3.1 Removal of organic matter in presence of TiO2 NPs
The removal of organic matter in the three types of wastewater was evaluated by relating the initial and final concentration (C/C0) of sCOD and dTOC (Fig. 1 ), which serves as indicative such removal in presence of TiO2 NPs. According to these results, the control groups presented removal of sCOD higher than 70% for the three types of wastewater (Fig. 1A), which corresponds to the typical removal for activated sludge systems (van Loosdrecht et al. 2016). However, the impact of TiO2 NPs on sCOD removal was different for all types of wastewater. Activated sludge treating SWW was not affected in the presence of all TiO2 NPs concentrations, in terms of sCOD removal, which were statistically similar to its control group. Conversely, in the case of RWW and FWW, the removal of sCOD decreased linearly as the concentration of TiO2 NPs increased, reaching 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 TiO2 NPs over sCOD removal can be attributed to the type of wastewater and concentration of NPs. Although the lowest sCOD removal was observed for real wastewater without suspended solids (FWW), there was no effect for assays with SWW at the experimental concentration of NPs. The non-influence of the TiO2 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, TiO2 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 presence of high load of TiO2 NPs, this removal is sensible to the wastewater characteristics, including the suspended solids content.
The removal of dTOC in the control reactors was at least 70% for the three types of wastewater (Fig. 1B). However, the organic carbon removal was most affected for RWW containing concentrations higher than 1000 mg/L of TiO2 NPs, which affected the removal of dTOC up to 6%. In contrast, the removal of organic carbon in experiments using SWW and FWW was not affected at all. This means that the presence of high concentrations of TiO2 NPs in activated sludge process can have a slight effect on the removal of organic matter. 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 TiO2 NPs and induced a deficiency of 7% in the organic matter removal.
The removal of sCOD was more affected than the dTOC removal by the presence of TiO2 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 occurrence of TiO2 NPs. The poor performance of aerobic microorganisms can be confirmed by previous experiments treating real wastewater in presence of TiO2 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 TiO2 NPs in the RWW or FWW could affect the microorganisms responsible for the oxidation of the inorganic compounds measured as sCOD in wastewater. This is in line with previous reports which has found a decrease of the viable bacteria of the genera related to the aerobic activity in activated sludge due to the presence of TiO2 NPs (Ma et al. 2015). Another study reported changes in bacterial diversity of activated sludge exposed to 1 mg/L of TiO2 NPs for 40 days, which decreased from the second day of exposure to NPs (Qiu et al. 2016). The effect of TiO2 NPs in the activated sludge is a decrease in the sCOD removal when real wastewater is used, which could be attributed to the damage of aerobic microorganisms.
Besides the type of wastewater, the 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 TiO2 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 TiO2 NPs, organic colloids, and suspended solids has 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, the formation of small and stable heteroaggregates in real wastewaters may explain a potential 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 plays an important role when evaluating toxicity by NPs.
3.2 Ammoniacal nitrogen removal in presence of TiO2 NPs
The ammoniacal nitrogen removal in activated sludge is mainly via nitrification-denitrification. In the control experiments using the three types of wastewater, the ammoniacal nitrogen removals were 64% for RWW and FWW, and 76% for SWW (Fig. 2). These removal percentages are commonly observed in activated sludge reactors treating domestic wastewater. However, when the activated sludge was exposed to TiO2 NPs, the ammoniacal nitrogen removal was negatively affected for the three types of wastewater, with the only exception of FWW containing 500 mg/L that presented similar removal than control group (Fig. 2).
The removal of ammoniacal nitrogen in SWW containing TiO2 NPs was less affected than for the other types of wastewater, decreasing the removal between 5% and 10% for all TiO2 NPs concentrations (Fig. 2). Interestingly, the highest inhibition (10%) of ammoniacal nitrogen removal was observed in the presence of 500 mg/L of TiO2 NPs and decreased as the TiO2 NPs content increased. Conversely, experiments with RWW and FWW indicated that inhibition of ammoniacal nitrogen removal increased as the concentration of TiO2 NPs in activated sludge increased. In experiments with RWW, ammoniacal nitrogen removal decreased 22% in the presence of 2000 mg/L of TiO2 NPs, while for FWW, the removal decreased 12% for the same concentrations of NPs. These results clearly indicate that the effect of TiO2 NPs on ammoniacal nitrogen removal was more severe when real wastewater with suspended solids is used.
In order to assess the ammoniacal nitrogen oxidation process, NO2-N and NO3-N concentrations were determined at the end of the macronutrient removal tests (Figs. 3A and 3B, respectively). In the case of SWW trials, the NO2-N formation in presence of TiO2 NPs were similar to the control. However, the NO3-N formation decreased as the concentration of TiO2 NPs increased. Hence, for SWW containing TiO2 NPs, the main effect occurred in the second step of the nitrification process.
Formation of NO2-N and NO3-N from RWW containing TiO2 NPs was higher or similar to the control group, e.g., NO3-N production increased 7% in presence of 2000 mg/L of TiO2 NPs compared to control group. Conversely, in experiments treating FWW with TiO2 NPs, the production of NO2-N and NO3-N by activated sludge was affected for the NPs. This means that TiO2 NPs in wastewater without TSS may damage the aerobic oxidizing bacteria (AOB) of the activated sludge, which has been recently demonstrated by sequencing the activated sludge exposed to TiO2 NPs (Li et al. 2020). Li et al. (2020) performed a quantitative polymerase chain reaction (qPCR) tests followed by Illumina high-throughput sequencing of the activated sludge exposed to a high cumulative concentration of TiO2 NPs (450–900 mg/L) and found that AOB are mainly affected by acute exposition to these NPs.
In general, ammoniacal nitrogen removal was affected in the presence of TiO2 NPs regardless of the type of wastewater. However, first and second steps of nitrification in presence of TiO2 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 TiO2 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 TiO2 NPs. Adsorption of nitrogen compounds in SWW has been reported for CeO2 NPs (Gómez-Rivera et al. 2012), which are non-soluble NPs such as TiO2 NPs at the experimental pH (7–8). In general, the nitrogen removal processes performed by activated sludge exposed to TiO2 NPs have been affected regardless of the type of wastewater. Although NPs can damage the nitrification processes, especially for synthetic media and real wastewater without suspended solids, the nitrogen removal could be carried out by physicochemical mechanisms, such as adsorption of nitrogen compounds on TiO2 NPs.
3.3 Orthophosphate removal in presence of TiO2 NPs
The orthophosphate removal in the activated sludge process containing TiO2 NPs was enhanced for all three types of wastewater (Fig. 4). In experiments with SWW, the highest PO4 − 3 removal was observed in the presence of 1000 mg/L of TiO2 NPs, with 34% higher removal than the control group. However, the most noticeable improvement was observed for experiments with real wastewater, which increased the removal of PO4 − 3 as the concentration of TiO2 NPs 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 PO4 − 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 TiO2 NPs may affect the bacterial performance, the possible explanation of phosphate removal could be the adsorption and co-precipitation of PO4 − 3 adsorbed by TiO2 NPs (Qian et al. 2017; Zhang et al. 2019). The ability of TiO2 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), 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 7-7.8, and the sources of PO43− in real wastewater are commonly detergents, the TiO2 NPs could be an adsorbent agent during wastewater treatment.
The removal of orthophosphate via TiO2 NPs could be considered as a positive effect that is due to potential adsorption. However, the affinity between TiO2 NPs and orthophosphate should be proven through measuring orthophosphate adsorption capacity by TiO2 NPs, and other parameters that complement the characterization of NPs, such as zeta potential, specific surface area, among others. Considering that TiO2 NPs are widely investigated for water treatment due their photocatalytic activity, and their potential contribution to the extracellular electron transfer (Wang et al. 2016), the ability of TiO2 NPs to remove orthophosphate from wastewater could promote their use during biological wastewater treatment.
3.4 Effect of TiO2 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 Fig. 5. For all different types of wastewater, the flocs size of activated sludge was not affected by the presence of TiO2 NPs in the reactors. However, the average values of SVI in presence of TiO2 NPs were 50% lower than control SVI values for all types of wastewater (Fig. 6), 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 TiO2 NPs regardless of the type of wastewater used (Fig. 6). These findings suggest that the flocs exposed to TiO2 NPs kept the same size but had a higher density, settling faster and gaining greater compaction of biomass in reactors with the highest concentration of NPs. Despite of the compaction of flocs by the potential adsorption of NPs, the microorganisms present in the activated sludge and exposed to TiO2 NPs performed metabolic activities that allowed the treatment of wastewater regardless of the type of substrate, as indicated by the results about the removal of macronutrients. In previous experiments, TiO2 NPs at cumulative concentrations of (nearly 120 mg/L) have decreased the SVI of activated sludge in a sequential batch reactor (Qiu et al. 2016). However, some other nanomaterials have induced changes in size and stability of flocs such as CuO (Hou et al. 2015), CeO2 (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 TiO2 NPs arrive to WWTP, especially to the primary and secondary clarifiers.
3.5 Morphological interactions between TiO2 NPs and activated sludge
The way TiO2 NPs interact with microorganisms was elucidated by electron microscopy. TEM imaging was performed for embedded samples of activates sludge exposed to TiO2 NPs, treating all three types of wastewater. During TEM observation, electrodense and particulate material (black color) was observed attached to the membrane of microorganisms (Fig. 7), presumably TiO2 NPs. This electrodense material presented aggregates smaller than 500 nm, and in some of the samples were surrounded by organic material (grey 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 (Fig. 7A and B), without internalized electro-dense material. A similar pattern was observed for the sample of the experiment treating FWW (Fig. 7D). However, in the case of the experiment treating RWW (Fig. 7C), electrodense material as crystals and abundant cellular detritus were observed. This indicates that, in addition to the TiO2 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. In order to confirm that electrodense particulate material correspond to TiO2 NPs, elemental mapping was performed in the sample of activated sludge treating RWW.
In the elemental mapping of activated sludge exposed to TiO2 NPs treating RWW, the electrodense material was confirmed to correspond to Ti (Fig. 8). By overlapping the layers of Ti-K, Ti-L, and O-K, it can be concluded that Ti corresponds to TiO2 at the nanoscale. The distribution of Ti in the micrograph captured in HAADF-STEM confirmed that this material is attached to the cell membrane (Fig. 8). Therefore, one of the mechanisms by which TiO2 NPs could harm microorganisms is through damage to the cell membrane, such as disruption. Some studies about the toxicity of TiO2 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). Moreover, TiO2 NPs have biocide effect when exposed to UV radiation as demonstrated by previous studies (Cai et al. 2014; Qian et al. 2017). Since our experiments were performed without exposition to UV light, the main mechanism of damage from TiO2 NPs to microorganisms of activated sludge possibly occurs through physical interactions conducting to the cell membrane rupture (Ma and Lin 2013). This would lead to lysis of microorganisms present in activated sludge, affecting the processes involved in the biological macronutrient removal from wastewater.