Accepted Manuscript Responses of anaerobic granule and flocculent sludge to ceria nanoparticles and toxic mechanisms Jingyun Ma, Xiangchun Quan, Xiurong Si, Yachuan Wu PII: DOI: Reference:

S0960-8524(13)01512-5 http://dx.doi.org/10.1016/j.biortech.2013.09.080 BITE 12445

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

8 July 2013 16 September 2013 18 September 2013

Please cite this article as: Ma, J., Quan, X., Si, X., Wu, Y., Responses of anaerobic granule and flocculent sludge to ceria nanoparticles and toxic mechanisms, Bioresource Technology (2013), doi: http://dx.doi.org/10.1016/ j.biortech.2013.09.080

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Responses of anaerobic granule and flocculent sludge to ceria nanoparticles and toxic mechanisms Jingyun Ma, Xiangchun Quan, Xiurong Si, Yachuan Wu Key Laboratory of Water and Sediment Sciences of Ministry of Education/State Key Laboratory of Water Environment Simulation, School of Environment, Beijing Normal University, Beijing 100875, P.R. China. Abstract Effects of CeO2-NPs on anaerobic fermentation were investigated from the processes of acidification and methanation with anaerobic granule sludge and anaerobic flocculent sludge as the targets. Results showed that acidification process was more sensitive to CeO2-NPs than methanation process. Both types of sludge produced less short-chain fatty acid compared to the control, with a reduction of 15-19% for the flocculent sludge at the dosage of 5, 50 and 150 mg CeO2-NPs/g-VSS, and a reduction of 35% for the granular sludge at 150 mg CeO2-NPs/g-VSS. CeO2-NPs caused no inhibition to methanation process. Most of CeO2-NPs distributed on the surface of sludge as revealed by fluorescence labeled CeO2-NPs. The toxicity of CeO2-NPs to anaerobic sludge did not result from reactive oxygen species. Physical penetration and membrane reduction may be important toxic mechanisms. Keywords: ceria nanoparticles; anaerobic granule sludge; anaerobic flocculent sludge; acidification; methanation 

Corresponding author. Address: School of Environment, Beijing Normal University, No.19, Xinjiekouwai Street, Haidian District, Beijing 100875, P.R. China. Tel.: 86-10-58802374; fax: 86-10-58802374. E-mail address: [email protected] (X. Quan). 1

1. Introduction

Ceria is an important rare-earth oxide and ceria nanoparticles (CeO2-NPs ) has been widely used as an abrasive, fuel additive, ultraviolet (UV) light absorber or an antioxidant to protect cells against radiation damage in many industries (Laberty-Robert et al., 2006; Perez et al., 2008; Tarnuzzer et al., 2005). With increasing production and application of CeO2-NPs, there would be increasing possibility of these particles released into sewage pipes, wastewater treatment plants (WWTPs) and finally entering aquatic environment (Boxall et al., 2007). CeO2-NPs have already been detected in surface water environment, and they were reported to produce adverse effects on organisms at environment relevant concentrations (1nmol/L-100 nmol/L) (Tiede et al., 2009; Zhang et al., 2011). Wastewater treatment plants play an important role in pollutants removal and a large part of nanoparticles is removed from a biological wastewater treatment system through adsorption to sludge (Limbach et al., 2008). Anaerobic treatment is often used as the pre-treatment for some complex wastewater and the method for excess sludge treatment. Therefore, there are significant chances for CeO2-NPs to interact with anaerobic sludge and influence anaerobic process. Several researches have studied the toxicity and possible toxic mechanisms of CeO2-NPs to some aquatic organisms and microorganisms. Rogers et al. (2010) found that CeO2-NPs displayed a significant

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toxicity to a freshwater alga (Pseudokirchneriella subcapitata) with a median effective concentration (EC50) of 10.3  1.7 mg/L. Thill et al. (2006) reported that CeO2-NPs was lethal to Escherichia coli. The toxicity of CeO2-NPs came from the production of harmful reactive oxygen species (Park et al., 2008) or the damage of cell membrane due to an oxidative reaction (Thill et al., 2006). Most of current researches focus on the toxicity of CeO2-NPs to a single species or pure strains under aerobic conditions, however, effects of CeO2-NPs on anaerobic mixed cultures were seldom reported. García et al. (2012) first reported that CeO2-NPs could also cause inhibition to thermophilic and mesophilic anaerobic bacteria under anaerobic conditions. Anaerobic sludge often exists in planktonic form such as flocculent sludge or aggregated form such as granular sludge. Granular sludge has a denser and multi-layer structure, with microbes closely attached to each other and embedded in an extracellular matrix (Liu et al., 2009), and different functional microbial populations located in different spaces (Subramanyam, 2013), while flocculent sludge shows a much looser and more homogenous structure. For anaerobic sludge existing in different forms (flocculent and aggregated form), the problem how CeO2-NPs will influence their performance during anaerobic process and possible toxic mechanism is not clear and deserves further study. The aims of this study were to reveal responses of different types of anaerobic sludge (flocculent and granular sludge) to CeO2-NPs during the separated process of acidification and methanation and their integration, and to explore possible toxic mechanisms of CeO2-NPs to anaerobic sludge under anaerobic conditions.

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2. Materials and Methods

2.1 Seeding sludge and nanoparticles

Anaerobic granule sludge used in this study was collected from an upflow anaerobic sludge blanket (UASB) reactor treating brewery wastewater (Beijing, China), which had a mean diameter of 720 ± 55 μm. Anaerobic flocculent sludge was collected from a secondary sedimentation tank of a municipal wastewater treatment plant (Beijing, China). Before exposition to ceria nanoparticles, both types of sludge was acclimated to synthetic wastewater at 35  1 ℃ for about one month until gas productions reached stable. The ceria nanoparticles used in this study was purchased from Sigma Aldrich (St. Louis, MO) with an average size less than 25 nm and purity higher than 99.9%. The suspensions of 300 mg/L nanoparticles stock were prepared by adding 300mg nanoparticles to 1.0 L distilled water (pH 6.9  0.1), followed by 1 h of ultrasonication (40 kHz, 200 W).

2.2 Effects of ceria nanoparticles on the acid- and methane- producing processes

Three dosages (5, 50 and 150 mg/g-volatile suspended solid (VSS)) of CeO2-NPs were used to investigate their impacts on anaerobic sludge in this paper considering

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different accumulation levels of nanoparticles in sludge and their potential toxicity (Nyberg et al., 2008;Gomez-Rivera et al., 2012). Effects of CeO2-NPs on anaerobic treatment process were assessed from the productions of short-chain fatty acid (SCFA) during the acidification process and methane during the methanation process. Acidification reaction was conducted in 50 mL of serum bottles (a reaction volume of 30 mL) filled with synthetic wastewater (glucose as carbon source), granular or flocculent sludge (2 g-VSS /L) and spiked with CeO2-NPs. Chloroform (15 μL) was also added to restrain the activity of methanogens. All bottles were flushed with nitrogen gas for 5 min to remove oxygen, and then sealed with butyl rubber stoppers and incubated in a shaker (180 rpm) at 35  1 ℃. The productions of short-chain fatty acid (SCFA) were detected after one-day reaction to assess the effects of CeO2-NPs on the acidification process. Methanation and accumulative methane production experiments were same to the acidification experiment except that they were conducted in 250 mL of serum bottles (a reaction volume of 50 mL) with sodium acetate used as the carbon source for methanation, glucose for accumulative methane assay and no chloroform added. The pH in each bottle was adjusted to 7.0 with NaHCO3 or HCl. Methane productions were measured at certain time intervals during a 6-day reaction. All the above experiments were carried out in triplicate. Synthetic wastewater with the COD:N:P proportion of 200:5:1 was used throughout this study, and the synthetic wastewater contains (mg/L): 2000 chemical oxygen demand (COD), 190 NH4Cl, 44 KH2PO4, 200 yeast extract, 6 CaCl2, 11.5 MgSO4·7H2O, 2 FeCl3·4H2O, 2 CoCl2·6H2O,

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0.4 MnSO4·H2O, 0.1 ZnSO4·7H2O, 0.09 (NH4)6Mo7O24·4H2O, 0.05 NiSO4·6H2O and 0.04 CuSO4·5H2O.

2.3 Distributions of CeO2-NPs in sludge

In order to visualize the distribution of CeO2-NPs in sludge, some CeO2-NPs were labeled with a fluorescence dye, fluorescein isothiocyanate (FITC), according to the methods described by Xia et al. (2008). Briefly, the surface of CeO2-NPs was first attached by alkoxy silane groups and then by FITC molecules. The detailed steps involved: CeO2-NPs (40 mg) was first dispersed in anhydrous dimethylformamide (DMF) (30 mL) and then added by 5 μL aminopropyl triethoxy silane (APTS) solution diluted in 250 μL DMF; the particle suspension was sonicated and stirred under nitrogen at room temperature for 20 h, and then collected by centrifugation; the modified nanoparticles was re-suspended in 5 mL of DMF and mixed with 5 mL of FITC solution (2 mg/mL) under stirring conditions for 4 h. The FITC-labeled CeO2-NPs was finally dried under vacuum to remove the organic solvent and stored as dry powders. Some flocculent and granular sludge samples were collected and observed with a fluorescence microscope (FM) after exposing to the fluorescence labeled CeO2-NPs (150 mg CeO2-NPs/g-VSS) for 6 days. Fluorescence was detected at an excitation and emission wavelengths of 488 nm and 500-550 nm. The green particles in FM images were FITC-labeled CeO2-NPs. The results were shown in Figure S1 (Supplementary

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material). At least ten samples under each condition were observed.

2.4 Distributions of live/dead cells in anaerobic granule sludge

To investigate the microbial survival status in anaerobic granule sludge after exposing to 150 mg/g-VSS of CeO2-NPs (6 days), granular sludge was stained with fluorescence labeled probes to discern the distribution of total cells (stained by Syto 63) and dead cells (stained by Sytox Blue). All probes were purchased from Invitrogen (Carlsbad, California, USA). The anaerobic granular sludge was stained as described by Chen et al. (2007). First, Syto 63 (20 μmol/L, 100 μL) was mixed with the sludge samples and incubated in a rotary shaker (100 rpm) for 30 min, then the excess dye solution was removed by 0.1 mol/L buffer (pH 7.2). Next, the Sytox Blue solution (2.5 μmol/L, 100 μL) was incubated with the samples for 5 min without further washing. The stained granules were sliced into 30 μm sections after freezing at −20 ℃. The sections around the core of granules were mounted onto microscopic slides and observed with a confocal laser scanning microscope (CLSM, Carl Zeiss LSM 510, Germany). Ten random cryosections under each condition (with or without CeO2-NPs) were observed. The CLSM images were displayed in Figure S2 (Supplementary material). The number of live/dead cells in sludge was quantified by calculating the integrated density of each image using the software ImageJ.

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2.5 Intracellular reactive oxygen species and lactate dehydrogenase concentration determination

To explore possible toxic mechanisms of CeO2-NPs to sludge, the productions reactive oxygen species (ROS) and lactate dehydrogenase activity (LDH) after exposure to CeO2-NPs were measured. The production of intracellular ROS was measured using dichlorodihydrofluorescein diacetate (H2DCF-DA, Molecular Probes, Invitrogen) according to the reference (Mu and Chen, 2011). Briefly, the sludge pretreated with CeO2-NPs washed with PBS and then incubated with 25 μmol/L H2DCF-DA at 35  1 ℃ in darkness for 30 min. The pellets collected by centrifugation were resuspended in PBS and transferred into a 96-well plate. Fluorescence was then read at an excitation and emission wavelengths of 485 and 520 nm using a microplate reader (Tecan Infinite M200, Switzerland). LDH was determined according to the following procedure (Han et al., 2011): the supernatant was separated from the sludge mixture after exposition experiments and mixed with sodium pyruvate (0.75 mmol/L), nicotinamide ade-nine dinucleotide (NADH, 0.15 mmol/L, preheating at 25 ℃) and potassium phosphate buffer (70 mmol/L), and then incubated at 37 ℃ for 30 min. The above mixture was then transferred into a 96-well plate and measured at 340 nm and LDH activity was obtained by measuring the decreasing rate of NADH absorbance.

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Each experiment was carried out in triplicate. ROS and LDH values of the treated groups were expressed as the percentage of control groups that were assumed to be 100%.

2.6 Other analytical methods and statistical analysis

The extracellular polymeric substances (EPS) in anaerobic sludge were extracted by heating at 80 ℃ for 30 min and measured according to the methods used by Adav and Lee (2008). The determinations of SCFA and VSS were the same as the methods described by Yuan et al. (2006). The total SCFA was calculated as the sum of measured acetic, propionic and butyric acid. The values of total SCFA, methane, ROS and LDH of CeO2-NPs-treated groups were expressed as the percentage of control groups, which were assumed to be 100%, and presented as the mean  standard deviation of three separate experiments. A normal distribution of these relative values (percentages), individual SCFA and EPS productions were firstly validated by a test of normal distribution and then one-way ANOVA (analysis of variance) followed by multiple comparison test was used to test the significance of each result (p < 0.05 was considered to be statistically significant).

3. Results and discussion

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3.1 Effects of CeO2-NPs on the productions of short-chain fatty acid and methane

Impacts of CeO2-NPs on acidification and methanation, the two main processes of anaerobic fermentation, were investigated with anaerobic granule sludge and flocculent sludge as the targets (Fig. 1). Results showed that the total SCFA productions during acidification were significantly affected for both granular and flocculent sludge at CeO2-NPs dosages of 5, 50 and 150 mg/g-VSS (p < 0.05). The production of total SCFA by granular sludge increased by 33-36% at CeO2-NPs dosages of 5 and 50 mg/g-VSS, but decreased by 35% at 150 mg/g-VSS compared to the control, while the corresponding parts by flocculent sludge decreased by 15-19% at the above tested dosages (Fig. 1a). The data indicated that flocculent sludge was more sensitive to the presence of CeO2-NPs as it was inhibited at all the tested concentrations, while anaerobic granular sludge was only inhibited at relatively higher concentrations. This difference may be due to the different structures of flocculent and granular sludge. Flocculent sludge has a larger specific surface and looser structure than granular sludge, which favors the adsorption of CeO2-NPs to sludge surface and increases the chance of microbes contacting and interacting with CeO2-NPs. On the contrary, anaerobic granule sludge has a dense and multilayer structure, which not only provides a shelter for bacteria inside to resist against a harsh environment, but also reduces the opportunity of microorganisms contacting with CeO2-NPs. For granular sludge, an enhancement of SCFA production at the dose of 5 and 50 mg/g-VSS was observed, which may be due to

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that a low toxic stress sometimes may stimulate bioactivity of some bacteria. The variation of individual SCFA after exposure to CeO2-NPs was also measured and presented in Fig. 1c and d. For granular sludge, butyric acid production increased at 5 and 50 mg/g-VSS of CeO2-NPs, but acetic and propionic acid productions were reduced at 150 mg/g-VSS. For flocculent sludge, the production of butyric acid decreased significantly at all dosages of CeO2-NPs, suggesting butyric acid mainly contributed the reduction of SCFA caused by CeO2-NPs. As for the process of methane production, it could be found that methane productions were not significantly influenced for both granular and flocculent sludge at above different CeO2-NPs dosages (Fig. 1b), indicating that ceria NPs exposition did not inhibit the process of methane productions. The different effects of CeO2-NPs on the individual process of acidification and methanation may be attributed to the different characteristics of the functional microbial populations and their different space distribution in sludge. Different to acidification microbial population, methanogens generally exist inside sludge clusters or granular sludge, and therefore have relatively less opportunity to contact with the nanoparticles absorbed on sludge surface (Hribersek et al., 2011; Wu and He, 2010). In addition, methanogens may restore bioactivity after a period of adaption to the presence of certain toxic compounds, which may be another reason for the no inhibition effects (Yan et al., 2008). #Figure 1 The above data showed impacts of CeO2-NPs on two independent processes of

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acidification and methanation. In fact, for anaerobic fermentation, acidification and methanation are not separated but an integrated process. Effects of CeO2-NPs on the whole process of anaerobic fermentation were assayed by measuring accumulative methane production verse operation time. It could be found that CeO2-NPs did not influence the final methane production rate (Fig. 2). The observation that CeO2-NPs inhibited a separated acidification process but not inhibit final methane production rate for a complete fermentation process may be due to the fact that acidification rate is much faster than methanation rate and methane production is a rate-limited step, so less variations of SCFA production may not influence the methanation process. Overall, for both types of anaerobic sludge, acidification process was more sensitive than methanation process to the presence CeO2-NPs. #Figure 2 Effects of CeO2-NPs on anaerobic digestion was also investigated by García et al. (2012), who found that CeO2-NPs caused a great inhibition to biogas production after long-term (about 50 days) operation. Different to his research, no inhibition of CeO2-NPs to methane production was found in this study, which may be due to that easily biodegradable carbon sources such as acetate sodium and glucose and short-term exposition (6 days) were used here to assay the inhibition effects. Similar to this result, Mu et al (2011) investigated other metal oxide nanoparticles on anaerobic digestion and found that Nano-TiO2, nano-Al2O3 and nano-SiO2 in doses up to 150 mg/g-TSS (total suspended solids) did not influence methane production.

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3.2 Distributions of ceria particles in sludge and effects on cell viability

According to the aforementioned data, effects of CeO2-NPs on the processes of acidification and methanation may be associated with their space distributions in sludge. Fluorescence labeled CeO2-NPs coupled with FM was used to track CeO2-NPs distribution in sludge. For flocculent sludge, CeO2-NPs distributed evenly in sludge (Supplementary Figure S1a), and they may diffuse to deeper position due to the loose structure. For granular sludge, CeO2-NPs focused on the surface or out layers (Supplementary Figure S1b and c) indicating that CeO2-NPs can hardly move into the interior of granules. This can be explained by the compact structure of granular sludge and formation of larger CeO2-NPs for particles aggregation. It was reported that the nanoparticles with a size larger than 66 nm would be completely excluded from biofilms (Golmohamadi et al., 2013). Mu et al. (2012) also reported that large amounts of ZnO nanoparticles were mainly absorbed on the surface of anaerobic granule sludge. To further explore effects of CeO2-NPs on cell viability, distributions of live and dead cells in granular sludge were investigated. From the typical CLSM image, it could find that the density of both dead cells (blue regions in Supplementary Figure S2b1 and b2) and live cells (red regions in Supplementary Figure S2d1 and d2) declined from periphery to inner part of granules, while relatively more dead cells were found on the surface of granular sludge after exposition to CeO2-NPs. According to the calculated

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results of integrated density, the ratio of dead cells accounting for the total cells was increased by 2-8% after exposition to CeO2-NPs, validating that CeO2-NPs was toxic to the microbes (most acid-producing bacteria) located on the surface of granules. Considering the distributions of CeO2-NPs and live/dead cell in granular sludge and the different inhibition effects of CeO2-NPs to acidification and methanation processes together, it could be concluded that direct physical contacts between particles and bacteria were an important precondition for the inhibition of CeO2-NPs to the microbes in sludge and the similar results were also reported by Thill et al. (2006).

3.3 Effects of ceria nanoparticles on extracellular polymeric substances secretion

EPS, as an important component of sludge, plays a key role in protecting microorganisms against environmental stress (Henriques and Love, 2007). The secretion of EPS for the two different types of sludge in the presence of CeO2-NPs was investigated. Results showed that for both granular sludge and flocculent sludge, exoprotein (PN) production kept relatively stable, while exopolysaccharide (PS) production was significantly changed (Table 1). The productions of PS in granular sludge increased by 31% and 39% at CeO2-NPs dosages of 5 and 150 mg/g-VSS respectively compared to the control, while those in flocculent sludge declined by 34% and 27% respectively. The different responds of granular and flocculent sludge on PS productions could be explained from following aspects: flocculent sludge has a large

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surface and a loose structure, which is beneficial for the adsorption and transport of nanoparticles into sludge and thus results in a strong toxicity; granule sludge has a dense structure with more microbes encapsulated inside the granule, which limited the contact of CeO2-NPs with sludge and thus leads to a less toxicity; sludge would accumulate more EPS (especially exopolysaccharides) under a low toxicity condition as a protective response to toxicants but reduce EPS production under a high toxicity condition for the loss of microbial activity (Zou et al., 2009). As EPS matrix serves as an important barrier to protect the bacteria inside activated sludge against toxic shocks (Henriques and Love, 2007), the production of more polysaccharide in granular sludge increased the ability to resist toxic or harsh environment. In addition, polysaccharides could increase the hydrodynamic diameter of nanoparticles and promote their aggregation (Joshi et al., 2012), which may be another important reason for the enhanced resistance to the toxicity of CeO2-NPs with more polysaccharides, because larger CeO2-NPs displayed less toxicity than smaller ones and can hardly move from granular surface to interior. #Table 1

3.4 Possible toxic mechanisms of CeO2-NPs under anaerobic conditions

Reactive oxygen species induced by nanoparticles have been one paradigm for explanation of toxic mechanisms of many nanoparticles (Niazi and Gu, 2009).

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Production of harmful ROS has been found in human cells or freshwater alga after exposition to CeO2-NPs (Park et al., 2008; Rogers et al., 2010). However, the response of ROS production for anaerobic sludge exposure to CeO2-NPs under anaerobic conditions was still not clear, and therefore it was measured in this study. It was found that CeO2-NPs did not result in an obvious increase in intracellular ROS for both granular and flocculent sludge, and even led to ROS reduction for flocculent sludge at the dosage of 150 mg/g-VSS (Fig. 3). The result suggests that the toxicity of CeO2-NPs under anaerobic conditions did not result from ROS. It has been regarded that that the toxicity of metal/metal oxide nanoparticles to aerobic bacteria mostly resulted from ROS generated through their reactions with molecular oxygen and/or light (Niazi and Gu, 2009). CeO2-NPs did not cause ROS increase in the tested anaerobic sludge in this study, which might be due to the fact that all these assays were conducted under anaerobic conditions without the presence of oxygen and light. Ce3+ usually exists on the surface of cerium oxide particle due to the variation of lattice parameters (Tsunekawa et al., 1999), or the reduction of CeO2 by the outer membrane of bacteria after a close contact (Zeyons et al., 2009). Ce3+ could act as an anti-oxidant to scavenge free radicals (hydroxyl radicals) from cultures (Das et al., 2007), and more Ce3+ may be produced on the surface of the flocculent sludge compared to the granular sludge due to the larger specific surfaces and stronger adsorption of CeO2, which may explain the reduction of ROS for flocculent sludge at 150 mg/g-VSS of ceria NP, but that for the granular sludge was not obviously influenced. Overall, under anaerobic conditions,

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CeO2-NPs did not induce the increase of ROS and even may act as a free radical scavenger, which might be a reason for the low cytotoxicity of CeO2-NPs to anaerobic sludge. LDH release was measured as an indicator of cell-membrane damage. For flocculent sludge, a significant increase in LDH was observed at the dosages of 5 mg/g-VSS and 150 mg/g-VSS, while for granular sludge, it was only observed at the high dosage of 150 mg/g-VSS (Fig. 3). Interactions of nanoparticles with cell membrane are complex. Some nanoparticles such as fullerene work on membrane by altering fatty acid compositions (Fang et al., 2007), some may increase membrane permeability by physical penetration (Park et al.,2008 ), and some may disrupt membrane by redox (Thill et al., 2006). For CeO2, besides the factor of physical penetration of small size nanoparticles into cell membrane, an oxidative stress to outer membrane triggered by the oxidative power of Ce4+ may be also an important factor for the membrane damage and LDH release (Zeyons et al., 2009). For many metal/metal oxides nanoparticles, metal ion released from nanopartilces is regarded as a key factor determining toxicity. As CeO2-NPs are insoluble and can hardly release cerium ion, the effects of cerium ion on cytotoxicity of CeO2-NPs can be neglected (Zhang et al., 2011). Besides the above factors, other unknown factors may also exist and determine the toxicity of CeO2-NPs. Based on current research results, a possible toxic mechanism of CeO2-NPs under anaerobic conditions could be summarized as follows: CeO2-NPs is

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adsorbed to sludge surface, followed by physical penetration and oxidation behavior of Ce4+ to the outer membrane of bacteria, and finally leads to the damage and death of cells. #Figure 3

4. Conclusions

Effects of ceria nanoparticles on anaerobic granular and flocculent sludge were investigated from two independent and whole fermentation processes. Acidification process was more sensitive to ceria nanoparticles than methanation process, with an obvious inhibition to SCFA production but no inhibition to methane production. Granule demonstrated a higher resistance to ceria nanoparticles than flocculent sludge due to its dense and multi-layer structure as well as secretion of more exopolysaccharides. The toxicity of ceria nanoparticles to anaerobic sludge was not caused by ROS. Physical penetration and membrane reduction through direct contacts and interactions with cell membrane may be important toxic mechanisms.

Acknowledgements

This research was supported by “National Natural Science Foundation of China” (No. 51178049).

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Figure Captions Fig. 1 Effects of ceria nanoparticles exposure to anaerobic sludge on the production of total short-chain fatty acid (a), methane (b) and individual SCFA (c, anaerobic granule sludge; d, anaerobic flocculent sludge). Lower case indicator letters mean p < 0.05. Indicator letters in common denote a lack of significant. The methane productions did not show significant differences between groups (p  0.05). Error bars represent standard deviations of triplicate tests. Fig. 2 Effects of ceria nanoparticles on accumulative methane production by anaerobic granule sludge (a) and flocculent sludge (b). All groups treated with CeO2-NPs (5, 50, 150 mg/g-VSS) did not show significant differences from the control (p  0.05). Error bars represent standard deviations of triplicate tests. Fig. 3 Relative ROS productions and LDH activities for anaerobic sludge at different dosages of CeO2-NPs. Asterisks indicate statistical differences (p < 0.05) from the controls. Error bars represent standard deviations of triplicate tests.

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Table 1 Effects of CeO2-NPs on the compositions of EPSa in anaerobic granular and flocculent sludge. Ceria nanoparticles (mg/g-VSS) Anaerobic granular sludge Anaerobic flocculent sludge

0

5

150

Exopolysaccharide

21.90  0.6

28.76  1.2

30.50  1.11 b

Exoprotein Exopolysaccharide

28.09  2.19 32.35  3.61

26.94  0.45 23.56  1.56 b

27.37  0.51 22.14  0.8 b

b

26.29  0.61 28.64  3.62 23.63  2.05 The data reported are the averages and their standard deviations in triplicate tests, and the unit is mg/g-VSS. bThe data reported are statistical differences (p < 0.05) from the control according to one-way ANOVA followed by multiple comparison test. Exoprotein

a

25

Figure 1

26

Figure 2

27

Figure 3

28

Highlights



Toxicity of CeO2-NPs to different types of anaerobic sludge was investigated.



Acidification process was more sensitive to CeO2-NPs than methanation process.



Granular sludge displayed a higher resistance to CeO2-NPs than flocculent sludge.



Direct contacts with sludge were a precondition for the cytotoxicity of CeO2-NPs.



The toxicity of CeO2-NPs did not result from ROS under anaerobic conditions.