TiO2 NPs and the activated sludge flocs was the adsorption of NPs on cell membrane. This means that NPs can be attached to cell membrane during aerobic wastewater treatment, and potentially disrupt this membrane. The effects of TiO2 NPs on macronutrient removal clearly depended on wastewater characteristics; hence, the use of realistic media is highly encouraged for ecotoxicological experiments involving NPs.


Introduction
TiO 2 nanoparticles (TiO 2 NPs) are present in multiple everyday products, such as food, toothpaste, and sunscreens (Weir et al. 2012;Adam et al. 2018;Polesel et al. 2018). According to the life cycle assessment studies (Keller and Lazareva 2013;Adam et al. 2018;), unintentional release of TiO 2 NPs to the environmental matrices, such as air, soil, or water, during or after the use of such products is expected. Therefore, some concerns have raised due to the potential effects of TiO 2 NPs in such matrices. Water streams have been identified as the main transport media of NP S in the environment owing to the connection between treated or non-treated effluents and water bodies (Brar et al. 2010;Kunhikrishnan et al. 2015). Some studies have reported the presence of TiO 2 NPs in wastewater streams (Kiser et al. 2009;Tuoriniemi et al. 2012), and in the sludge of the bioreactors applied for wastewater treatment (Polesel et al. 2018;Huang et al. 2020;Cervantes-Avilés and Keller 2021). This confirms that NPs present in Responsible Editor: Philippe Garrigues * Pabel Cervantes-Avilés pabel.cervantes@tec.mx * Germán Cuevas-Rodríguez german28@ugto.mx wastewater are interacting with microorganisms of bioreactors, which can induce some effects on the performance of wastewater treatment processes. Some effects of TiO 2 NPs during aerobic wastewater treatment are already reported, including toxicity to some microorganisms of the aerobic processes (Li et al. 2017) and inhibition of oxygen consumption (Supha et al. 2015;Zhou et al. 2015). This deficiency in oxygen consumption by aerobic microorganisms could negatively affect removal of carbon, nitrogen, and phosphorus in the aerobic bioreactors. Gartiser et al. (2014) exposed activated sludge to a synthetic wastewater (SWW) containing TiO 2 NPs during a period of 32 days, and found that the cumulative effect of up to 840 mg/L of TiO 2 NPs negatively affected the removal of organic matter.
Studies about the effects of TiO 2 NPs on the removal of ammoniacal nitrogen and phosphorus in activated sludge systems are limited. In the few studies, no impact of TiO 2 NPs on nitrogen and phosphorus removal was reported in activated sludge process exposed to 1, 10, and 50 mg/L of NPs in SWW during 24 days (Supha et al. 2015). In contrast, for higher concentration of TiO 2 NPs and also using SWW, an inhibitory effect on nitrogen removal has been observed, which was attributed to the inhibition of microorganism related to nitrification-denitrification processes (Li et al. 2020). In the experiment of García et al. (2012), a synthetic media, such as ammonium chloride, was also used to evaluate the nitrification process, which was inhibited 5% after 4 h in a system containing suspended biomass. Higher inhibition of the nitrification process was observed in long-term experiments performed by Zheng et al. (2011). In these experiments, 50 mg/L of TiO 2 NPs in SWW were added to activated sludge bioreactors, decreasing the ammoniacal nitrogen removal from 80.3 to 24.4% after 70 days. The loss of nitrification function of activated sludge exposed to a synthetic media was linked to a decrease in nitrifying bacteria (Ma et al. 2015). Moreover, adverse effects of TiO 2 NPs over organic matter and nitrogen removal were mainly related to the stability of the NPs (Zhou et al. 2015), which can change according to wastewater characteristics (Keller et al. 2010;Gartiser et al. 2014;Cervantes-Avilés et al. 2017). Considering that most of the experiments testing effects of TiO 2 NPs on activated sludge were performed with SWW, such effects should be evaluated using real wastewater to determine similarities and differences between wastewater types.
Previous findings suggest that the effects of TiO 2 NPs on organic matter and nitrogen removal depends on several factors, such as the accumulated concentration of TiO 2 NPs in bioreactor and the exposure time (Zheng et al. 2011;García et al. 2012;Li et al. 2020). Moreover, given the influence that physicochemical characteristics of wastewater has on the stability of NPs (Keller et al. 2010;Gartiser et al. 2014), type of wastewater can be also considered as important factor when testing the performance of activated sludge exposed to NPs. Currently, there are not studies that evaluate the influence of the type of wastewater in the effects that NPs can cause over activated sludge. Therefore, the aim of this work was to evaluate the effect of TiO 2 NPs in the removal of macronutrients, such as carbon, nitrogen, and phosphorus, by activated sludge, when the NPs are in three types of domestic wastewaters: synthetic, raw, and filtered. Floc size, compaction of the activated sludge, and the morphological interaction between TiO 2 NPs and microorganisms were also evaluated for all types of wastewaters.
Evaluation of macronutrients removal was performed in glass reactors of 2 L, which were covered from light to avoid photolysis induced by TiO 2 NPs. Each reactor was equipped with air diffusers and an air pump that provided air flux at 4 m 3 air /h·m 3 reactor . Commercial tests (Hach-Lange) were used to measure the concentration of chemical oxygen demand (COD), soluble COD (sCOD), ammoniacal nitrogen (NH 3 -N), nitrate-nitrogen (NO 3 -N), nitrite-nitrogen (NO 2 -N), and orthophosphate (PO 4 3− ). The dissolved total organic carbon (dTOC) was measured in a TOC-L (Shimadzu). Nylon filters (Whatman) with a pore size of 0.45 μm were used to remove the suspended solids before analysis. Glass fiber filters (Whatman) with a pore size of 0.7 μm were used to determine the concentration of total suspended solids (TSS) and volatile suspended solids (VSS) according to the standard methods (APHA 2005).

Wastewater characterization
Three types of wastewaters, namely SWW, raw wastewater (RWW), and filtered wastewater (FWW), were tested separately in the reactors. SWW was prepared based on the constituents and procedure reported in materials section. RWW was collected in the septic tank of a house without access to sanitation services in Guanajuato, Mexico. FWW consisted of RWW without TSS; hence, the collected RWW was passed through 0.45-μm nylon filters. The three types of wastewaters were characterized to determine COD, sCOD, BOD, NH 3 -N, NO 3 -N, NO 2 -N, PO 4 3− , TSS, and VSS ( Table 1).

Characterization of the TiO 2 NPs
Stock suspension of NPs was prepared with cleaned TiO 2 NPs for characterization. TiO 2 NPs were cleaned to remove impurities by washing three times with absolute ethanol and then dried at 90°C overnight. Stock suspension of TiO 2 NPs (2 g/L) was prepared in Milli-Q water and then ultrasonicated during 1 h at 40 kHz and 200 W, as recommended to promote dispersibility for ecotoxicological assessment (Taurozzi et al. 2012). The primary size, shape, purity, phase, and UV spectrum were determined in a previous study (Cervantes-Avilés et al. 2018). Briefly, TiO 2 NPs were spherical with mean diameter ranging from 3 to 10 nm and were found in the anatase phase. Energy-dispersive X-ray spectroscopy (EDS) confirmed the purity, detecting Ti and O in the NPs. Finally, the localized surface plasmon resonance was detected at 295 nm. Before spiking the experiments, TiO 2 NPs stock suspension was ultrasonicated again during 1 h at same frequency and intensity.

Macronutrient removal tests in activated sludge
The reactors contained three main components: 0.842 L of inoculum of activated sludge, 0.842 L of wastewater, and 0.316 L of TiO 2 NPs suspension or Milli-Q water for controls. Inoculum of activated sludge had a concentration of 3120 ± 210 mg/L of SSV and was collected from a pilot plant fed with SWW and operated in the laboratory during 60 days before the experiment. The inoculum was exposed to final concentrations of 500, 1000, 1500, and 2000 mg/L of TiO 2 NPs. All concentrations of TiO 2 NPs and controls were performed at the same time with three replicates for each type of wastewater. The physicochemical parameters such as sCOD, dTOC, NH 3 -N, NO 3 -N, NO 2 -N, PO 4 3− , and the sludge volumetric index (SVI) were determined per triplicate at the end of the tests in all systems. The time of exposure of activated sludge to NPs was 8 h, which is the typical hydraulic retention (HRT) time of the conventional secondary treatment at WWTPs. The results of the physicochemical parameters were processed by ANOVA one factor. Dunnett's test was applied to determine the significant difference between groups. Moreover, statistical analysis was performed for each type of wastewater. Results with p value < 0.05 were considered statistically significant. Variation partitioning analysis (VPA) was performed for data of ammoniacal nitrogen removal and explanatory data Table 1 Physicochemical characteristics of the three types of wastewaters: synthetic wastewater (SWW), raw wastewater (RWW), and filtered wastewater (FWW). SD, standard deviation; "Min," minimum, and "Max", maximum; BOD 5 , biological oxygen demand at 5 days of TiO 2 NPs concentration and physicochemical characteristics of wastewater. Procedure was based according public script in R language (Borcard et al. 2011).

Interactions between TiO 2 NPs and activated sludge
Morphological interactions between flocs of activated sludge and NPs were studied at the end of the macronutrient removal tests by measuring size of the flocs, and by imaging of flocs exposed to TiO 2 NPs through TEM and high-angle annular dark field scanning-transmission electron microscopy (HAADF-STEM). Size of the flocs was determined by static light scattering (SLS, Microtrac S3500), applying a value of 1.81 as the refractive index of the flocs. For TEM imaging, samples of activated sludge (1.5 mL) were collected immediately after macronutrient removal tests and centrifuged at 9000g. Supernatant was replaced by 2.5% glutaraldehyde, and pellets were resuspended and kept at room temperature for 2 h. Then, samples were centrifuged to replace glutaraldehyde with 1% osmium in sodium cacodylate 0.1 M, keeping the samples at room temperature for 1 h. After this period, the samples were dehydrated gradually with ethanol absolute, starting at 10% (v/v) up to ethanol 100%. Finally, samples were embedded in an epoxy resin (EPON812) to form blocks of resin containing the samples. The resin blocks were cut in thin sections, between 60 and 90 nm, by using an ultramicrotome (MTX-RMC). TEM imaging was conducted at 80 kV in a JEOL-1010, while HAADF-STEM imaging was performed at 300 kV in a microscope FEI-Titan 80-300.

Removal of organic matter in presence of TiO 2 NPs
The removal of organic matter in the three types of wastewaters was evaluated by relating the initial and final concentration (C/C 0 ) of sCOD and dTOC (Figure 1), which serves as indicative of such removal in the presence of TiO 2 NPs. According to these results, the control groups presented removal of sCOD higher than 70% for the three types of wastewater ( Figure 1A), which corresponds to the typical removal for activated sludge systems (van Loosdrecht et al. 2016). However, impact of TiO 2 NPs on sCOD removal was different for all types of wastewaters. In the presence of SWW, TiO 2 NPs concentrations did not affect sCOD removal, which was statistically similar for all concentrations to control group. Conversely, in the case of RWW and FWW, removal of sCOD decreased linearly as the concentration of TiO 2 NPs increased, decreasing up to 10% and 14% lower removal for RWW and FWW, respectively. Since experimental conditions were similar for all three wastewaters, the negative effect of TiO 2 NPs over sCOD removal can be attributed to the type of wastewater and concentration of NPs. In this way, real wastewater without suspended solids (FWW) showed the higher potential effect on sCOD removal, while a synthetic medium such as SWW did not present any effect at the experimental concentrations of NPs. The non-influence of the TiO 2 NPs suspended in synthetic substrates on the removal of organic matter during activated sludge experiments has been already reported (García et al. 2012;Qiu et al. 2016). In contrast, TiO 2 NPs in real wastewater have decreased the oxygen uptake during aerobic experiments (Zhou et al. 2015), limiting the oxidation of organic matter and leading to a decrease in the removal of sCOD. Although the removal of sCOD from RWW, which is more realistic matrix, was affected only 10% in the presence of high load of TiO 2 NPs, this removal is sensible to the wastewater characteristics, including the suspended solids content. In this experiment, a difference on the chemical nature of the contained substrates in wastewaters (e.g., glucose for SWW, and proteins, fat, carbohydrates, among other compounds for real wastewaters), can play an important role in the effect of TiO 2 NPs over activated sludge culture. The removal of dTOC in the control reactors was at least 70% for the three types of wastewaters ( Figure 1B). However, organic carbon removal was most affected when more than 1000 mg/L of TiO 2 NPs was contained in RWW, decreasing the removal of dTOC up to 6%. In contrast, removal of organic carbon in experiments using SWW and FWW was not affected at all. This means that the presence of high concentrations of TiO 2 NPs in activated sludge process can have a slight effect on the removal of organic matter, which is also a fraction of the measured sCOD. A minimum adverse effect on organic matter removal was also found by a similar study performed by Gartiser et al. (2014), who fed synthetic wastewater based on peptone and meat extract, and exposed an activated sludge reactor to 840 mg/L of TiO 2 NPs, inducing a deficiency of 7% in the organic matter removal.
Removal of sCOD was more affected than the dTOC removal by the presence of TiO 2 NPs. In the experiments with real wastewaters (RWW and FWW), the most adverse effects were observed on sCOD removal. This means that the oxidizable (bio or chemically) inorganic compounds were not removed by the aerobic microorganisms present in the activated sludge due to the presence of TiO 2 NPs. The poor performance of aerobic microorganisms can be confirmed by previous experiments treating real wastewater in the presence of TiO 2 NPs, which inhibited the oxygen consumption by activated sludge in higher levels than SWW (Cervantes-Avilés et al. 2017). Therefore, since dTOC was not affected considerably in this experiment, the TiO 2 NPs present in real wastewaters could affect the microorganisms responsible for the oxidation of the inorganic compounds in wastewaters, commonly measured as sCOD. Although in this study there was no bacterial diversity analysis, there are a couple of studies that reported decrease in viable aerobic bacteria. Ma et al. reported a decrease of aerobic activity in activated sludge due to shifts in microbial community composition due to the presence of TiO 2 NPs (Ma et al. 2015). Another study reported a decrease in bacterial diversity of activated sludge exposed to 1 mg/L of TiO 2 NPs for 40 days, which was affected from the second day of exposure to NPs (Qiu et al. 2016). The effect of TiO 2 NPs in the activated sludge is a decrease in sCOD removal when real wastewaters are used, which could be attributed to the damage of aerobic microorganisms capable of oxidizing inorganic compounds.
Besides the type of wastewater, sCOD removal alterations also depended on the concentration of NPs in the performed experiments. The wastewater characteristics and the concentration of NPs are related to the particle stability in aqueous media (Keller et al. 2010;Zhou et al. 2015). This may result in stable dispersion or formation of aggregates. Some studies have reported that TiO 2 NPs may form heteroaggregates (e.g., suspended solids of the wastewater and organic colloids linked to NPs) (Gartiser et al. 2014), which could be deposited in activated sludge flocs and induce a toxic effect on bacteria, such as physical or chemical damage in the cell membrane. The formation of heteroaggregates containing TiO 2 NPs, organic colloids, and suspended solids has also been related to the carbon/nitrogen (C/N) ratio in wastewater. The most stable and small heteroaggregates have been found at low C/N ratio is close to 1 (Hotze et al. 2010;Keller et al. 2010). In this work, the C/N ratio for RWW and FWW was 1.27 and 1.22, respectively, while the C/N ratio for the SWW was 8.18. Thus, formation of small and stable heteroaggregates in real wastewaters could potentially explain a damage in aerobic microorganisms. Besides, the presence of TSS may alter the effects of NPs on sCOD and dTOC removal. This means that the type of organic substrate and suspended solids content in wastewater, combined with NPs concentration can play an important role when evaluating toxicity by NPs.

Ammoniacal nitrogen removal in the presence of TiO 2 NPs
In the control experiments using the three types of wastewaters, the ammoniacal nitrogen removals were 64% for RWW and FWW, and 76% for SWW (Figure 2). These removal percentages are commonly observed in activated sludge reactors treating domestic wastewater. However, when the activated sludge was exposed to TiO 2 NPs, the ammoniacal nitrogen removal was negatively affected for the three types of wastewaters, with the only exception of FWW containing 500 mg/L that presented statistically similar removal than control group (Figure 2).
Removal of ammoniacal nitrogen in SWW containing TiO 2 NPs was less affected than that for the other types of Fig. 1 Effect of TiO 2 NPs in the organic matter removal from synthetic wastewater (SWW), raw wastewater (RWW), and filtered wastewater (FWW), measured as A soluble chemical oxygen demand (sCOD) and B dissolved total organic carbon (dTOC). Note: "*" means that there was no significant difference to the control group Fig. 2 Ammoniacal nitrogen removal in the activated sludge process exposed to different concentrations of TiO 2 NPs expressed as the ratio between final (C) and initial concentration (C 0 ) of ammoniacal nitrogen after 8 h of treatment of synthetic wastewater (SWW), raw wastewater (RWW), and filtered wastewater (FWW). Note: "*" means that there was no significant difference to the control group wastewaters, decreasing the removals between 5 and 10% for all TiO 2 NPs concentrations ( Figure 2). Experiments with RWW and FWW indicated that inhibition of ammoniacal nitrogen removal increased as concentration of TiO 2 NPs in activated sludge increased. In experiments with RWW, ammoniacal nitrogen removal decreased up to 22% in the presence of 2000 mg/L of TiO 2 NPs, while for FWW, the removal decreased up to 12% for the same concentrations of NPs. These results clearly indicate that the effect of TiO 2 NPs on ammoniacal nitrogen removal was more severe when real wastewater with suspended solids is used.
Ammoniacal nitrogen removal in activated sludge is mainly via nitrification-denitrification. To assess nitrification (ammoniacal oxidation processes), NO 2 -N and NO 3 -N were determined at the end of the macronutrient removal tests ( Figures 3A, B, respectively). In the case of SWW, NO 2 -N formation in presence of TiO 2 NPs was similar to the control. However, the NO 3 -N formation decreased as the concentration of TiO 2 NPs increased. Hence, for SWW containing TiO 2 NPs, the main effect occurred in nitrate formation, which is the second step of the nitrification process.
Formation of NO 2 -N and NO 3 -N from RWW containing TiO 2 NPs was higher or similar to control group, e.g., NO 3 -N production increased 7% in the presence of 2000 mg/L of TiO 2 NPs compared to control group. Conversely, in experiments treating FWW with TiO 2 NPs, the production of NO 2 -N and NO 3 -N by activated sludge was affected for the NPs. This means that TiO 2 NPs in wastewater without TSS may damage the aerobic oxidizing bacteria (AOB) of the activated sludge. Li et al. (2020) performed a quantitative polymerase chain reaction (qPCR) test followed by Illumina highthroughput sequencing of the activated sludge exposed to a high cumulative concentration of TiO 2 NPs (450-900 mg/L) and found that AOB can be mainly affected by acute exposition to these NPs.
In general, ammoniacal nitrogen removal was affected in the presence of TiO 2 NPs regardless of the type of wastewater.
However, first and second steps of nitrification in the presence of TiO 2 NPs were better performed using RWW than FWW. Considering that the only difference between FWW and RWW is the TSS content, the presence of suspended solids may contribute to the formation of heteroaggregates less toxic for nitrifying bacteria (Nitrobacter, Nitrosospira, and Nitrosomonas). In contrast, for the experiments using the SWW and FWW, deficiencies in the production of nitrite and nitrate were found under the presence of TiO 2 NPs, which could be attributed to damage in the nitrifying bacteria. The limited production of nitrite and nitrate in experiments treating SWW and FWW suggests that the overall removal of ammoniacal nitrogen in the presence of NPs could occur due to the adsorption of nitrogen onto TiO 2 NPs.
To explore influencing factors in ammoniacal nitrogen removal, VPA was performed as function of TiO 2 NPs concentration and physicochemical parameters of all three types of wastewaters ( Figure 4). During this analysis, collinearity > 10 was found for almost all parameters of wastewaters, except for BOD 5 and NH 3 -N, which were then used for VPA. Combined effect of NPs concentration and content of both BOD 5 and NH 3 -N in each type of wastewater were dominant factors (0.61) in ammoniacal nitrogen removal, followed by residuals influence (e.g., experimental conditions or others nonmonitored parameters) and concentration effect. This analysis confirms that effect of different concentrations of TiO 2 NP on nitrogen removal is strong dependent on organic matter and ammonia content.

Orthophosphate removal in presence of TiO 2 NPs
The orthophosphate removal in the activated sludge process containing TiO 2 NPs was enhanced for all three types of wastewaters ( Figure 5). In experiments with SWW, the highest PO 4 3− removal was observed in the presence of 1000 mg/L of TiO 2 NPs, with 34% higher removal than the control group. However, the most noticeable improvement Fig. 3 Effect of TiO 2 NPs in the formation of A Nitrite (NO 2 -N) and B Nitrate (NO 3 -N), which were measured at the end of the treatment of synthetic wastewater (SWW), raw (RWW), and filtered (FWW). Note: "*" means that there was no significant difference to the control group was observed for experiments with real wastewater, which increased the removal of PO 4 3− as the concentration of TiO 2 NPs also increased. The percentages of enhancement when compared to their respective control groups were 16% and 55% for RWW and FWW, respectively. This suggests a direct action of NPs over PO 4 3− , such as adsorption or precipitation. Phosphorus removal during activated sludge process is developed by polyphosphate-accumulating organisms (PAO), which capture the chains of polyphosphates under aerobic conditions. Since TiO 2 NPs may affect the bacterial performance, the possible explanation of phosphate removal could be adsorption and co-precipitation of PO 4 3− adsorbed by TiO 2 NPs. The ability of TiO 2 NPs for adsorbing compounds has been already reported (Cervantes-Avilés et al. 2017), including those measured as sCOD such as the polyphosphate chains present in activated sludge reactors. The adsorption of phosphate in NPs surface is related to the pH of the aqueous medium and pH between 7 and 8; the typical of activated sludge is the most favored (Rathnayake et al. 2014). Moreover, the type of substrates in wastewater containing phosphate could also be an influencing factor (Neale et al. 2015;Zhang et al. 2019); hence, further evaluation of NPs for phosphorus removal or recovery should be addressed. Since the pH of the experiments in the present study was between 7 and 7.8, and the sources of PO 4 3− in real ) removal as the ratio of final (C) and initial (C 0 ) concentration of PO 4 3− after 8 h of treatment by activated sludge of synthetic wastewater (SWW), raw wastewater (RWW), and filtered wastewater (FWW) exposed to TiO 2 NPs. Note: "*" means that there was no significant difference to the control group Fig. 6 Size distribution of activated sludge flocs exposed to TiO 2 NPs during treatment of A synthetic wastewater (SWW), B raw wastewater (RWW), and C filtered wastewater (FWW) wastewater are commonly detergents, the TiO 2 NPs could be an adsorbent agent during wastewater treatment.
The removal of orthophosphate via TiO 2 NPs could be considered as a positive effect that is due to potential adsorption. However, the affinity between TiO 2 NPs and orthophosphate should be proven through measuring orthophosphate adsorption capacity by TiO 2 NPs, and other parameters that complement the characterization of NPs, such as zeta potential and specific surface area. Considering that TiO 2 NPs are widely investigated for water treatment due to their photocatalytic activity, and their potential contribution to the extracellular electron transfer , the ability of TiO 2 NPs to remove orthophosphate from wastewater could promote their use during biological wastewater treatment.
Effect of TiO 2 NPs in the floc size and SVI of activated sludge The distribution of floc size of activated sludge in the three wastewater types is observed in Figure 6. For all different types of wastewaters, the flocs size of activated sludge was not affected by the presence of TiO 2 NPs in the reactors. However, the average values of SVI in the presence of TiO 2 NPs were 50% lower than control SVI values for all types of wastewaters (Figure 7), improving the compaction of activated sludge.
The activated sludge compaction during settling was dependent on the NPs content. This was observed in the SVI values, which were inversely proportional to the concentration of TiO 2 NPs regardless of the type of wastewater used (Figure 7). These findings suggest that the flocs exposed to TiO 2 NPs kept the same size but had higher Fig. 7 Sludge volume index (SVI) of activated sludge exposed to TiO 2 NPs during the treatment of synthetic (SWW), raw wastewater (RWW), and filtered wastewater (FWW) Fig. 8 Transmission electron microscopy (TEM) imaging of microorganisms present in aerobic wastewater treatment exposed to TiO 2 NPs. A Control treating RWW. B SWW + 1000 mg/L of TiO 2 NPs. C RWW + 1000 mg/L of TiO 2 NPs. D FWW + 2000 mg/ L of TiO 2 NPs density, settling faster and gaining greater compaction of biomass as concentration of NPs is also higher. Despite of the compaction of flocs by the potential adsorption of NPs, the microorganisms present in the activated sludge and exposed to TiO 2 NPs performed metabolic activities that allowed, at some level, the treatment of wastewater regardless of the type of substrate, as indicated by the results about the removal of macronutrients. These adsorption has been observed for some other nanomaterials such as CuO (Hou et al. 2015), CeO 2 (You et al. 2016), and graphene-oxide (Ahmed and Rodrigues 2013), which led to an increased in both SVI and turbidity values. The improvement in the biomass compaction can be considered as positive effect when TiO 2 NPs arrive to WWTP, especially to the primary and secondary clarifiers.

Morphological interactions between TiO 2 NPs and activated sludge
The way TiO 2 NPs interacted with microorganisms was elucidated by electron microscopy. TEM imaging was performed for embedded samples of activated sludge exposed to TiO 2 NPs, treating all three types of wastewaters. During TEM observation, electrodense and particulate material (black color) was observed attached to the membrane of microorganisms (Figure 8), presumably TiO 2 NPs. This electrodense material presented aggregates smaller than 500 nm, and in some of the samples were surrounded by organic material (gray color). These aggregates are consistent with the description of heteroaggregates proposed by Sani-Kast et al. (2015) and Dale et al. (2015). The integrity of the cell membrane of microorganisms in the control and SWW imaging is similar ( Figure 7A, B), without internalized electro-dense material. A similar pattern was observed for the sample of the experiment treating FWW ( Figure 7D). However, in the case of the experiment treating RWW ( Figure 7C), electrodense material as crystals and abundant cellular detritus was observed. This indicates that, in addition to the TiO 2 NPs supplied in the experiments, the RWW already contains water-insoluble metallic material. Although internalization of some NPs such as ZnO (Sirelkhatim et al. 2015;Cervantes-Avilés et al. 2016) and CuO (Perreault et al. 2012) has previously been considered as a pattern that leads to apoptosis of cells, the internalization of electrodense material in cells was not observed for the samples analyzed. Electrodense material was frequently observed attached to the cell membrane. To confirm that electrodense particulate material correspond to TiO 2 NPs, elemental mapping was performed in the sample of activated sludge treating RWW.
Electrodense material was confirmed to correspond to Ti (Figure 9). Overlapping the layers of Ti-K, Ti-L, and O-K, can be concluded that Ti corresponds to TiO 2 at the nanoscale. Distribution of Ti in image captured in HAADF-STEM confirmed that this material is attached to the cell membrane ( Figure 9). Therefore, one of the mechanisms by which TiO 2 NPs could harm microorganisms is through damage to the cell membrane, such as disruption. Some studies about the toxicity of TiO 2 NPs in isolated microorganisms reported the chemical activity of these particles on the phospholipid membrane of cells (Ma and Lin 2013;Neale et al. 2015). Since our experiments were performed without exposition to UV light, the main mechanism of damage from TiO 2 NPs to microorganisms of activated sludge occurs potentially through physical interactions conducting to the cell membrane rupture. This would lead to lysis of microorganisms present in activated sludge, affecting the processes involved in the biological macronutrient removal from wastewater.

Conclusions
In this work, the effects of the type of wastewater such as synthetic, raw, and filtered, in the removal of macronutrients by the activated sludge process exposed to TiO 2 NPs were evaluated. The most representative impact of TiO 2 NPs was the inhibition of up to 22% of ammoniacal nitrogen removal for the highest load of TiO 2 NPs. Although this inhibition was related to the combined effect of NPs concentration and content of BOD 5 and NH 3 -N, there is evidence of alterations of nitrification process when real wastewater is used. Organic matter removal was not substantially affected; however, oxidation of the inorganic compounds in real wastewaters, commonly measured as sCOD can be affected by the presence of TiO 2 NPs. Phosphate removal and compaction of the sludge are positive effects when TiO 2 NPs are in activated sludge reactors, which can be explored carefully to avoid overload of NPs in activated sludge reactors.
The use of real wastewater in ecotoxicological experiments of this and other NPs is highly encouraged due to effects of NPs were limited for synthetic substrate. Moreover, the most adverse effects were detected for real matrices of wastewater. Furthermore, although activated sludge still removes macronutrients in the presence of high load of TiO 2 NPs, the removal mechanisms could be physicochemical rather than biological.
Author contribution P. Cervantes-Avilés: conceptualization, methodology, investigation, writing of original draft; writing review and edition, funding acquisition; A.N. Saber: data curation, formal analysis, writing of original draft, writing review and edition; A. Mora Polanco: formal analysis, writing review and edition; conceptualization; J. Mahlknecht: formal analysis, writing review and edition; G. Cuevas-Rodríguez: data curation, resources, supervision, funding acquisition, project administration, writing review and edition. Fig. 9 Elemental mapping of S-K, P-K, C-K, O-K, Ti-K, and Ti-L in activated sludge exposed to raw wastewater (RWW) containing 1000 mg/ L of TiO 2 NPs Funding This work was supported by the Research Direction and Support to Postgraduate of the University of Guanajuato. Pabel Cervantes-Avilés received funding from National Council of Science and Technology of Mexico and from Sciences Department of Tecnologico de Monterrey Campus Puebla.
Data availability The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

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Competing interests The authors declare no competing interests.