A comparative study on the chronic responses of titanium dioxide nanoparticles on aerobic granular sludge and algal-bacterial granular sludge processes

A comparative study on the chronic responses of titanium dioxide nanoparticles on aerobic granular sludge and algal-bacterial granular sludge processes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A comparative study on the chronic responses of titanium dioxide nanoparticles on aerobic granular sludge and algal-bacterial granular sludge processes Alfonz Kedves, Henrik Haspel, Çağdaş Yavuz, Bence Kutus, Zoltán Kónya This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4629286/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Nov, 2024 Read the published version in Environmental Science and Pollution Research → Version 1 posted 5 You are reading this latest preprint version Abstract The chronic effects of titanium dioxide nanoparticles (TiO 2 NPs) on aerobic granular sludge (AGS) and algal-bacterial granular sludge (ABGS) was examined in this study. Sequencing batch bioreactors (SBRs) and photo sequencing batch bioreactors (PSBRs) were operated with synthetic wastewater containing 0, 1, 5, 10, 20, 30, and 50 mg L − 1 TiO 2 NPs for 10 days. Nanoparticles at concentrations of 1 and 5 mg L − 1 did not impact nutrient removal but led to an increase in extracellular polymeric substances (EPSs), primarily in protein (PN). With increasing nanoparticle concentration, the negative effect became more pronounced, mainly in the AGS SBRs. At 50 mg L − 1 TiO 2 , chemical oxygen demand (COD), ammonia-nitrogen (NH 3 -N), and phosphorus (PO 4 3− ) removal decreased by 20.9%, 12.2%, and 35.1% in AGS, respectively, while in ABGS, they reached only 13.4%, 5.7%, and 14.2%. ABGS exhibited steady-state nutrient removal at 30 and 50 mg L − 1 TiO 2 NPs after around 5 days. The higher microbial activity and EPS content in the sludge, coupled with the symbiotic relationship between algae and bacteria, contributed to the higher tolerance of ABGS to nanoparticles. Finally, although nanoparticles reduced biomass in both types of bioreactors, the accumulation of TiO 2 NPs in the sludge, confirmed by Energy-dispersive X-ray spectroscopy analysis, and the absence of detectable titanium concentrations in the effluent wastewater, measured by Inductively-coupled plasma mass spectrometry, may be attributed to the specific operational conditions of this study, including the relatively short operation period (10 days) and high initial MLSS concentration (6 g L − 1 ). titanium dioxide nanoparticles aerobic granular sludge algal-bacterial granular sludge microbial activity chronic response Figures Figure 1 Figure 2 Figure 3 Figure 4 Highlights The long-term impact of TiO 2 NPs on AGS and ABGS bioreactors was investigated. NPs were enriched in both sludges, and showed higher toxicity on AGS than on ABGS. 1 and 5 mg L -1 TiO 2 NPs had no adverse impact on nutrient removal. Higher protein content reduced NPs toxicity on ABGS at > 5 mg L -1 concentrations. Filamentous microorganisms reduced the settleability of ABGS. 1. Introduction Engineered nanoparticles (NPs) have found extensive applications due to their unique physical and chemical attributes in various fields including catalysts (Hou et al., 2019 ), electronics (Qamar et al., 2023 ), textiles (Rashid et al., 2021 ), and others. Titanium dioxide (TiO 2 ) NPs, as a commercially significant and widely utilized nanoparticle, have gained prominence for their versatile applications such as in personal skincare products (Kumari and Virdi, 2023 ), toothpastes (Al-Salman Fadheela et al., 2020 ), and as a white pigment material (Mohammadparast and Mallard, 2023 ) owing to their excellent stability and photocatalytic activity. TiO 2 NPs have emerged as one of the most extensively manufactured nanomaterials (> 10,000 tons/year), it raises concerns regarding their potential environmental release, posing risks to microorganisms in the ecosystems (Kedves and Kónya, 2024 ). Numerous studies have demonstrated the potential negative effects of these NPs on various organisms including algae (Natarajan et al., 2022 ), bacteria (Tahir et al., 2023 ), and fungi (Najibi Ilkhechi et al., 2021 ), underscoring the need for a comprehensive assessment and management of their environmental impact. The widespread use of nanoparticles has led to their significant presence in wastewater. TiO 2 nanoparticles were found both in sewage and sludge at concentrations up to 3 mg L − 1 (Wang et al., 2017 ) and 23 mg kg − 1 (Gottschalk et al., 2009 ), respectively. During biological wastewater treatment, TiO 2 NPs could have numerous negative effect on bioreactors efficiency. Yuan et al. ( 2021 ) reported that the specific resistance to filtration of activated sludge (AS) increased after introducing 1 mg L − 1 TiO 2 NPs, while in another study the microbial diversity slightly shifted at 2 mg L − 1 TiO 2 NPs after 8 hours (Cervantes-Avilés et al., 2021 ). In studies, where nanoparticle concentrations were increased to 5 or 10 mg L − 1 , either significant decrease in the removal rates of total nitrogen and phosphate after 6–8 days of exposure (Li et al., 2017 ; Zheng et al., 2011 ), or a decline in floc stability was observed (Zhou et al., 2019 ). Mishima and Nakamura ( 1991 ) were the first to report on the aerobic granular sludge (AGS) wastewater treatment process, which has emerged as a promising technology for treating municipal and industrial waters. AGS offers advantages over activated sludge, such as greater stability, higher biomass content in the bioreactor, smaller footprint, and higher tolerance to toxic substances (Q. Jiang et al., 2022 ; Zhou et al., 2022 ). Consequently, it has become one of the most extensively studied technologies in wastewater treatment over the past 30 years. Recently, algal-bacterial granular sludge (ABGS) has garnered attention due to its strong symbiotic relationship between algae and bacteria, resulting in excellent pollutant removal capabilities from wastewater (Fard and Wu, 2023 ; Liu et al., 2022 ). Both AGS and ABGS have demonstrated the ability to simultaneously remove organic matter, phosphorus, and nitrogen from high-strength industrial wastewater (Li et al., 2022 ; Lochmatter et al., 2013 ), treat leachate (Ilmasari et al., 2023 ), and remove heavy metals (Kedves et al., 2024 ; Purba et al., 2020 ). These two technologies are suitable for widespread applications due to the structure of the granules and the high amount of extracellular polymeric substances (EPS) they contain. EPS consists of two main components: polysaccharides and proteins. While the former predominantly forms the outer part of the granules, the proteins typically constitute the main component of the granules’ inner layer (Nuramkhaan et al., 2019 ; Samaei et al., 2023 ). Since AGS technology is already used on an industrial scale for the treatment of industrial and municipal wastewater (Hamza et al., 2022 ), several studies examined the impact of the increasingly produced nanoparticles on the AGS wastewater treatment. In wastewater treatment systems, mixed liquor suspended solids (MLSS) are directly related to the system resilience against contaminants (Wang et al., 2023 ). Accordingly, where possible, the amount of nanoparticles introduced in the various experiments was also calculated based on MLSS. Quan et al. ( 2015 ) observed a decrease in biomass and in microbial activity during the long-term exposure of silver NPs of 5 and 50 mg L − 1 (1.67 and 16.67 mg gMLSS − 1 ). In another studies, the increase of cupric oxide (CuO) NPs concentration from 5 to 50 mg L − 1 (1.67 to 16.67 mg gMLSS − 1 ) significantly reduced the removal of phosphorus (Zheng et al., 2017 ), while Cu NPs at 5 mg L − 1 (0.38 mg gMLSS − 1 ) inhibited the nitrogen removal capacity by 51.9% during long-term exposure (Y. F. Cheng et al., 2019 ). The shock loading of zinc oxide (ZnO) NPs at 1-100 mg L − 1 (0.23–22.73 mg gMLSS − 1 ) caused acute toxic effect on microbial activity (He et al., 2017b ), whilst the nitrification and denitrification processes were inhibited even at 20 mg L − 1 in the long term (He et al., 2017a ). Moreover, nanoparticles tend to enrich in the sludge, as Xiao et al. ( 2024 ) found the 95% of ZnO NPs in the sludge when the wastewater contained 10 mg L − 1 NPs. Another study showed no harmful effects of ZnO NPs up to 1 mg L − 1 (0.17 mg gMLSS − 1 ), whereas the ammonia and phosphorus removal significantly decreased at 10 mg L − 1 (1.67 mg gMLSS − 1 ) (Xiao et al., 2022 ). So far, only one study examined the effect of TiO 2 NPs on AGS at a single concentration of 50 mg L − 1 (Y. Jiang et al., 2022 ), while in the case of ABGS, the effect of TiO 2 NPs on granule formation was studied (B. Li et al., 2015 ). Considering that i) previous studies covered the effects of Zn and Cu-based NPs, ii) TiO 2 NPs are present in wastewater, iii) AGS is used on full-scale, and iv) the future industrial-scale utilization of ABGS is highly likely, the investigation of the effects of TiO 2 NPs on AGS and ABGS became vital. Herein, we report the chronic impact (over 10 days) of TiO 2 NPs at a series of concentrations (0, 1, 5, 10, 20, 30, and 50 mg L − 1 , corresponding to 0.17, 0.83, 1.65, 3.31, 4.96, and 8.26 mg gMLSS − 1 ) on the nutrient removal efficiency, the microbial activity, and the extracellular polymeric substances of both, the AGS and ABGS processes. These findings provide fundamental insights into the TiO 2 nanoparticle tolerance on aerobic granular and algal-bacterial aerobic granular sludge, and thus are expected to contribute to the knowledge on the operation of AGS and ABGS in treating TiO 2 NPs contaminated wastewater. 2. Materials and methods 2.1. Experimental All bioreactors were inoculated with aerobic granular sludge and algal-bacterial granular sludge freshly collected from mother reactors operated for over half a year in our laboratory. The AGS sequencing batch reactors (SBRs) and ABGS photo-sequencing batch reactors (PSBRs) were continuously fed with synthetic wastewater (SWW) ( Table S1 ) containing 1, 5, 10, 20, 30, and 50 mg L − 1 TiO 2 NPs, every four hours for 10 days. The hydraulic retention time of SWW in the bioreactors was 8 hours with an effective volume of 1.4 L. The average mixed liquor suspended solids and sludge volume index (MLSS and SVI 5 ) were 6.05 ± 0.2 g L − 1 and 24.6 ± 0.1 mL g − 1 , respectively. Further details on the components of the bioreactors, the configuration of SBR and PSBR, and the constituents of SWW are provided in the Supplementary Information . TiO 2 nanoparticles were synthesized through a modified nonaqueous solvothermal process (Z. Q. Li et al., 2015 ), and were characterized by using a Rigaku Miniflex-II X-ray diffractometer ( Fig. S1 a ), a Bruker Vertex 70 FT-IR instrument ( Fig. S1 b ), and a Hitachi S-4700 Type II scanning electron microscope (SEM) with 10 kV accelerating voltage equipped ( Fig. S1 d ) with a Röntec QX2 energy dispersive X-ray spectrometer (EDX) ( Fig. S1 c , see Supporting Information ). 2.2. Analysis of effluent water, sludge properties, and microbial activity Every 12 hours over a period of 10 days, the concentration of chemical oxygen demand (COD), phosphorus (PO 4 3− ), nitrate-nitrogen (NO 3 -N), nitrite-nitrogen (NO 2 -N), and ammonia nitrogen (NH 3 -N) in the effluent of the SBRs and PSBRs was measured using Hanna kits (HI93754B-25, HI93717-01, HI93728-01, HI93708-01, and HI93715-01) with a spectrophotometer HI83399. Additionally, titanium content in the effluent wastewater was measured via Inductively-coupled plasma mass spectrometry (ICP-MS) using an Agilent 7900 instrument with 15.0 L min − 1 Ar carrier gas. Samples were filtered through a 0.45 µm syringe filter and stored at − 4°C, and prior to analysis cc. HNO 3 (NORMATOM by VWR Chemicals, final concentration: 1 wt%) and solutions of the internal standards 45 Sc and 89 Y (ARISTAR by VWR Chemicals, final concentration: 100 ppb) were added. Calibration was performed between 0 and 50 ppb Ti using the same procedure, signals of the 47 Ti, 48 Ti, and 49 Ti isotopes were monitored with and without using He cell collision mode. On the tenth day of the experiments, the MLSS, SVI 5 , and EPS content of both granular sludges were determined. Additionally, to assess the impact of TiO 2 NPs on the microbial activity, specific phosphorus uptake rate (SPUR), specific ammonia, nitrite, and nitrate uptake rates (SAUR, SNIUR, and SNUR) were determined. Finally, the effect of TiO 2 NPs on the structure of the aerobic and algal-bacterial granular sludge was examined using a Hitachi S-4700 Type II scanning electron microscope (SEM) and EDX. Detailed descriptions of the MLSS, SVI 5 , EPS, SPUR, SAUR, SNIUR, and SNUR measurements, as well as the preparation of sludge samples for SEM investigation, can be found in the Supplementary Information . 3. Results and discussion 3.1. Sludge properties and EPS production in AGS and ABGS Compared to activated sludge (AS), both AGS SBRs and ABGS PSBRs contain a significant amount of biomass, along with high levels of extracellular polymeric substances (EPSs), primarily consisting of protein (PN) and polysaccharide (PS). EPS not only protect microorganisms from harmful substances, such as nanoparticles, but also contribute to good settling ability and possess biosorption properties (Hakim et al., 2023 ; Zheng et al., 2019 ). At the beginning of each experiment, the MLSS and SVI 5 of the sludges were approximately 6.05 ± 0.2 g L − 1 and 24.6 ± 0.1 mL g − 1 , while the EPS content in AGS and ABGS was 106.5 ± 6.1 and 129.6 ± 6.7 mg g − 1 MLVSS, respectively. As shown in Fig. 1 a, numerous microorganisms were embedded in the polymer matrix on the surface of the aerobic granules, whereas algae can be seen alongside other microorganisms within the exterior of the algal-bacterial sludge (Fig. 1 b) (Salimon et al., 2020 ). Based on the sludge EDX analysis, the main differences between the types of sludge are nitrogen and phosphorus contents. In AGS 8.78 and 0.58 at%, in ABGS 9.14 and 0.88 at% nitrogen and phosphorus were found, respectively ( Fig. S2 ). The difference originates from the presence of algae, as these microorganisms are capable of accumulating high amounts of these elements (Kube et al., 2018 ). TiO 2 NPs (with spherical morphology of a diameters ranging between 30 and 130 nm, see Fig. S1 ) at concentrations of 5 and 10 mg L − 1 led to an increase in EPS in both granular sludges. This increase was accompanied by the rise in the PN/PS ratio, attributed to a higher volume of PN secretion after 10 days of exposure (Fig. 2 ). At 10 mg L − 1 NPs, the PN content increased by 24% from 59.8 ± 5.1 to 74.2 ± 5 mg g − 1 MLVSS, and by 60% from 82.3 ± 4.7 to 132.3 ± 4.3 mg g − 1 MLVSS in AGS and ABGS, respectively. These results suggest that ABGS has a higher tolerance to the negative effects of nanoparticles compared to AGS, as it exhibited a greater capacity for protein secretion. In two previous studies, He et al. (2017) and Xiao et al. ( 2022 ) investigated the chronic effects of zinc oxide nanoparticles on AGS and ABGS. During the experiments, it was observed that ZnO NPs caused a reduction in the amount of sludge EPS at concentrations as low as 10 mg L − 1 , significantly impacting the efficiency of the reactors. In our case, however, following the administration of 10 mg L − 1 of TiO 2 , the quantity of polymer materials was higher than in the initial sludge. The low toxicity promoted bacterial production of polymer materials in the sludge, whereas higher toxicity reduced its quantity (Quan et al., 2015 ). We, therefore, presume that zinc oxide nanoparticles may be more toxic to the granular sludge. The EPS content declined, and sludge properties changed with increasing nanoparticle content (20, 30, and 50 mg L − 1 TiO 2 NPs) in AGS. The polymer volume decreased to 97.4, 83.2, and 68.3 mg g − 1 MLVSS, and the PN/PS ratio dropped to 1.22, 1.1, and 0.99, below those of the initial sludge (Fig. 2 a). Simultaneously, the MLSS decreased to 5.96, 5.52, and 4.83 g L − 1 , while significant changes were not observed in the SVI 5 . This data suggests that the persistent presence of TiO 2 NPs in the influent led to a decrease in biomass production in the AGS reactor. The long-term presence of low concentration (5 and 10 mg L − 1 ) CuO NPs had a similar effect on AGS, i.e., the secretion of EPS was promoted, while a decrease in biomass and EPS content was observed at higher concentrations (50 mg L − 1 ) (Zheng et al., 2017 ). The decreasing amount of biomass can be explained by the decrease in the size of the granules, since their average diameter declined from 600 to 350 µm at 50 mg L − 1 NPs after 10 days. Along with the size reduction, the external structure of AGS also changed (Fig. 1 c), as the polymer layer disappeared and rod-shaped microorganisms can be seen. The latter suggests a significant change in the microbial structure. The addition of TiO 2 NPs at 20, 30, and 50 mg L − 1 had distinct effects in PSBRs and SBRs. Although significant changes in sludge properties were not observed until the day 10 upon the addition of 20 and 30 mg L − 1 NPs, the PN content remained higher compared to that of the initial ABGS with a PN/PS ratio of 2.45 and 2.11, respectively. In contrast, the PN and PS decreased from 82.3 ± 4.7 and 47.3 ± 3.9 mg g − 1 MLVSS to 72.7 ± 5.1 and 40.7 ± 4.1 mg g − 1 MLVSS at 50 mg L − 1 TiO 2 NPs, respectively (Fig. 3 b). Simultaneously, the MLSS decreased to 3.69 ± 0.5 g L − 1 , and the SVI 5 increased to 165.9 ± 7.6 mL g − 1 . The significant change occurred because filamentous microorganisms appeared on the outer part of the granules (Fig. 1 d), leading to a loose structure with poorer settling ability and an increase in the granular sludge size from 0.6 mm to 2–8 mm. 3.2. Performance on nutrients removal of AGS and ABGS In order to characterize the efficiency of a biological wastewater treatment, the removal of organic matter, nitrogen, and phosphate need to be measured. The performance of AGS and ABGS bioreactors was assessed by recording the COD, NH 3 -N, NO 2 -N, NO 3 -N, and PO 4 3− every third cycle (every 12 h) in each experiment over 10 days (Fig. 3 ). Although the removal of nutrients remained stable after introducing the nanoparticles at concentrations as low as 1 and 5 mg L − 1 , increasing NP content resulted in a gradually decreasing removal efficiency. COD Upon the introduction of 10 and 20 mg L − 1 nanoparticles into the AGS-SBR, the COD in the effluent began to increase after 5 and 2 days, and rising from 79 ± 2.1 to 132 ± 4.2 mg L − 1 , and 156.9 ± 3.7 mg L − 1 on day 10, respectively (Fig. 3 a). In contrast, in the ABGS PSBRs, the COD started to increase after 7 and 5 days with levels changing from 75 ± 1.4 to 101 ± 1.9 mg L − 1 , and 120 ± 1.7 mg L − 1 by day 10, respectively. At concentration of 50 mg L − 1 , the COD removal rate declined after just half a day due to the shock loads of titanium. In the AGS, a continuous increase in COD was measured in the effluent reaching 331.5 ± 4.9 mg L − 1 by day 10, while in the ABGS, COD removal reached a steady state after 6 days. Overall, the observations indicated that TiO 2 nanoparticles had a lower impact on the heterotrophic microbial community in algal-bacterial sludge compared to other studies. In previous research, where the long-term impact of TiO 2 nanoparticles on activated sludge was investigated, a decline in COD removal was already observed at concentrations as low as 1 or 2 mg L − 1 (Cervantes-Avilés et al., 2021 ; Li et al., 2019 ). This suggests that heterotrophic microorganisms in AGS and ABGS are more tolerant to titanium dioxide nanoparticles, likely due to their robust structure and high EPS content. NH3 Here, we observed a decrease in NH 3 -N removal after 5-2.5, and 8.5–7.5 days upon the exposure of the AGS and ABGS by 10 and 20 mg L − 1 NP, respectively. The further increase in the TiO 2 NPs content (30–50 mg L − 1 ) caused significant inhibition of the aerobic sludge within half a day, with removal efficiency decreasing from 99.94–92.87%, and 88.33% by day 10 (Fig. 3 b). The negative impact of nanoparticles on ammonia removal was less pronounced in ABGS. Although efficiency declined after the first day at 30 and 50 mg L − 1 NPs, levels stabilized around days 6 and 5 with the removal rates reaching 96.31% and 94.26% by day 10, respectively. NO2 The NO 2 -N contents in the effluent (Fig. 3 c) exhibited similar variations to ammonia-nitrogen, suggesting that nanoparticles had a negative effect on both aerobic ammonia and nitrite oxidizing microorganisms. These microbes are predominantly located in the outer part of the granules, allowing TiO 2 NPs to potentially attach to their surface and inhibit their microbial activity. Zheng et al. ( 2011 ) also observed a decreased removal rate of both ammonia and nitrite in activated sludge exposed to 50 mg L − 1 TiO 2 NPs, along with a drastically dropping nitrogen elimination to 24.4%. Throughout the experiments, the NO 3 -N content in the effluent remained constant in the algal-bacterial sludge. In the AGS, on the other hand, it increased to 10.2 and 36.7 mg L − 1 as a response to the introduction of 30 and 50 mg L − 1 TiO 2 NPs, respectively. PO When considering phosphorus removal, notable distinctions between AGS and ABGS are evident (Fig. 3 d). At 10 and 20 mg L − 1 NPs, phosphorus removal in ABGS began to decrease after 9 and 6 days with effluent PO 4 3− contents of 0.84 ± 0.05 and 1.36 ± 0.12 mg L − 1 after 10 days, respectively. However, in AGS, the efficiency declined after 3 and 2 days, resulting in PO 4 3− amounts of 1.69 ± 0.8 and 3.06 ± 0.16 mg L − 1 at day 10, respectively. Additionally, when titanium content of the influent wastewater was 30 and 50 mg L − 1 , the PO 4 3− content in the effluent stabilized after 7 and 6 days in ABGS, respectively. By day 10, however, the phosphate concentration was similar or lower than that in AGS in response to 20 mg L − 1 TiO 2 NPs. Upon further increasing the TiO 2 concentration, the phosphorus content increased from 0.51 ± 0.05 to 7.52 ± 0.39 mg L − 1 in the latter along with a drop in the removal rate to 62.4%. In contrast, phosphorus concentration increased from 0.35 ± 0.05 to 3.18 ± 0.13 mg L − 1 , leading to a reduction in removal efficiency to 84.1% in ABGS. Li et al. ( 2017 ) also observed a decrease in phosphorus removal efficiency in AS, where it reduced from 89–68%, suggesting that granular sludges exhibit significantly higher resistance against titanium-dioxide. A correlation between the PO 4 3− , and the NH 3 -N and NO 2 -N nitrogen removal results was found. The correlation is partly due to the fact that microorganisms that are sensitive to higher concentrations of ammonia and nitrite in the wastewater are also the ones capable of accumulating and removing phosphate, as previously observed (Zheng et al., 2017 ). The better nutrient removal efficiency of ABGS may also originate from the algae's high tolerance to heavy metals and their ability to remove nutrients from wastewater (Nguyen et al., 2022 ; Priyadarshini et al., 2019 ). In AGS, chronic exposure to titanium dioxide nanoparticles led to a more pronounced decrease in nutrient removal efficiency, resulting in a lower biomass quantity compared to that in the initial sludge after 10 days. This reduction in biomass could have potentially contributed to the release of nutrients from the polymer matrix, exacerbating the effluent's COD, nitrogen, and phosphorus content. Continuous monitoring of the titanium content in the effluent wastewater was conducted throughout the experiments, whose presence could not be detected. This in turn highlights the importance of preventing the pollutant release into the environment, if it accumulates in the sludge. The titanium content in AGS and ABGS was 8.25 and 2.66 at% at 50 mg L − 1 TiO 2 NP concentration ( Fig. S3 ). The difference is likely due to the low penetration depth of the EDX sludge analysis. Since numerous filamentous microorganisms covered the surface of the granules in ABGS (Fig. 1 d), only part of the elemental information comes from the Ti-rich granules, which in turn results in a lower measured titanium content. 3.3. Influence of TiO 2 NPs on the microbial activity of AGS and ABGS As shown in Fig. 4 a, the specific phosphorus uptake rate (SPUR) of AGS remained unaffected after the addition of TiO 2 NPs at 1 and 5 mg L − 1 . However, with further increase the concentration to 10, 20, 30, and 50 mg L − 1 , the SPUR decreased from 12.23 to 11.86, 11.58, 10.65, and 9.34 mg P (g MLVSS∙h) −1 , respectively. Similar results were observed in previous studies, where increasing Ag, ZnO, and CeO 2 NPs concentrations in AGS resulted in a significant reduction in phosphorus uptake, leading to an increased phosphate volume in the effluent water (Wang et al., 2016b , 2016a ; Xu et al., 2017 ). Unlike AGS, the phosphorus uptake of ABGS remained stable at ≤ 20 mg L − 1 NPs, and then declining from 13.07 to 12.15 and 11.72 mg P/(g MLVSS∙h) −1 after the addition of 30 and 50 mg L − 1 nanoparticles. These results coincide with the trend found in phosphorus removal, where the PO 4 3− amount in effluent was consistently lower in ABGS, than in AGS at the same contaminant concentration. This indicates that TiO 2 had a greater impact on SPUR in aerobic sludge, and on the microbial community in ABGS. This in turn plays an important role in phosphorus removal and exhibited a better tolerance against the nanoparticles. The smaller change in SPUR can be attributed to i) their higher resilience against metal contaminants (Xiao et al., 2023 ), and ii) the better phosphorus uptake capacity of algae (Boelee et al., 2011 ). At concentrations of ≥ 20 mg L − 1 TiO 2 NPs, both AGS and ABGS exhibited a major drop in SAUR. The microbial activity declined by 7.49%, 17.34%, and 36.18% in AGS, and 4.21%, 19.04%, and 31.31% in ABGS. Interestingly, despite the significant decrease in microbial activity in the algal-bacterial sludge, it showed a higher ammonia removal compared to that in AGS as evidenced by the higher ammonia uptake value in ABGS (Fig. 4 b). Quan et al. ( 2015 ) observed a similar significant reduction in SAUR in AGS, where the ammonia uptake dropped by 28% along with a 50 mg L − 1 Ag NPs concentration in the effluent. A similar trend could be observed in the case of SNIUR (Fig. 4 c). The nitrite uptake of AGS decreased by 10.70%, 20.89%, and 29.76%, while in ABGS, it dropped by 2.65%, 7.95%, and 17.66% after the introduction of TiO 2 NPs at concentrations of 20, 30, and 50 mg L − 1 , respectively. The changes were lower in the SNIUR of the algal-bacterial sludge, resulting in lower NO 2 -N content in the effluent. These findings are consistent with prior reports, wherein an increase in ammonia and nitrite contents were found in the effluent, when the SAUR and SNIUR were lower compared to the initial granular sludge (Wang et al., 2016b , 2015 ). Since nitrite-oxidizing microorganisms are typically found in the outer layers of the granules, the decrease in SNIUR can be lower in ABGS than in AGS. This can be due to i) the higher amount of EPS (Fig. 2 ) and ii) the filamentous microorganisms, which increase the surface area and reduce the nanoparticle exposure of the initial granules. The SNUR in the initial ABGS was 16.6 mg N/(g MLVSS∙h) −1 and remained unchanged during the experiments. This finding indicates that nitrate removal in algal-bacterial sludge remained constant, suggesting that the elevated titanium dioxide content did not hinder the microbial activity of denitrifying bacteria. The nitrate uptake in the control AGS was 15.1 mg N/(g MLVSS∙h) −1 , and no significant change was observed at TiO 2 NP concentrations ≤ 20 mg L − 1 . However, when the nanoparticle concentration was increased to 30 and 50 mg L − 1 , the SNUR in AGS declined to 14.85 and 14.53 N/(g MLVSS∙h) −1 , respectively. This observation is consistent with the elevated nitrate level in the effluent and suggests that the nanoparticles were able to penetrate the interior of the granules, thereby reducing the microbial activity of the denitrifying anaerobic microorganisms. This reduction may have occurred due to a significant decrease in the quantity of extracellular polymeric substances (Fig. 2 a), which normally protect the cells. 3.4. Possible mechanisms at TiO 2 NP concentrations of 1 and 5 mg L − 1 , no negative effects were observed on the microbial activity of AGS, while the EPS content increased and nutrient removal efficiency did not decrease. In contrast, both the microbial activity and EPS amount of ABGS increased even after the addition of 10 mg L − 1 TiO 2 NPs, and nutrient removal remained stable. This slight "positive" effect can be attributed to the fact that low amounts of heavy metals can stimulate the enzymatic activity of microorganisms and their EPS secretion (Y.-F. Cheng et al., 2019 ), while the extended tolerance threshold of ABGS may be due to the tolerance of algae to heavy metals. based on the ICP measurements, TiO 2 NPs exhibited a poor ability to release Ti 1+ , with only 2% being released after 4 hours and just 5% after 24 hours. This small amount of Ti 1+ and TiO 2 NPs could penetrate through the EPS and infiltrate the microorganisms, altering their selective permeability and leading to cell death (Kedves and Kónya, 2024 ). However, the released ions were unable to penetrate the granular sludge, resulting in stable nitrate removal in both types of sludge. EPS serve as the initial protective barrier for microbial aggregates, enabling them to come into direct contact with and adsorb metal ions and nanoparticles present in wastewater (Y.-F. F. Cheng et al., 2019 ). When TiO 2 NPs and Ti 1+ attached to the surface or penetrated the interior of the granules at a tolerable amount (in AGS it was 5–10 mg L − 1 , while in ABGS 10–20 mg L − 1 TiO 2 NPs), the cells responded by producing more EPS, which increased by 18% in AGS and by 41% in ABGS, as a protective mechanism to prevent entry and mitigate excessive oxidative stress. the response of microbial activity and EPS production differed between ABGS and AGS. The algal-bacterial consortia in ABGS secreted a higher amount of EPS to mitigate the harmful effects of TiO 2 NPs, while AGS produced less EPS. Consequently, microbial activity and nutrient removal efficiency remained higher in ABGS. This EPS-driven defense mechanism may be linked to the energy dynamics of ABGS, where the symbiotic relationship between algae and bacteria promotes the secretion of more polymers for protection against NPs. In contrast, AGS may direct additional energy toward detoxification or cellular repair processes, such as actively expelling excess NPs or ions from sensitive cell areas (Zhang et al., 2018 ). the contact between TiO 2 NPs and the microalgae in the outer layer of the granules caused limited light availability in case of ABGS. This reduction in light exposure diminished the viability of algae viability, subsequently leading to decreased nitrogen and phosphorus absorption (Xiao et al., 2022 ). Furthermore, the proliferation of filamentous microorganisms on the surface of the sludge, following the addition of 50 mg L − 1 TiO 2 NPs, could also cause a shading effect on the algae. 4. Conclusions Titanium dioxide (TiO 2 ) nanoparticles (NPs) are widely utilized in versatile applications in various fields of our everyday life, which makes them one of the most extensively manufactured nanomaterials. Still, their potential environmental effect on the microorganisms in biological wastewater treatments has not been examined before. In this study, the chronic effects of TiO 2 NPs on aerobic granular sludge (AGS) and algal-bacterial aerobic granular sludge (ABGS) biological wastewater treatment processes were investigated over a 10-day period. At low nanoparticle concentrations (1 and 5 mg L − 1 ), the performance of the bioreactors remained unaffected, whereas at higher concentrations (10, 20, 30, and 50 mg L − 1 ) a decreased nutrient removal efficiency was found. ABGS exhibited higher tolerance to elevated nanoparticle concentrations than that found in AGS, hence maintaining a more stable treatment efficiency and microbial activity. Some minor changes were observed in the algal-bacterial sludge structure, characterized by filamentous microorganisms surrounding the granules and reducing its settling ability. In contrast, TiO 2 NPs have a more pronounced AGS showed negative effects at a concentration of 10 mg L − 1 , with decreasing efficiency in nutrient removal, microbial activity, and EPS quantity compared to ABGS. These findings suggest a strong symbiotic relationship between algae and bacteria in the algal-bacterial granular sludge, enabling the development of a more effective defense mechanism against nanoparticles. Despite the reduction in performance observed in both bioreactors, nanoparticles were not detectable in the effluent wastewater, as they accumulated in the sludge. Declarations Data availability The authors confirm that the data supporting the findings of this study are available within the article and extra information can be obtained by emailing the corresponding author, upon reasonable request. Acknowledgement H.H. acknowledges financial support of the János Bolyai Research Grant of the Hungarian Academy of Sciences (BO/682/22) and the New National Excellence Program of the Ministry for Culture and Innovation from NKFI Fund (ÚNKP-22-5-SZTE-574). Project no. RRF-2.3.1-21-2022-00009 (National Laboratory for Renewable Energy) has been implemented with the support provided by the Recovery and Resilience Facility of the European Union within the framework of Programme Széchenyi Plan Plus. This project has received funding from the HUN-REN Hungarian Research Network. Z.K. is grateful for K 21 138714 and SNN_135918 project from the source of the National Research, Development and Innovation Fund. TKP2021-NVA-19 under the TKP2021-NVA funding scheme of the Ministry for Innovation and Technology are acknowledged. Funding Open access funding provided by University of Szeged. Authors and Affiliations Department of Applied and Environmental Chemistry, University of Szeged, 6720, Szeged, Hungary Alfonz Kedves, Henrik Haspel, Çağdaş Yavuz, Zoltán Kónya Department of Molecular and Analytical Chemistry, University of Szeged, 6720, Szeged, Hungary Bence Kutus Authors Contributions Alfonz Kedves : conceptualization, data curation, formal analysis, validation, investigation, methodology, visualization, software, writing - original draft. Henrik Haspel : formal analysis, funding acquisition, project administration, resources, writing - review & editing, supervision. Çagdas Yavuz : data curation, investigation, methodology, software, visualization, formal analysis, writing - original draft. Bence Kutus : investigation, methodology. Zoltán Kónya : formal analysis, resources, writing - review & editing, supervision, project administration, and funding acquisition. All authors have given approval to the final version of the manuscript. 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Technol. 365, 128165. https://doi.org/10.1016/j.biortech.2022.128165 Supplementary Files Kedvesetal.Supplementary.docx Cite Share Download PDF Status: Published Journal Publication published 20 Nov, 2024 Read the published version in Environmental Science and Pollution Research → Version 1 posted Editorial decision: Accept 13 Nov, 2024 Reviewers agreed at journal 06 Nov, 2024 Reviewers invited by journal 06 Nov, 2024 Editor invited by journal 05 Nov, 2024 First submitted to journal 05 Nov, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4629286","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":374930471,"identity":"f6f94439-d6eb-41bc-b382-5c3326062a65","order_by":0,"name":"Alfonz Kedves","email":"data:image/png;base64,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","orcid":"https://orcid.org/0009-0001-6885-9001","institution":"University of Szeged: Szegedi Tudomanyegyetem","correspondingAuthor":true,"prefix":"","firstName":"Alfonz","middleName":"","lastName":"Kedves","suffix":""},{"id":374930472,"identity":"c06ef361-b71b-439f-9421-ed8bc227e5fd","order_by":1,"name":"Henrik Haspel","email":"","orcid":"","institution":"University of Szeged: Szegedi Tudomanyegyetem","correspondingAuthor":false,"prefix":"","firstName":"Henrik","middleName":"","lastName":"Haspel","suffix":""},{"id":374930473,"identity":"0b99cf7d-597c-4709-ab06-62525a11c660","order_by":2,"name":"Çağdaş Yavuz","email":"","orcid":"","institution":"University of Szeged: Szegedi Tudomanyegyetem","correspondingAuthor":false,"prefix":"","firstName":"Çağdaş","middleName":"","lastName":"Yavuz","suffix":""},{"id":374930474,"identity":"303d72e3-a036-421f-99dd-677da1860bde","order_by":3,"name":"Bence Kutus","email":"","orcid":"","institution":"University of Szeged: Szegedi Tudomanyegyetem","correspondingAuthor":false,"prefix":"","firstName":"Bence","middleName":"","lastName":"Kutus","suffix":""},{"id":374930475,"identity":"d1463a61-9749-4548-8ec3-4bdc5f6ddea2","order_by":4,"name":"Zoltán Kónya","email":"","orcid":"","institution":"University of Szeged: Szegedi Tudomanyegyetem","correspondingAuthor":false,"prefix":"","firstName":"Zoltán","middleName":"","lastName":"Kónya","suffix":""}],"badges":[],"createdAt":"2024-06-24 10:03:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4629286/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4629286/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11356-024-35581-z","type":"published","date":"2024-11-20T15:57:25+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":69620858,"identity":"f3dbec2a-665a-4acb-921f-a7d7d56a5fda","added_by":"auto","created_at":"2024-11-22 10:07:43","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1696399,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron microscope (SEM) images of the investigated sludges. The surface of the initial \u003cstrong\u003ea)\u003c/strong\u003e aerobic granular sludge (AGS), and the \u003cstrong\u003eb)\u003c/strong\u003e algal-bacterial granular sludge (ABGS). The exterior of the \u003cstrong\u003ec)\u003c/strong\u003e AGS, and the \u003cstrong\u003ed) \u003c/strong\u003eABGS after the introduction of 50 mg L\u003csup\u003e-1\u003c/sup\u003e TiO\u003csub\u003e2\u003c/sub\u003e NPs\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4629286/v1/5138cb9620d835e53a12c51a.jpg"},{"id":69621542,"identity":"b44a64d1-f366-444b-86db-9dedcf71233d","added_by":"auto","created_at":"2024-11-22 10:15:43","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":976546,"visible":true,"origin":"","legend":"\u003cp\u003eEPS (PN+PS) secretion in AGS and ABGS processes after addition of TiO\u003csub\u003e2\u003c/sub\u003e NPs. EPS volume in \u003cstrong\u003ea)\u003c/strong\u003e AGS and \u003cstrong\u003eb)\u003c/strong\u003e ABGS after addition of TiO\u003csub\u003e2\u003c/sub\u003e NPs\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4629286/v1/c74eb3c5282e1ad4492e1b9f.jpg"},{"id":69620860,"identity":"d4fc9192-7dd2-4a9f-a94a-979742596b05","added_by":"auto","created_at":"2024-11-22 10:07:43","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":756475,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of titanium dioxide nanoparticles (TiO\u003csub\u003e2\u003c/sub\u003e NPs) on the removal of nutrients. \u003cstrong\u003ea)\u003c/strong\u003e COD, \u003cstrong\u003eb)\u003c/strong\u003e NH\u003csub\u003e3\u003c/sub\u003e-N, \u003cstrong\u003ec)\u003c/strong\u003e NO\u003csub\u003e2\u003c/sub\u003e-N, and \u003cstrong\u003ed)\u003c/strong\u003e PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3-\u003c/sup\u003e contents\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4629286/v1/9e16c1bd80278b3b2407282a.jpg"},{"id":69620862,"identity":"d4ef610f-872d-4a80-a452-35359edf3950","added_by":"auto","created_at":"2024-11-22 10:07:43","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1453299,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of titanium dioxide nanoparticles (TiO\u003csub\u003e2\u003c/sub\u003e NPs) on the microbial activities of aerobic granular sludge (AGS) and algal-bacterial granular sludge (ABGS). \u003cstrong\u003ea) \u003c/strong\u003especific phosphorus uptake rate (SPUR), \u003cstrong\u003eb) \u003c/strong\u003especific ammonia uptake rate (SAUR), \u003cstrong\u003ec) \u003c/strong\u003especific nitrite uptake rate (SNIUR), and \u003cstrong\u003ed) \u003c/strong\u003especific nitrate uptake rate (SNUR)\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4629286/v1/8b76ad6353a1b63c3dee129f.jpg"},{"id":69834844,"identity":"d7d99652-a037-42ce-8ea2-468f11262a13","added_by":"auto","created_at":"2024-11-25 16:09:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5553946,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4629286/v1/5f61775c-9264-49be-aee1-4cb22b8bd634.pdf"},{"id":69620861,"identity":"7cd4c16b-b8bf-4d07-b1aa-356618eb2c46","added_by":"auto","created_at":"2024-11-22 10:07:43","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":681964,"visible":true,"origin":"","legend":"","description":"","filename":"Kedvesetal.Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-4629286/v1/3c67392a4c48cf4a9aec09c9.docx"}],"financialInterests":"","formattedTitle":"A comparative study on the chronic responses of titanium dioxide nanoparticles on aerobic granular sludge and algal-bacterial granular sludge processes","fulltext":[{"header":"Highlights","content":"\u003cul\u003e\n \u003cli\u003eThe long-term impact of TiO\u003csub\u003e2\u003c/sub\u003e NPs on AGS and ABGS bioreactors was investigated.\u003c/li\u003e\n \u003cli\u003eNPs\u0026nbsp;were enriched in both sludges, and showed higher toxicity on AGS than on ABGS.\u003c/li\u003e\n \u003cli\u003e1 and 5 mg L\u003csup\u003e-1\u003c/sup\u003e TiO\u003csub\u003e2\u003c/sub\u003e NPs had no adverse impact on nutrient removal.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eHigher protein content reduced NPs toxicity on ABGS at \u0026gt; 5 mg L\u003csup\u003e-1\u003c/sup\u003e concentrations.\u003c/li\u003e\n \u003cli\u003eFilamentous microorganisms reduced the settleability of ABGS.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eEngineered nanoparticles (NPs) have found extensive applications due to their unique physical and chemical attributes in various fields including catalysts (Hou et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), electronics (Qamar et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), textiles (Rashid et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and others. Titanium dioxide (TiO\u003csub\u003e2\u003c/sub\u003e) NPs, as a commercially significant and widely utilized nanoparticle, have gained prominence for their versatile applications such as in personal skincare products (Kumari and Virdi, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), toothpastes (Al-Salman Fadheela et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and as a white pigment material (Mohammadparast and Mallard, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) owing to their excellent stability and photocatalytic activity. TiO\u003csub\u003e2\u003c/sub\u003e NPs have emerged as one of the most extensively manufactured nanomaterials (\u0026gt;\u0026thinsp;10,000 tons/year), it raises concerns regarding their potential environmental release, posing risks to microorganisms in the ecosystems (Kedves and K\u0026oacute;nya, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Numerous studies have demonstrated the potential negative effects of these NPs on various organisms including algae (Natarajan et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), bacteria (Tahir et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and fungi (Najibi Ilkhechi et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), underscoring the need for a comprehensive assessment and management of their environmental impact.\u003c/p\u003e \u003cp\u003eThe widespread use of nanoparticles has led to their significant presence in wastewater. TiO\u003csub\u003e2\u003c/sub\u003e nanoparticles were found both in sewage and sludge at concentrations up to 3 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Wang et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and 23 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Gottschalk et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), respectively. During biological wastewater treatment, TiO\u003csub\u003e2\u003c/sub\u003e NPs could have numerous negative effect on bioreactors efficiency. Yuan et al. (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) reported that the specific resistance to filtration of activated sludge (AS) increased after introducing 1 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e TiO\u003csub\u003e2\u003c/sub\u003e NPs, while in another study the microbial diversity slightly shifted at 2 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e TiO\u003csub\u003e2\u003c/sub\u003e NPs after 8 hours (Cervantes-Avil\u0026eacute;s et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In studies, where nanoparticle concentrations were increased to 5 or 10 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, either significant decrease in the removal rates of total nitrogen and phosphate after 6\u0026ndash;8 days of exposure (Li et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Zheng et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), or a decline in floc stability was observed (Zhou et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMishima and Nakamura (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1991\u003c/span\u003e) were the first to report on the aerobic granular sludge (AGS) wastewater treatment process, which has emerged as a promising technology for treating municipal and industrial waters. AGS offers advantages over activated sludge, such as greater stability, higher biomass content in the bioreactor, smaller footprint, and higher tolerance to toxic substances (Q. Jiang et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhou et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Consequently, it has become one of the most extensively studied technologies in wastewater treatment over the past 30 years. Recently, algal-bacterial granular sludge (ABGS) has garnered attention due to its strong symbiotic relationship between algae and bacteria, resulting in excellent pollutant removal capabilities from wastewater (Fard and Wu, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Both AGS and ABGS have demonstrated the ability to simultaneously remove organic matter, phosphorus, and nitrogen from high-strength industrial wastewater (Li et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Lochmatter et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), treat leachate (Ilmasari et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and remove heavy metals (Kedves et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Purba et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThese two technologies are suitable for widespread applications due to the structure of the granules and the high amount of extracellular polymeric substances (EPS) they contain. EPS consists of two main components: polysaccharides and proteins. While the former predominantly forms the outer part of the granules, the proteins typically constitute the main component of the granules\u0026rsquo; inner layer (Nuramkhaan et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Samaei et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Since AGS technology is already used on an industrial scale for the treatment of industrial and municipal wastewater (Hamza et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), several studies examined the impact of the increasingly produced nanoparticles on the AGS wastewater treatment.\u003c/p\u003e \u003cp\u003eIn wastewater treatment systems, mixed liquor suspended solids (MLSS) are directly related to the system resilience against contaminants (Wang et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Accordingly, where possible, the amount of nanoparticles introduced in the various experiments was also calculated based on MLSS. Quan et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) observed a decrease in biomass and in microbial activity during the long-term exposure of silver NPs of 5 and 50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (1.67 and 16.67 mg gMLSS\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). In another studies, the increase of cupric oxide (CuO) NPs concentration from 5 to 50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (1.67 to 16.67 mg gMLSS\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) significantly reduced the removal of phosphorus (Zheng et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), while Cu NPs at 5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (0.38 mg gMLSS\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) inhibited the nitrogen removal capacity by 51.9% during long-term exposure (Y. F. Cheng et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The shock loading of zinc oxide (ZnO) NPs at 1-100 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (0.23\u0026ndash;22.73 mg gMLSS\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) caused acute toxic effect on microbial activity (He et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017b\u003c/span\u003e), whilst the nitrification and denitrification processes were inhibited even at 20 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the long term (He et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017a\u003c/span\u003e). Moreover, nanoparticles tend to enrich in the sludge, as Xiao et al. (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) found the 95% of ZnO NPs in the sludge when the wastewater contained 10 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NPs. Another study showed no harmful effects of ZnO NPs up to 1 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (0.17 mg gMLSS\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), whereas the ammonia and phosphorus removal significantly decreased at 10 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (1.67 mg gMLSS\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (Xiao et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). So far, only one study examined the effect of TiO\u003csub\u003e2\u003c/sub\u003e NPs on AGS at a single concentration of 50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Y. Jiang et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), while in the case of ABGS, the effect of TiO\u003csub\u003e2\u003c/sub\u003e NPs on granule formation was studied (B. Li et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Considering that i) previous studies covered the effects of Zn and Cu-based NPs, ii) TiO\u003csub\u003e2\u003c/sub\u003e NPs are present in wastewater, iii) AGS is used on full-scale, and iv) the future industrial-scale utilization of ABGS is highly likely, the investigation of the effects of TiO\u003csub\u003e2\u003c/sub\u003e NPs on AGS and ABGS became vital.\u003c/p\u003e \u003cp\u003eHerein, we report the chronic impact (over 10 days) of TiO\u003csub\u003e2\u003c/sub\u003e NPs at a series of concentrations (0, 1, 5, 10, 20, 30, and 50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, corresponding to 0.17, 0.83, 1.65, 3.31, 4.96, and 8.26 mg gMLSS\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) on the nutrient removal efficiency, the microbial activity, and the extracellular polymeric substances of both, the AGS and ABGS processes. These findings provide fundamental insights into the TiO\u003csub\u003e2\u003c/sub\u003e nanoparticle tolerance on aerobic granular and algal-bacterial aerobic granular sludge, and thus are expected to contribute to the knowledge on the operation of AGS and ABGS in treating TiO\u003csub\u003e2\u003c/sub\u003e NPs contaminated wastewater.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Experimental\u003c/h2\u003e \u003cp\u003eAll bioreactors were inoculated with aerobic granular sludge and algal-bacterial granular sludge freshly collected from mother reactors operated for over half a year in our laboratory. The AGS sequencing batch reactors (SBRs) and ABGS photo-sequencing batch reactors (PSBRs) were continuously fed with synthetic wastewater (SWW) (\u003cem\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/em\u003e) containing 1, 5, 10, 20, 30, and 50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e TiO\u003csub\u003e2\u003c/sub\u003e NPs, every four hours for 10 days. The hydraulic retention time of SWW in the bioreactors was 8 hours with an effective volume of 1.4 L. The average mixed liquor suspended solids and sludge volume index (MLSS and SVI\u003csub\u003e5\u003c/sub\u003e) were 6.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 24.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 mL g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. Further details on the components of the bioreactors, the configuration of SBR and PSBR, and the constituents of SWW are provided in the \u003cem\u003eSupplementary Information\u003c/em\u003e. TiO\u003csub\u003e2\u003c/sub\u003e nanoparticles were synthesized through a modified nonaqueous solvothermal process (Z. Q. Li et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), and were characterized by using a Rigaku Miniflex-II X-ray diffractometer (\u003cem\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea\u003c/em\u003e), a Bruker Vertex 70 FT-IR instrument (\u003cem\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb\u003c/em\u003e), and a Hitachi S-4700 Type II scanning electron microscope (SEM) with 10 kV accelerating voltage equipped (\u003cem\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ed\u003c/em\u003e) with a R\u0026ouml;ntec QX2 energy dispersive X-ray spectrometer (EDX) (\u003cem\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ec\u003c/em\u003e, see \u003cem\u003eSupporting Information\u003c/em\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Analysis of effluent water, sludge properties, and microbial activity\u003c/h2\u003e \u003cp\u003eEvery 12 hours over a period of 10 days, the concentration of chemical oxygen demand (COD), phosphorus (PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e), nitrate-nitrogen (NO\u003csub\u003e3\u003c/sub\u003e-N), nitrite-nitrogen (NO\u003csub\u003e2\u003c/sub\u003e-N), and ammonia nitrogen (NH\u003csub\u003e3\u003c/sub\u003e-N) in the effluent of the SBRs and PSBRs was measured using Hanna kits (HI93754B-25, HI93717-01, HI93728-01, HI93708-01, and HI93715-01) with a spectrophotometer HI83399. Additionally, titanium content in the effluent wastewater was measured via Inductively-coupled plasma mass spectrometry (ICP-MS) using an Agilent 7900 instrument with 15.0 L min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Ar carrier gas. Samples were filtered through a 0.45 \u0026micro;m syringe filter and stored at \u0026minus;\u0026thinsp;4\u0026deg;C, and prior to analysis cc. HNO\u003csub\u003e3\u003c/sub\u003e (NORMATOM by VWR Chemicals, final concentration: 1 wt%) and solutions of the internal standards \u003csup\u003e45\u003c/sup\u003eSc and \u003csup\u003e89\u003c/sup\u003eY (ARISTAR by VWR Chemicals, final concentration: 100 ppb) were added. Calibration was performed between 0 and 50 ppb Ti using the same procedure, signals of the \u003csup\u003e47\u003c/sup\u003eTi, \u003csup\u003e48\u003c/sup\u003eTi, and \u003csup\u003e49\u003c/sup\u003eTi isotopes were monitored with and without using He cell collision mode.\u003c/p\u003e \u003cp\u003eOn the tenth day of the experiments, the MLSS, SVI\u003csub\u003e5\u003c/sub\u003e, and EPS content of both granular sludges were determined. Additionally, to assess the impact of TiO\u003csub\u003e2\u003c/sub\u003e NPs on the microbial activity, specific phosphorus uptake rate (SPUR), specific ammonia, nitrite, and nitrate uptake rates (SAUR, SNIUR, and SNUR) were determined. Finally, the effect of TiO\u003csub\u003e2\u003c/sub\u003e NPs on the structure of the aerobic and algal-bacterial granular sludge was examined using a Hitachi S-4700 Type II scanning electron microscope (SEM) and EDX. Detailed descriptions of the MLSS, SVI\u003csub\u003e5\u003c/sub\u003e, EPS, SPUR, SAUR, SNIUR, and SNUR measurements, as well as the preparation of sludge samples for SEM investigation, can be found in the \u003cem\u003eSupplementary Information\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Sludge properties and EPS production in AGS and ABGS\u003c/h2\u003e \u003cp\u003eCompared to activated sludge (AS), both AGS SBRs and ABGS PSBRs contain a significant amount of biomass, along with high levels of extracellular polymeric substances (EPSs), primarily consisting of protein (PN) and polysaccharide (PS). EPS not only protect microorganisms from harmful substances, such as nanoparticles, but also contribute to good settling ability and possess biosorption properties (Hakim et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zheng et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). At the beginning of each experiment, the MLSS and SVI\u003csub\u003e5\u003c/sub\u003e of the sludges were approximately 6.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 24.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 mL g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, while the EPS content in AGS and ABGS was 106.5\u0026thinsp;\u0026plusmn;\u0026thinsp;6.1 and 129.6\u0026thinsp;\u0026plusmn;\u0026thinsp;6.7 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e MLVSS, respectively. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, numerous microorganisms were embedded in the polymer matrix on the surface of the aerobic granules, whereas algae can be seen alongside other microorganisms within the exterior of the algal-bacterial sludge (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) (Salimon et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Based on the sludge EDX analysis, the main differences between the types of sludge are nitrogen and phosphorus contents. In AGS 8.78 and 0.58 at%, in ABGS 9.14 and 0.88 at% nitrogen and phosphorus were found, respectively (\u003cem\u003eFig. S2\u003c/em\u003e). The difference originates from the presence of algae, as these microorganisms are capable of accumulating high amounts of these elements (Kube et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTiO\u003csub\u003e2\u003c/sub\u003e NPs (with spherical morphology of a diameters ranging between 30 and 130 nm, see \u003cem\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/em\u003e) at concentrations of 5 and 10 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e led to an increase in EPS in both granular sludges. This increase was accompanied by the rise in the PN/PS ratio, attributed to a higher volume of PN secretion after 10 days of exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). At 10 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NPs, the PN content increased by 24% from 59.8\u0026thinsp;\u0026plusmn;\u0026thinsp;5.1 to 74.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e MLVSS, and by 60% from 82.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.7 to 132.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e MLVSS in AGS and ABGS, respectively. These results suggest that ABGS has a higher tolerance to the negative effects of nanoparticles compared to AGS, as it exhibited a greater capacity for protein secretion. In two previous studies, He et al. (2017) and Xiao et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) investigated the chronic effects of zinc oxide nanoparticles on AGS and ABGS. During the experiments, it was observed that ZnO NPs caused a reduction in the amount of sludge EPS at concentrations as low as 10 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, significantly impacting the efficiency of the reactors. In our case, however, following the administration of 10 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of TiO\u003csub\u003e2\u003c/sub\u003e, the quantity of polymer materials was higher than in the initial sludge. The low toxicity promoted bacterial production of polymer materials in the sludge, whereas higher toxicity reduced its quantity (Quan et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). We, therefore, presume that zinc oxide nanoparticles may be more toxic to the granular sludge.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe EPS content declined, and sludge properties changed with increasing nanoparticle content (20, 30, and 50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e TiO\u003csub\u003e2\u003c/sub\u003e NPs) in AGS. The polymer volume decreased to 97.4, 83.2, and 68.3 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e MLVSS, and the PN/PS ratio dropped to 1.22, 1.1, and 0.99, below those of the initial sludge (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Simultaneously, the MLSS decreased to 5.96, 5.52, and 4.83 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, while significant changes were not observed in the SVI\u003csub\u003e5\u003c/sub\u003e. This data suggests that the persistent presence of TiO\u003csub\u003e2\u003c/sub\u003e NPs in the influent led to a decrease in biomass production in the AGS reactor. The long-term presence of low concentration (5 and 10 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) CuO NPs had a similar effect on AGS, i.e., the secretion of EPS was promoted, while a decrease in biomass and EPS content was observed at higher concentrations (50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (Zheng et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The decreasing amount of biomass can be explained by the decrease in the size of the granules, since their average diameter declined from 600 to 350 \u0026micro;m at 50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NPs after 10 days. Along with the size reduction, the external structure of AGS also changed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), as the polymer layer disappeared and rod-shaped microorganisms can be seen. The latter suggests a significant change in the microbial structure.\u003c/p\u003e \u003cp\u003eThe addition of TiO\u003csub\u003e2\u003c/sub\u003e NPs at 20, 30, and 50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e had distinct effects in PSBRs and SBRs. Although significant changes in sludge properties were not observed until the day 10 upon the addition of 20 and 30 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NPs, the PN content remained higher compared to that of the initial ABGS with a PN/PS ratio of 2.45 and 2.11, respectively. In contrast, the PN and PS decreased from 82.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.7 and 47.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e MLVSS to 72.7\u0026thinsp;\u0026plusmn;\u0026thinsp;5.1 and 40.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e MLVSS at 50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e TiO\u003csub\u003e2\u003c/sub\u003e NPs, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Simultaneously, the MLSS decreased to 3.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and the SVI\u003csub\u003e5\u003c/sub\u003e increased to 165.9\u0026thinsp;\u0026plusmn;\u0026thinsp;7.6 mL g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The significant change occurred because filamentous microorganisms appeared on the outer part of the granules (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed), leading to a loose structure with poorer settling ability and an increase in the granular sludge size from 0.6 mm to 2\u0026ndash;8 mm.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Performance on nutrients removal of AGS and ABGS\u003c/h2\u003e \u003cp\u003eIn order to characterize the efficiency of a biological wastewater treatment, the removal of organic matter, nitrogen, and phosphate need to be measured. The performance of AGS and ABGS bioreactors was assessed by recording the COD, NH\u003csub\u003e3\u003c/sub\u003e-N, NO\u003csub\u003e2\u003c/sub\u003e-N, NO\u003csub\u003e3\u003c/sub\u003e-N, and PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e every third cycle (every 12 h) in each experiment over 10 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Although the removal of nutrients remained stable after introducing the nanoparticles at concentrations as low as 1 and 5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, increasing NP content resulted in a gradually decreasing removal efficiency.\u003c/p\u003e \u003cp\u003eCOD\u003c/p\u003e \u003cp\u003eUpon the introduction of 10 and 20 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e nanoparticles into the AGS-SBR, the COD in the effluent began to increase after 5 and 2 days, and rising from 79\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1 to 132\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 156.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e on day 10, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). In contrast, in the ABGS PSBRs, the COD started to increase after 7 and 5 days with levels changing from 75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4 to 101\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 120\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e by day 10, respectively. At concentration of 50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the COD removal rate declined after just half a day due to the shock loads of titanium. In the AGS, a continuous increase in COD was measured in the effluent reaching 331.5\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e by day 10, while in the ABGS, COD removal reached a steady state after 6 days. Overall, the observations indicated that TiO\u003csub\u003e2\u003c/sub\u003e nanoparticles had a lower impact on the heterotrophic microbial community in algal-bacterial sludge compared to other studies. In previous research, where the long-term impact of TiO\u003csub\u003e2\u003c/sub\u003e nanoparticles on activated sludge was investigated, a decline in COD removal was already observed at concentrations as low as 1 or 2 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Cervantes-Avil\u0026eacute;s et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This suggests that heterotrophic microorganisms in AGS and ABGS are more tolerant to titanium dioxide nanoparticles, likely due to their robust structure and high EPS content.\u003c/p\u003e \u003cp\u003eNH3\u003c/p\u003e \u003cp\u003eHere, we observed a decrease in NH\u003csub\u003e3\u003c/sub\u003e-N removal after 5-2.5, and 8.5\u0026ndash;7.5 days upon the exposure of the AGS and ABGS by 10 and 20 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NP, respectively. The further increase in the TiO\u003csub\u003e2\u003c/sub\u003e NPs content (30\u0026ndash;50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) caused significant inhibition of the aerobic sludge within half a day, with removal efficiency decreasing from 99.94\u0026ndash;92.87%, and 88.33% by day 10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). The negative impact of nanoparticles on ammonia removal was less pronounced in ABGS. Although efficiency declined after the first day at 30 and 50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NPs, levels stabilized around days 6 and 5 with the removal rates reaching 96.31% and 94.26% by day 10, respectively.\u003c/p\u003e \u003cp\u003eNO2\u003c/p\u003e \u003cp\u003eThe NO\u003csub\u003e2\u003c/sub\u003e-N contents in the effluent (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec) exhibited similar variations to ammonia-nitrogen, suggesting that nanoparticles had a negative effect on both aerobic ammonia and nitrite oxidizing microorganisms. These microbes are predominantly located in the outer part of the granules, allowing TiO\u003csub\u003e2\u003c/sub\u003e NPs to potentially attach to their surface and inhibit their microbial activity. Zheng et al. (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) also observed a decreased removal rate of both ammonia and nitrite in activated sludge exposed to 50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e TiO\u003csub\u003e2\u003c/sub\u003e NPs, along with a drastically dropping nitrogen elimination to 24.4%. Throughout the experiments, the NO\u003csub\u003e3\u003c/sub\u003e-N content in the effluent remained constant in the algal-bacterial sludge. In the AGS, on the other hand, it increased to 10.2 and 36.7 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e as a response to the introduction of 30 and 50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e TiO\u003csub\u003e2\u003c/sub\u003e NPs, respectively.\u003c/p\u003e \u003cp\u003ePO\u003c/p\u003e \u003cp\u003eWhen considering phosphorus removal, notable distinctions between AGS and ABGS are evident (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). At 10 and 20 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NPs, phosphorus removal in ABGS began to decrease after 9 and 6 days with effluent PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e contents of 0.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 and 1.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e after 10 days, respectively. However, in AGS, the efficiency declined after 3 and 2 days, resulting in PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e amounts of 1.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 and 3.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at day 10, respectively. Additionally, when titanium content of the influent wastewater was 30 and 50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e content in the effluent stabilized after 7 and 6 days in ABGS, respectively. By day 10, however, the phosphate concentration was similar or lower than that in AGS in response to 20 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e TiO\u003csub\u003e2\u003c/sub\u003e NPs. Upon further increasing the TiO\u003csub\u003e2\u003c/sub\u003e concentration, the phosphorus content increased from 0.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 to 7.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the latter along with a drop in the removal rate to 62.4%. In contrast, phosphorus concentration increased from 0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 to 3.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, leading to a reduction in removal efficiency to 84.1% in ABGS. Li et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) also observed a decrease in phosphorus removal efficiency in AS, where it reduced from 89\u0026ndash;68%, suggesting that granular sludges exhibit significantly higher resistance against titanium-dioxide.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA correlation between the PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e, and the NH\u003csub\u003e3\u003c/sub\u003e-N and NO\u003csub\u003e2\u003c/sub\u003e-N nitrogen removal results was found. The correlation is partly due to the fact that microorganisms that are sensitive to higher concentrations of ammonia and nitrite in the wastewater are also the ones capable of accumulating and removing phosphate, as previously observed (Zheng et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The better nutrient removal efficiency of ABGS may also originate from the algae's high tolerance to heavy metals and their ability to remove nutrients from wastewater (Nguyen et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Priyadarshini et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In AGS, chronic exposure to titanium dioxide nanoparticles led to a more pronounced decrease in nutrient removal efficiency, resulting in a lower biomass quantity compared to that in the initial sludge after 10 days. This reduction in biomass could have potentially contributed to the release of nutrients from the polymer matrix, exacerbating the effluent's COD, nitrogen, and phosphorus content. Continuous monitoring of the titanium content in the effluent wastewater was conducted throughout the experiments, whose presence could not be detected. This in turn highlights the importance of preventing the pollutant release into the environment, if it accumulates in the sludge. The titanium content in AGS and ABGS was 8.25 and 2.66 at% at 50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e TiO\u003csub\u003e2\u003c/sub\u003e NP concentration (\u003cem\u003eFig. S3\u003c/em\u003e). The difference is likely due to the low penetration depth of the EDX sludge analysis. Since numerous filamentous microorganisms covered the surface of the granules in ABGS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed), only part of the elemental information comes from the Ti-rich granules, which in turn results in a lower measured titanium content.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Influence of TiO\u003csub\u003e2\u003c/sub\u003e NPs on the microbial activity of AGS and ABGS\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, the specific phosphorus uptake rate (SPUR) of AGS remained unaffected after the addition of TiO\u003csub\u003e2\u003c/sub\u003e NPs at 1 and 5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. However, with further increase the concentration to 10, 20, 30, and 50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the SPUR decreased from 12.23 to 11.86, 11.58, 10.65, and 9.34 mg P (g MLVSS∙h) \u003csup\u003e\u0026minus;1\u003c/sup\u003e, respectively. Similar results were observed in previous studies, where increasing Ag, ZnO, and CeO\u003csub\u003e2\u003c/sub\u003e NPs concentrations in AGS resulted in a significant reduction in phosphorus uptake, leading to an increased phosphate volume in the effluent water (Wang et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016b\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2016a\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eUnlike AGS, the phosphorus uptake of ABGS remained stable at \u0026le;\u0026thinsp;20 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NPs, and then declining from 13.07 to 12.15 and 11.72 mg P/(g MLVSS∙h) \u003csup\u003e\u0026minus;1\u003c/sup\u003e after the addition of 30 and 50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e nanoparticles. These results coincide with the trend found in phosphorus removal, where the PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e amount in effluent was consistently lower in ABGS, than in AGS at the same contaminant concentration. This indicates that TiO\u003csub\u003e2\u003c/sub\u003e had a greater impact on SPUR in aerobic sludge, and on the microbial community in ABGS. This in turn plays an important role in phosphorus removal and exhibited a better tolerance against the nanoparticles. The smaller change in SPUR can be attributed to i) their higher resilience against metal contaminants (Xiao et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and ii) the better phosphorus uptake capacity of algae (Boelee et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). At concentrations of \u0026ge;\u0026thinsp;20 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e TiO\u003csub\u003e2\u003c/sub\u003e NPs, both AGS and ABGS exhibited a major drop in SAUR. The microbial activity declined by 7.49%, 17.34%, and 36.18% in AGS, and 4.21%, 19.04%, and 31.31% in ABGS. Interestingly, despite the significant decrease in microbial activity in the algal-bacterial sludge, it showed a higher ammonia removal compared to that in AGS as evidenced by the higher ammonia uptake value in ABGS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Quan et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) observed a similar significant reduction in SAUR in AGS, where the ammonia uptake dropped by 28% along with a 50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Ag NPs concentration in the effluent.\u003c/p\u003e \u003cp\u003eA similar trend could be observed in the case of SNIUR (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). The nitrite uptake of AGS decreased by 10.70%, 20.89%, and 29.76%, while in ABGS, it dropped by 2.65%, 7.95%, and 17.66% after the introduction of TiO\u003csub\u003e2\u003c/sub\u003e NPs at concentrations of 20, 30, and 50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. The changes were lower in the SNIUR of the algal-bacterial sludge, resulting in lower NO\u003csub\u003e2\u003c/sub\u003e-N content in the effluent. These findings are consistent with prior reports, wherein an increase in ammonia and nitrite contents were found in the effluent, when the SAUR and SNIUR were lower compared to the initial granular sludge (Wang et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016b\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Since nitrite-oxidizing microorganisms are typically found in the outer layers of the granules, the decrease in SNIUR can be lower in ABGS than in AGS. This can be due to i) the higher amount of EPS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and ii) the filamentous microorganisms, which increase the surface area and reduce the nanoparticle exposure of the initial granules.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe SNUR in the initial ABGS was 16.6 mg N/(g MLVSS∙h) \u003csup\u003e\u0026minus;1\u003c/sup\u003e and remained unchanged during the experiments. This finding indicates that nitrate removal in algal-bacterial sludge remained constant, suggesting that the elevated titanium dioxide content did not hinder the microbial activity of denitrifying bacteria. The nitrate uptake in the control AGS was 15.1 mg N/(g MLVSS∙h) \u003csup\u003e\u0026minus;1\u003c/sup\u003e, and no significant change was observed at TiO\u003csub\u003e2\u003c/sub\u003e NP concentrations\u0026thinsp;\u0026le;\u0026thinsp;20 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. However, when the nanoparticle concentration was increased to 30 and 50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the SNUR in AGS declined to 14.85 and 14.53 N/(g MLVSS∙h) \u003csup\u003e\u0026minus;1\u003c/sup\u003e, respectively. This observation is consistent with the elevated nitrate level in the effluent and suggests that the nanoparticles were able to penetrate the interior of the granules, thereby reducing the microbial activity of the denitrifying anaerobic microorganisms. This reduction may have occurred due to a significant decrease in the quantity of extracellular polymeric substances (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), which normally protect the cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Possible mechanisms\u003c/h2\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eat TiO\u003csub\u003e2\u003c/sub\u003e NP concentrations of 1 and 5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, no negative effects were observed on the microbial activity of AGS, while the EPS content increased and nutrient removal efficiency did not decrease. In contrast, both the microbial activity and EPS amount of ABGS increased even after the addition of 10 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e TiO\u003csub\u003e2\u003c/sub\u003e NPs, and nutrient removal remained stable. This slight \"positive\" effect can be attributed to the fact that low amounts of heavy metals can stimulate the enzymatic activity of microorganisms and their EPS secretion (Y.-F. Cheng et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), while the extended tolerance threshold of ABGS may be due to the tolerance of algae to heavy metals.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003ebased on the ICP measurements, TiO\u003csub\u003e2\u003c/sub\u003e NPs exhibited a poor ability to release Ti\u003csup\u003e1+\u003c/sup\u003e, with only 2% being released after 4 hours and just 5% after 24 hours. This small amount of Ti\u003csup\u003e1+\u003c/sup\u003e and TiO\u003csub\u003e2\u003c/sub\u003e NPs could penetrate through the EPS and infiltrate the microorganisms, altering their selective permeability and leading to cell death (Kedves and K\u0026oacute;nya, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, the released ions were unable to penetrate the granular sludge, resulting in stable nitrate removal in both types of sludge.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eEPS serve as the initial protective barrier for microbial aggregates, enabling them to come into direct contact with and adsorb metal ions and nanoparticles present in wastewater (Y.-F. F. Cheng et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). When TiO\u003csub\u003e2\u003c/sub\u003e NPs and Ti\u003csup\u003e1+\u003c/sup\u003e attached to the surface or penetrated the interior of the granules at a tolerable amount (in AGS it was 5\u0026ndash;10 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, while in ABGS 10\u0026ndash;20 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e TiO\u003csub\u003e2\u003c/sub\u003e NPs), the cells responded by producing more EPS, which increased by 18% in AGS and by 41% in ABGS, as a protective mechanism to prevent entry and mitigate excessive oxidative stress.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003ethe response of microbial activity and EPS production differed between ABGS and AGS. The algal-bacterial consortia in ABGS secreted a higher amount of EPS to mitigate the harmful effects of TiO\u003csub\u003e2\u003c/sub\u003e NPs, while AGS produced less EPS. Consequently, microbial activity and nutrient removal efficiency remained higher in ABGS. This EPS-driven defense mechanism may be linked to the energy dynamics of ABGS, where the symbiotic relationship between algae and bacteria promotes the secretion of more polymers for protection against NPs. In contrast, AGS may direct additional energy toward detoxification or cellular repair processes, such as actively expelling excess NPs or ions from sensitive cell areas (Zhang et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003ethe contact between TiO\u003csub\u003e2\u003c/sub\u003e NPs and the microalgae in the outer layer of the granules caused limited light availability in case of ABGS. This reduction in light exposure diminished the viability of algae viability, subsequently leading to decreased nitrogen and phosphorus absorption (Xiao et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Furthermore, the proliferation of filamentous microorganisms on the surface of the sludge, following the addition of 50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e TiO\u003csub\u003e2\u003c/sub\u003e NPs, could also cause a shading effect on the algae.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eTitanium dioxide (TiO\u003csub\u003e2\u003c/sub\u003e) nanoparticles (NPs) are widely utilized in versatile applications in various fields of our everyday life, which makes them one of the most extensively manufactured nanomaterials. Still, their potential environmental effect on the microorganisms in biological wastewater treatments has not been examined before. In this study, the chronic effects of TiO\u003csub\u003e2\u003c/sub\u003e NPs on aerobic granular sludge (AGS) and algal-bacterial aerobic granular sludge (ABGS) biological wastewater treatment processes were investigated over a 10-day period. At low nanoparticle concentrations (1 and 5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), the performance of the bioreactors remained unaffected, whereas at higher concentrations (10, 20, 30, and 50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) a decreased nutrient removal efficiency was found. ABGS exhibited higher tolerance to elevated nanoparticle concentrations than that found in AGS, hence maintaining a more stable treatment efficiency and microbial activity. Some minor changes were observed in the algal-bacterial sludge structure, characterized by filamentous microorganisms surrounding the granules and reducing its settling ability. In contrast, TiO\u003csub\u003e2\u003c/sub\u003e NPs have a more pronounced AGS showed negative effects at a concentration of 10 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, with decreasing efficiency in nutrient removal, microbial activity, and EPS quantity compared to ABGS. These findings suggest a strong symbiotic relationship between algae and bacteria in the algal-bacterial granular sludge, enabling the development of a more effective defense mechanism against nanoparticles. Despite the reduction in performance observed in both bioreactors, nanoparticles were not detectable in the effluent wastewater, as they accumulated in the sludge.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm that the data supporting the findings of this study are available within the article and extra information can be obtained by emailing the corresponding author, upon reasonable request.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH.H. acknowledges financial support of the J\u0026aacute;nos Bolyai Research Grant of the Hungarian Academy of Sciences (BO/682/22) and the New National Excellence Program of the Ministry for Culture and Innovation from NKFI Fund (\u0026Uacute;NKP-22-5-SZTE-574). Project no. RRF-2.3.1-21-2022-00009 (National Laboratory for Renewable Energy) has been implemented with the support provided by the Recovery and Resilience Facility of the European Union within the framework of Programme Sz\u0026eacute;chenyi Plan Plus. This project has received funding from the HUN-REN Hungarian Research Network.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eZ.K. is grateful for K 21 138714 and SNN_135918 project from the source of the National Research, Development and Innovation Fund. TKP2021-NVA-19 under the TKP2021-NVA funding scheme of the Ministry for Innovation and Technology are acknowledged.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOpen access funding provided by University of Szeged.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Applied and Environmental Chemistry, University of Szeged, 6720, Szeged, Hungary\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAlfonz Kedves, Henrik Haspel, \u0026Ccedil;ağdaş Yavuz, Zolt\u0026aacute;n K\u0026oacute;nya\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Molecular and Analytical Chemistry, University of Szeged, 6720, Szeged, Hungary\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBence Kutus\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contributions\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAlfonz Kedves\u003c/strong\u003e:\u0026nbsp;conceptualization, data curation, formal analysis, validation, investigation, methodology, visualization, software, writing - original draft. \u003cstrong\u003eHenrik Haspel\u003c/strong\u003e:\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eformal analysis, funding acquisition, project administration, resources, writing - review \u0026amp; editing, supervision. \u003cstrong\u003e\u0026Ccedil;agdas Yavuz\u003c/strong\u003e: data curation, investigation, methodology, software, visualization, formal analysis, writing - original draft. \u003cstrong\u003eBence Kutus\u003c/strong\u003e:\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003einvestigation, methodology. \u003cstrong\u003eZolt\u0026aacute;n K\u0026oacute;nya\u003c/strong\u003e: formal analysis, resources, writing - review \u0026amp; editing, supervision, project administration, and funding acquisition. All authors have given approval to the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors have approved the manuscript for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAl-Salman Fadheela, Ali Redha Ali, Al-Shaikh Hawraa, Hazeem Layla, Taha Safa, 2020. 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Technol. 365, 128165. https://doi.org/10.1016/j.biortech.2022.128165\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"environmental-science-and-pollution-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"espr","sideBox":"Learn more about [Environmental Science and Pollution Research](https://www.springer.com/journal/11356)","snPcode":"11356","submissionUrl":"https://submission.nature.com/new-submission/11356/3","title":"Environmental Science and Pollution Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"titanium dioxide nanoparticles, aerobic granular sludge, algal-bacterial granular sludge, microbial activity, chronic response","lastPublishedDoi":"10.21203/rs.3.rs-4629286/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4629286/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe chronic effects of titanium dioxide nanoparticles (TiO\u003csub\u003e2\u003c/sub\u003e NPs) on aerobic granular sludge (AGS) and algal-bacterial granular sludge (ABGS) was examined in this study. Sequencing batch bioreactors (SBRs) and photo sequencing batch bioreactors (PSBRs) were operated with synthetic wastewater containing 0, 1, 5, 10, 20, 30, and 50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e TiO\u003csub\u003e2\u003c/sub\u003e NPs for 10 days. Nanoparticles at concentrations of 1 and 5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e did not impact nutrient removal but led to an increase in extracellular polymeric substances (EPSs), primarily in protein (PN). With increasing nanoparticle concentration, the negative effect became more pronounced, mainly in the AGS SBRs. At 50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e TiO\u003csub\u003e2\u003c/sub\u003e, chemical oxygen demand (COD), ammonia-nitrogen (NH\u003csub\u003e3\u003c/sub\u003e-N), and phosphorus (PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e) removal decreased by 20.9%, 12.2%, and 35.1% in AGS, respectively, while in ABGS, they reached only 13.4%, 5.7%, and 14.2%. ABGS exhibited steady-state nutrient removal at 30 and 50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e TiO\u003csub\u003e2\u003c/sub\u003e NPs after around 5 days. The higher microbial activity and EPS content in the sludge, coupled with the symbiotic relationship between algae and bacteria, contributed to the higher tolerance of ABGS to nanoparticles. Finally, although nanoparticles reduced biomass in both types of bioreactors, the accumulation of TiO\u003csub\u003e2\u003c/sub\u003e NPs in the sludge, confirmed by Energy-dispersive X-ray spectroscopy analysis, and the absence of detectable titanium concentrations in the effluent wastewater, measured by Inductively-coupled plasma mass spectrometry, may be attributed to the specific operational conditions of this study, including the relatively short operation period (10 days) and high initial MLSS concentration (6 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003c/p\u003e","manuscriptTitle":"A comparative study on the chronic responses of titanium dioxide nanoparticles on aerobic granular sludge and algal-bacterial granular sludge processes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-22 10:07:38","doi":"10.21203/rs.3.rs-4629286/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accept","date":"2024-11-14T03:50:22+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-11-07T01:36:53+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-06T15:36:28+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Environmental Science and Pollution Research","date":"2024-11-06T04:11:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Environmental Science and Pollution Research","date":"2024-11-05T05:06:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"environmental-science-and-pollution-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"espr","sideBox":"Learn more about [Environmental Science and Pollution Research](https://www.springer.com/journal/11356)","snPcode":"11356","submissionUrl":"https://submission.nature.com/new-submission/11356/3","title":"Environmental Science and Pollution Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8ac64b76-de39-431a-a73b-fb171c6ad96b","owner":[],"postedDate":"November 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-11-25T16:01:18+00:00","versionOfRecord":{"articleIdentity":"rs-4629286","link":"https://doi.org/10.1007/s11356-024-35581-z","journal":{"identity":"environmental-science-and-pollution-research","isVorOnly":false,"title":"Environmental Science and Pollution Research"},"publishedOn":"2024-11-20 15:57:25","publishedOnDateReadable":"November 20th, 2024"},"versionCreatedAt":"2024-11-22 10:07:38","video":"","vorDoi":"10.1007/s11356-024-35581-z","vorDoiUrl":"https://doi.org/10.1007/s11356-024-35581-z","workflowStages":[]},"version":"v1","identity":"rs-4629286","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4629286","identity":"rs-4629286","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}