Effects of CeO2 nanoparticles on production and physicochemical characteristics of extracellular polymeric substances in biofilms in sequencing batch biofilm reactor

Bioresource Technology 194 (2015) 91–98

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Effects of CeO2 nanoparticles on production and physicochemical characteristics of extracellular polymeric substances in biofilms in sequencing batch biofilm reactor Guoxiang You, Jun Hou ⇑, Yi Xu, Chao Wang, Peifang Wang, Lingzhan Miao, Yanhui Ao, Yi Li, Bowen Lv Key Laboratory of Integrated Regulation and Resources Development on Shallow Lakes, Ministry of Education, Hohai University, Nanjing 210098, People’s Republic of China College of Environment, Hohai University, Nanjing 210098, People’s Republic of China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 Studied effect of CeO2 NPs on EPS

production and properties in SBBR.  Protein production increased for

LB-EPS and TB-EPS after 50 mg/L CeO2 NP exposure.  A potential mechanism to explain the enhanced production of EPS.  –OH and –NH2 branches of hydroxyl and amine groups in EPS susceptible to CeO2 NPs.  Exposure to 50 mg/L CeO2 NP reduced flocculating capacity of LB-EPS and TB-EPS.

a r t i c l e

i n f o

Article history: Received 20 May 2015 Received in revised form 30 June 2015 Accepted 1 July 2015 Available online 8 July 2015 Keywords: CeO2 nanoparticle Extracellular polymeric substances (EPS) Production Physicochemical characteristics Sequencing batch biofilm reactor (SBBR)

a b s t r a c t Extracellular polymeric substances (EPS) are a major component of biofilms that act as a gel-like matrix, binding the cells together to form their three-dimensional structure. The effects of ceria nanoparticles (CeO2 NPs) on the production and physicochemical characteristics of EPS in biofilms in a sequencing batch biofilm reactor were investigated. Total EPS production, including loosely bound EPS (LB-EPS) and tightly bound EPS (TB-EPS), increased by 35.41% compared to in control tests without CeO2 NPs. Protein production increased by 47.02% (LB-EPS) and 58.83% (TB-EPS) after 50 mg/L CeO2 NP exposure. Three-dimensional excitation–emission fluorescence spectra revealed that tyrosine (LB-EPS) and aromatic (TB-EPS) protein-like substances formed after CeO2 NP exposure. Fourier transform infrared spectroscopy results indicated the susceptibility of –OH and –NH2 in EPS hydroxyl and amine groups to CeO2 NPs. Exposure to 50 mg/L CeO2 NPs reduced the flocculating capacity of LB-EPS (51.78%) and TB-EPS (17.14%), consistent with the decreased zeta potential. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Cerium oxide nanoparticles (CeO2 NPs) are widely applied in many commercial, industrial, and consumer products, such as

⇑ Corresponding author at: College of Environment, Hohai University, 1 Xikang Road, Nanjing 210098, People’s Republic of China. Tel./fax: +86 25 83787332. E-mail address: [email protected] (J. Hou). http://dx.doi.org/10.1016/j.biortech.2015.07.006 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.

polishing agents, personal care products, superconducting materials, fuel additives, and coating glass/ceramics (Trujillo-Reyes et al., 2013). Nevertheless, recent studies have shown that once CeO2 NPs are released into the environment, they could be absorbed and generate toxicity in algae, activated sludge, and zebrafish, owing to interactions with microorganisms (Thill et al., 2006; Limbach et al., 2008; Johnston et al., 2010), and pose a potential threat to both the ecosystem and human health (Wang et al., 2012; Lin

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G. You et al. / Bioresource Technology 194 (2015) 91–98

et al., 2006). It has demonstrated that CeO2 NPs have acute toxic effect to bacteria (Hou et al., 2015b), decrease cell viability of activated sludge (Ma et al., 2013), and cause oxidative stress in microalgae (Thill et al., 2006). Hence, there is an urgent need to understand the environmental impact of CeO2 NPs. Wastewater treatment plants (WWTPs) are crucial for blocking NPs from entering the environment (Hou et al., 2015a), and some reports have suggested that the presence of NPs may adversely affect the population of the microbial community, reduce the microbial diversity, and lead to a reduction in the efficiency of the activated sludge (García et al., 2012). In addition to activated sludge, biofilms are a major focus in wastewater treatment (Hou et al., 2014), and play a key role in adsorbing and removing contaminants (Sheng and Liu, 2011). In particular, extracellular polymeric substances (EPS), a major component in biofilms, act as a gel-like matrix that binds the cells together in order to form aggregates and provide protection for microorganisms against the harsh external environment (Sheng et al., 2010; Ma et al., 2013). In addition, EPS are usually thought to bind with toxic materials (Sheng et al., 2013), and can thus improve the resistance of the microbial community to NPs (Hou et al., 2014; Sheng and Liu, 2011). Studies have shown that in the presence of EPS, NPs might be prevented from diffusing into the wastewater biofilm (Hou et al., 2014), leading to biofilm bacteria more tolerant to TiO2 NPs and AgO NPs than planktonic bacteria (Liu et al., 2007; Sheng and Liu, 2011). Although certain researchers have noted that the characteristics of EPS were obviously influenced by NPs in activated sludge (Hou et al., 2015a; Sheng and Liu, 2011), few studies have been conducted to investigate the impacts of CeO2 NPs on the production and physicochemical characteristics of EPS in biofilms in a sequencing batch biofilm reactor (SBBR). The production and physicochemical characteristics of EPS in biofilms make great contributions to the formation process of a biofilm, as well as the growth and proliferation of bacteria (Sheng et al., 2010; Jing et al., 2014). They are mainly composed of high-molecular weight organic materials that are secreted by microbes, such as polysaccharides, proteins, lipids, DNA, and humic acids (Flemming and Wingender, 2010). EPS are also involved in the formation of microbial aggregates, adhesion to surfaces, and bioflocculation (Sheng and Yu, 2006). In recent studies, EPS were mainly separated into two groups, loosely bound EPS (LB-EPS) and tightly bound EPS (TB-EPS) (Li and Yang, 2007). The TB-EPS, which have a particular shape, are present in the inner layer of the biofilm, while the LB-EPS are in the outer layer, and are loose, without a distinct boundary (Sheng et al., 2010). It is reasonable to assume that EPS containing various fractions of TB-EPS and LB-EPS exhibit different physicochemical properties in protecting the bacteria from the hostile external environmental conditions (Zhang et al., 2014). Furthermore, the different compositions, with the various characteristics of each of the EPS fractions in terms of organic matter and cations, possess a diverse flocculability in their underlying bioflocculants (Yu et al., 2009). Unfortunately, with the emergence of CeO2 NPs, knowledge regarding the flocculation of EPS fractions in biofilms remains sparse. The aim of this study is to explore the effects of CeO2 NPs on the production and composition of EPS in biofilms, as well as the changes in the physicochemical characteristics of EPS induced by these effects, by answering the following questions: 1. How do different concentrations of CeO2 NPs affect the content of LB-EPS and TB-EPS in a biofilm, and what is the potential mechanism related to content? 2. What are the different responses of these two types of EPS to polysaccharides, proteins, and humic acid, and how do the functional groups in these compositions respond when faced with toxicity?

3. What is the potential impact of CeO2 NPs on the flocculation efficiency of EPS? 2. Methods 2.1. CeO2 NPs and the biofilm matrix CeO2 nanopowder was purchased from Sigma–Aldrich (St. Louis, MO). According to the CeO2-NP product description, the particle size is less than 50 nm, the density is 7.13 g/mL at 25 °C, and the specific surface area is 30 m2/g. A scanning electron microscope (SEM) image of the CeO2 NPs was obtained using a Hitachi S-4800 SEM to obtain a direct view of the NP shape (Fig. S1). Through the addition of 0.1 g of CeO2 NPs to 1 L of Milli-Q water (pH 7 ± 0.1), a 100 mg/L CeO2 NPs stock suspension was prepared prior to experiment, followed by 1 h of ultra-sonication (20 °C, 250 W, 40 kHz) (Keller et al., 2010). The particle size and zeta potential of the CeO2 NPs in stock suspension was then determined using Malvern Zetasizer Nano ZS90 equipment (Malvern Instruments, UK). The average hydrodynamic diameter of the CeO2 NPs was 290 ± 16 nm, and the zeta potential was 20 ± 4.21 mV (there were 5 samples). The SBBR operation and biofilm culturing were conducted according to the method described in our previous publication (Hou et al., 2015b). 2.2. Short-term exposure experiments In order to explore the effects of the CeO2 NPs on the production and physicochemical characteristics of the different EPS fractions, three different concentrations of CeO2 NPs were employed in this experiment. At the lowest level of concentration, a suspension of 1 mg/L of CeO2 NPs was prepared. This is the environmentally relevant concentration of CeO2 NPs in WWTPs, and this sample is denoted as SBBR1. Considering the rapid development and growth in the use of NPs, suspensions of 10 and 50 mg/L CeO2 NPs were also considered (denoted SBBR2 and SBBR3, respectively). To conduct the experiments, 0, 30, 300, and 1500 mL of the CeO2 NPs stock solution (100 mg/L) were injected into the control SBBR, SBBR1, SBBR2, and SBBR3, respectively, and then 1.5 L of the synthetic wastewater was fed into each reactor. Milli-Q water was added to make the final volume of each reactor to be 3 L. Aeration was started to maintain the DO at 0.1–0.45 mg/L for 6.5 h, after an initial water sample was withdrawn. Thereafter, the biofilm samples were removed, and the properties and composition of the EPS in the biofilms were determined. 2.3. EPS extraction and analysis Fresh biofilm samples were scraped from the carrier and the LB-EPS and TB-EPS were extracted using a process involving centrifugation, sonication, and thermal extraction (Hou et al., 2015a). Approximately 25 mL of biofilm samples were placed in 50 mL centrifuge tubes, mixed with distilled water to form a 50 mL suspension, and then centrifuged at 6000g for 10 min in order to remove the loose slime polymers found in a biofilm. The sediments at the bottom of the centrifuge tubes were resuspended in a 0.05% (w/w) NaCl solution, and then sonicated at 20 kHz for 2 min. The suspensions were horizontally vibrated in a thermostat incubator at 150 rpm for 15 min. The liquid was centrifuged at 8000g for 10 min and the supernatant was collected carefully for use in measuring the LB-EPS. The residual sediments were resuspended at their initial volume again using a 0.05% (w/w) NaCl solution, sonicated at 20 kHz for 2 min, then heated at 70 °C for 30 min. Finally, the suspension was centrifuged at 11,000g for 30 min, and the supernatant was collected as the TB-EPS. All the EPS fractions

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were filtered through 0.45 lm acetate cellulose membranes and stored at 20 °C before analysis. Both the extracted LB-EPS and TB-EPS fractions were analyzed for total organic carbon (TOC), polysaccharides (PS), proteins (PRO), and humic-like substances (HS). The TOC was determined using a TOC analyzer (Liqui TOC II, Elementar, Germany). The PS content was measured using the anthrone-sulfuric acid method, using glucose as the standard (Hou et al., 2015a). The PRO content and HS content were measured using the modified Lowry method, with bovine serum albumin (Sigma) and humic acid (Sigma) as the standards, respectively (Li and Yang, 2007). 2.4. FT-IR spectroscopy FT-IR spectroscopy of the LB-EPS and TB-EPS exposed to different concentrations of CeO2 NPs was performed using FT-IR equipment (Tensor 27, Bruker). The wet samples were freeze-dried prior to analysis. The dry powdered samples were mixed and ground using spectrometry grade KBr at a mass ratio of 1:100. The absorbance spectroscopy was recorded over the frequency range from 4000 to 600 cm1 at a resolution of 4 cm1, using 32 scans, co-added and averaged. Baseline shifts were used to correct the baseline to reduce systematic errors. 2.5. 3D-EEM fluorescence spectroscopy 3D-EEM fluorescence spectroscopy is a highly efficient, highly selective, and highly sensitive technique, which can be employed to distinguish fluorescence compounds from complex EPS mixtures (Sheng and Yu, 2006). All the EEM spectra were obtained using luminescence spectrometry (F-7000 FL, Hitachi, Japan). In this work, the EEM spectra are composed of a series of emission spectra from wavelengths of 210 to 600 nm at 2 nm sampling intervals over a range of excitation wavelengths from 200 to 400 nm at 2 nm increments. The scanning speed was maintained at 1200 nm/min, and each slit was set to 2 nm for all measurements. 2.6. EPS flocculation test and zeta potentials Bioflocculation has gained much attention, due to its safety and environmentally friendly nature (Wu and Ye, 2007). The flocculating efficiencies of various EPS fractions have been obtained in a kaolin suspension (Yu et al., 2009). All the EPS solutions in this study were prepared at 1 mg/mL in Milli-Q, then 0.1 mL of EPS solution, 0.25 mL of CaCl2 (90 mM), and 4.65 mL kaolin suspension (5 g/L) were mixed in a colorimetric tube. The contents of the tube were mixed using a vortex generator (WH-2, Shanghai huxi analysis instrument factory, China) for 30 s, and then kept still for 5 min. The absorbance of the sample solution (Abs550) and the control sample (Abs550,b) were measured at a wavelength of 550 nm using a UV–Vis spectrophotometer (TU-1901, Persee, China). All tests were conducted in triplicate. A percentage giving the flocculating efficiency can be determined as the difference between the Abs550 readings taken for the different samples according to the following equation:

Flocculating efficiency % ¼ ðAbs550;b  Abs550 Þ=Abs550;b

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2.7. Other analytical methods and statistical analysis The assessment of bacterial viability was conducted via a two-color fluorescence assay (the viable bacteria fluoresce green and the damaged bacteria fluoresce red) using confocal laser scanning microscopy (CLSM, Nikon A1, Japan). The CLSM images were used to observe the activity of the bacteria, details of which are available in the Supplemental material. The surface structure of the microorganisms in biofilms in the presence and absence of CeO2 NPs treatment was examined using SEM (Hitachi S-4800). Detailed results are provided in the Supplemental material. Reactive oxygen species (ROS) generation is currently considered to be an important indicator of cellular effects, and regarded as the most acceptable parameter to use in assessing the toxicity of different NPs released into the environment (Von Moos and Slaveykova, 2014). The level of extracellular lactate dehydrogenase (LDH) is a paradigm used to characterize the integrity of cell membranes (Zheng et al., 2011). These two parameters were studied in order to explore the possible effects of CeO2 on cellular viability. Details of the methods employed to measure these are available in the Supplemental material. All assays were performed in triplicate and the results are given in terms of their mean ± standard deviation. For statistical analysis, the experimental values were compared to their corresponding control values. Statistical analysis (t-test) was performed to test the significance of results and p < 0.05 was considered to be statistically significant, as similar described by Hou et al. (2014).

3. Results and discussion 3.1. Effects of CeO2 NPs on production of EPS EPS, as a protective layer for the bacteria in a biofilm, are considered to determine the physicochemical properties of the biofilm and determine its structural and functional integrity (Flemming and Wingender, 2010). The effects of CeO2 NPs on the secretion of EPS in biofilms were therefore studied. The content of LB-EPS and TB-EPS (expressed in terms of TOC), shown in Fig. 1, was subject to various amounts of increase after exposure to different concentrations of CeO2 NPs. As the concentration of CeO2 NPs

ð1Þ

The flocculating efficiency of EPS in biofilms varied relative to the changing trend of the zeta potential (Zhang et al., 2014). The zeta potentials of different EPS fractions were investigated using a Zetasizer Nano ZS90 (Malvern Instruments, UK). Both the TB-EPS and LB-EPS solutions were diluted at 1 mg/mL using Milli-Q before measurement. All assays were performed in triplicate.

Fig. 1. The amount of LB-EPS and TB-EPS (expressed in terms of TOC content) extracted from biofilms exposed to different concentrations of CeO2 NPs. Asterisks indicate the statistical difference (p < 0.05) from the control sample. Error bars represent standard deviations determined from measurements performed in triplicate.

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increased from 0 to 50 mg/L, the average content of LB-EPS increased from 28.51 to 36.40 mg TOC/g EPS. In addition, the average TB-EPS increased from 61.72 to 85.78 mg TOC/g EPS. The production of EPS increased by 35.41% (LB-EPS increased by 27.67% and TB-EPS increased by 38.98%) when the biofilm was exposed to a high concentration (50 mg/L) of CeO2 NPs, which demonstrates the fact that the addition of CeO2 NPs helped to produce more EPS. The enhanced EPS encapsulated the cells and reduced toxicity by inducing the aggregation of NPs (Joshi et al., 2012). However, the percentage of TB-EPS only ranged from 68.40% to 70.87%. These results indicate that EPS exposed to the different concentrations of CeO2 NPs exhibited various yields but similar fractions of each type of EPS. Polysaccharides, proteins, and humic acid are believed to be the major components in the EPS matrix (Li and Yang, 2007). These three ingredients were therefore measured in order to reveal the potential mechanisms for increasing the EPS. The PS, PRO, and HS contents are shown in Table 1. PS comprise the dominant component in EPS, which is much higher than the other two components in production. The presence of 1 mg/L CeO2 NPs did not significantly affect the production of PS, PRO, and HS in both LB-EPS and TB-EPS. As the concentration of CeO2 NPs increased to 10 mg/L, the average PS and PRO contents in the LB-EPS increased by 11.37% and 12.15%, respectively, and by approximately 23.52% and 5.56%, respectively, in TB-EPS, compared to the control. When the concentration of CeO2 NPs increased to 50 mg/L, there was a noticeable increase in the chemical constituents in the EPS, the PS content increased by 17.80% in the LB-EPS, and by 10.37% in the TB-EPS, whereas the PRO content increased by 47.02% (LB-EPS) and 58.83% (TB-EPS). A significant increase in the PRO (both in LB-EPS and TB-EPS) content was observed only at a CeO2 NPs dosage of 50 mg/L (p < 0.05), which indicate the PRO may play a more important role in inhibiting the toxicity of CeO2 NPs, through enhanced EPS secretion. This result was consistent with Li et al. (2015) that PRO in the single-wall carbon nanotube (SWCNT) treated granules were accumulated at the outer layer of the granules, probably providing defense against the intrusion of SWCNTs. However, the production of HS decreased as the biofilm was exposed to higher concentrations of CeO2 NPs (10 and 50 mg/L). HS probably originated from the decomposition of dead cells and macromolecular organics (Qu et al., 2012). This may be explained by the fact that it was difficult for the organic material to move into the biofilm and be adsorbed by cells with an increase in EPS, as mass transfer into a biofilm is driven by diffusion (Sheng and Liu, 2011). In addition, the death and lysis of bacteria (a source of HS) might cause the release of a comparable amount of organic material that may be utilized by living bacteria (Foladori et al., 2015). The response in terms of the production of EPS in a biofilm to exposure to CeO2 NPs may be attributed to the following two mechanisms (Sheng and Liu, 2011; Hou et al., 2014): (i) the protective role of EPS; (ii) the increasing activity of living bacteria in the deeper areas of the biofilm. EPS may function as a barrier against the NPs and other toxicants for the microbes inside the biofilm.

The LB-EPS in the outer layer acted as the first place at which the biofilm has contact and interacts with CeO2 NPs. The loose and porous structure of LB-EPS provided enough binding sites for the adsorption of CeO2 NPs (Jing et al., 2014). In this way, the bacteria in the biofilm accumulated more EPS under toxic conditions, as a protective response to toxicants (Sheng et al., 2010), while the tight and dense structure of the TB-EPS provided a shelter to protect the bacteria inside the biofilm from toxicity. The biofilm is therefore more tolerant to toxicants, due to the physical protection provided by the EPS. Furthermore, PS (the dominant component in the EPS) could increase the hydrodynamic diameter of the CeO2 NPs, and render them practically unable to move into the cells (Ma et al., 2013). Recently, it has been reported that the bacteria present in the deeper regions of biofilms became even more active after exposure to NPs (Hou et al., 2014). This may help to demonstrate the fact that highly active bacteria secrete more EPS. However, the production of more EPS not only protects the cells against the NPs but also prevents the organic material from reaching the inner regions of the cells. A key function of PRO in EPS is as enzymes that digest macromolecules and particulate material within the microenvironment of the embedded cells (Laspidou and Rittmann, 2002). The rapid increase of PRO revealed the fact that the dead cells might be decomposed into organic material, providing nutrients for the living cells with a high activity. As a result, more nutrients were taken in by the living cells so as to secret more EPS and resist the toxicity of CeO2 NPs. 3.2. Role of bacteria in EPS secretion of biofilms in the presence of CeO2 NPs Previous studies have reported that the adsorption of NPs on activated sludge can affect its structural features, which may be attributed to interactions among the NPs, microbes, and their induced EPS (Li et al., 2015). In this study, the spatial distribution of CeO2 NPs on the surface of biofilms and the surface morphology of the biofilms were obtained using SEM (Fig. S2). The SEM images show that CeO2 NPs were adsorbed on the surface, while the morphology of the outer layer of the biofilm thickened in the presence of CeO2 NPs, corresponding with the observations of Li et al. (2015). The increased production of EPS (Fig. 1) may explain the thicker surface of the biofilm, and may have provided more binding sites for CeO2 NPs, keeping them in the outer layer, which is consistent with the findings of a previous study (Jing et al., 2014). Studies have previously demonstrated that NPs such as CuO and Ag are able to depress bacterial viability, and thus influence the EPS content (Hou et al., 2015a; Sheng and Liu, 2011). The distribution of live/dead cells in a biofilm was explored by using CLSM to investigate the viability of bacteria. As shown in Fig. S4, dead cells were found after exposing the biofilm to 50 mg/L of CeO2 NPs, revealing that CeO2 NPs entered into the bacteria inside the biofilm and caused toxicity in cells. Although the EPS acted as a physical barrier, keeping CeO2 NPs from entering into the cells, some of the NPs can still reach the bacterial cell membranes and affect the viability of the bacteria. The content of ROS production and LDH

Table 1 Effect of CeO2 NP concentration on the production of protein, polysaccharides, and humic-like substances contained in EPSa (LB-EPS and TB-EPS).

a b

CeO2 NPs (mg/L)

LB-EPS

TB-EPS

PRO

PS

HS

PRO

PS

HS

0 1 10 50

33.26 ± 2.45 31.31 ± 2.23 37.04 ± 3.42 48.90 ± 3.87b

141.49 ± 8.64 146.36 ± 5.98 158.68 ± 5.23 166.68 ± 6.21

44.42 ± 3.13 43.36 ± 2.96 37.62 ± 2.85 30.12 ± 2.94b

63.17 ± 4.12 64.92 ± 3.55 78.03 ± 4.86b 100.33 ± 6.74b

262.08 ± 7.17 261.17 ± 6.92 276.65 ± 8.19 289.25 ± 11.34b

72.38 ± 6.38 73.54 ± 7.12 65.71 ± 6.37 56.52 ± 4.75

The data in the table gives the averages and their standard deviations, determined from triplicate measurements, in units of mg/g EPS. The data reported are statistical differences (p < 0.05) from the control.

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Fig. 2. EEM fluorescence spectra for the LB-EPS (a–c) and TB-EPS (d–f) extracted from biofilms at various CeO2 NP concentrations (0 mg/L: a and d, 10 mg/L: b and e, 50 mg/L: c and f).

release increased significantly (p < 0.05) at a concentration of 50 mg/L (Fig. S3), indicating that oxidative stress and cell lysis occurred. It confirmed that the toxicity of CeO2 NPs has an adverse influence on microcolonies inside biofilms. However, the production of EPS in biofilms increased after exposure to CeO2 NPs. A possible cause for this phenomenon could be the complicated behavior of the biofilm microbes in resisting the toxicity of the NPs (Gu et al., 2014). CeO2 NPs have toxic effects on the bacteria and induce cell death (Fig. S4). Apoptosis benefits the subpopulation of living cells, which undergo continued differentiation and dispersal in a biofilm (Webb et al., 2003). The equally important role of cell lysis, which causes the release of soluble organic materials, has been demonstrated by Foladori et al. (2015). In addition, the dual role of stress indication through the activation of cellular stress responses and defense mechanisms is assumed by ROS. At high concentrations, ROS were thought to be toxic by-products of biologically important oxygen metabolism, while at low concentrations, playing vital roles in cellular defense and the control of physiological cell processes such as gene expression, abiotic stress responses, and apoptosis (Von Moos and Slaveykova, 2014). In this work, the ROS production may facilitate the proliferation and the defense of living cells. As a result, the death and lysis of cells and ROS production resulting from the toxicity of CeO2 NPs may be advantageous in terms of the secretion of EPS by living bacteria. 3.3. Main composition analysis of EPS fractions 3.3.1. EPS characterization by 3D-EEM EPS samples contain large quantities of aromatic structures and unsaturated fatty chains with fluorescence characteristics, thus 3D-EEM fluorescence spectroscopy has been used for their characterization (Sheng et al., 2013). The EEM spectra of EPS (LB-EPS and TB-EPS) were recorded at various CeO2 NP concentrations (the EEM spectra of EPS exposure to 1 mg/L CeO2 NPs were similar to the control, therefore, not presented in the study). Each EEM spectrum gave information about the chemical compositions of the EPS samples. Three main peaks (A, B, C) (Fig. 2) were observed from the fluorescence spectra of the LB-EPS and TB-EPS, both with and without the presence of CeO2 NPs, and were identified according to

literature (Zhang et al., 2014; Sheng and Yu, 2006; Chen et al., 2003). The fluorescence peak positions and fluorescence intensity of the different EPS fractions in the 3D-EEM spectra are detailed in Table S1. Peak A was located at excitation/emission wavelengths (Ex/Em) of 236–244/385–395 nm, while Peak B was observed at an Ex/Em of 272–284/344–355 nm. These two peaks were assigned to the previously reported Fulvic acid-like substances (Peak A) and tryptophan protein-like substances (Peak B). Peak C, located at an Ex/Em of 220–224/304–310 nm, corresponds to tyrosin aromatic protein-like substances. Peak D was identified at an Ex/Em of 272–276/305–310 nm (tyrosine protein-like substances), which was only present in the LB-EPS at high CeO2 NP concentrations (10 and 50 mg/L). However, Peak E was located at an Ex/Em of 220/340–350 nm (aromatic protein-like substances), which barely occurred in the TB-EPS extracted at high CeO2 NP concentrations (10 and 50 mg/L). Slight shifts were observed for the peak locations of the EPS (LB-EPS and TB-EPS) at various CeO2 NP concentrations. This shift was also reported by Sheng and Yu (2006) and Zhang et al. (2014), and revealed the changes in the conformations of the EPS components at different CeO2 NP concentrations. 3D-EEM spectra can also be employed for quantitative comparison, according to peak intensity and the ratios between different peak intensities (Sheng and Yu, 2006; Sheng et al., 2013). Comparing the LB-EPS and TB-EPS, the peak intensities of the TB-EPS were much higher than those of the LB-EPS, indicating that the TB-EPS contents were higher than those of LB-EPS. Peak A was identified as corresponding to Fulvic acid-like substances, which are of a lower molecular weight and higher oxygen content than other humic acids (Chen et al., 2003). The intensities of Peak A were greatly decreased at high CeO2 NP concentrations (10 and 50 mg/L), which is consistent with the HS content given in Table 1. Only as the CeO2 NP concentrations reached levels of 10 and 50 mg/L, were peaks D and E present in the LB-EPS and TB-EPS spectra, respectively. In addition, Peak C in the TB-EPS spectrum suddenly vanished when the concentration of CeO2 NPs reached 50 mg/L, whereas its intensity increased in the LB-EPS. Such differences demonstrated that adding CeO2 NPs led to different compositions and amounts of EPS fraction production (Zhang et al., 2014). Both Peaks D and E correspond to protein-like substances, and the presence of these indicates that the EPS contained

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more abundant protein species at high CeO2 NP concentrations, also in accordance with an increased PRO content (Table 1) (Chen et al., 2003; Sheng et al., 2013). A high dosage of CeO2 NPs could reduce the production of Fulvic acid-like substances, while the living cells in a biofilm may be induced to proliferate and produce other neo-protein-like substances (such as tyrosine protein-like substances and aromatic protein-like substances as in this study) in order to inhibit the toxicity of CeO2 NPs. However, the potential mechanisms relating to these processes need to be further explored. 3.3.2. EPS characterization by FT-IR In order to identify the functional groups of the EPS at different concentrations of CeO2 NPs, FT-IR analysis was conducted using varying EPS fractions. The FT-IR spectrum of the EPS is shown in Fig. S5, where detailed information as to the bands in the spectrum that were assigned to the various groups, according to the wavenumbers, is provided in Table S2. The broad adsorption peak in the 3255–3351 cm1 region of the spectrum was attributed to the –OH and –NH2 stretching vibrations of the hydroxyl and amine groups of the EPS. The weak bands visible at approximately 2942 cm1 originated from a C–H stretching of alkyl chains, while the bands at approximately 1211 cm1 were related to the C–O deformation vibration of the carboxylic group. The strong bands at 1623–1639 cm1 were assigned to the C@O and C–N (amide I) stretching vibrations in the protein. The bands at 1457 cm1 and 1408 cm1 were a result of C–H bending and C–N stretching vibrations, respectively, which may arise from the amine III. The sharp peak at 1041 cm1 corresponded to the stretching vibration of the C-O-C in the polysaccharides. Some bands in the ‘‘fingerprint zone’’ (<1000 cm1) could be assigned to the functional groups of the phosphate and sulfur (Hou et al., 2015a; Comte et al., 2006). The FT-IR spectra of the EPS with and without the presence of CeO2 NPs were similar, as shown in Fig. S5. However, there are some qualitative differences between these spectra. After exposure to the CeO2 NPs, certain peaks shifted, some became weak, while other peaks disappeared altogether. The peak at 2942 cm1 in the FT-IT spectrum for the LB-EPS was gradually shifted to 2962 cm1 with increasing CeO2 NP concentrations, indicating that the CeO2 NPs had an impact on the C–H of alkyl chains in the LB-EPS. The peak at 3351 cm1 in the LB-EPS spectrum before exposure to 1 mg/L of CeO2 NPs was shifted to 3325 cm1. This may indicate that the –OH and –NH2 of the hydroxyl and amine groups in the LB-EPS were susceptible to the presence of CeO2 NPs. When the concentration of CeO2 NPs was increased to 50 mg/L, the bands of the LB-EPS at 1211 cm1 and in the fingerprint zone underwent a distinct change, whereas the bands at 1078 cm1 disappeared. This implies that the C–O of the carboxylic group and the C–O–C in the polysaccharides in the LB-EPS were mainly bonded to the CeO2 NPs at higher CeO2 NP concentrations (50 mg/L). In the TB-EPS, the peaks at 3270 and 3435 cm1 were assigned to the stretching vibrations of –OH and –NH2, which may also have originated from hydroxyl and amine groups, while the peaks became weak and disappeared with increasing CeO2 NP concentrations. The bands of the TB-EPS at 1457 cm1 disappeared, and the bands at 1246 cm1 became weak, at higher CeO2 NP concentrations (10 and 50 mg/L). Furthermore, the fingerprint zone of the TB-EPS spectrum also changed greatly after the exposure of the biofilm to higher concentrations of CeO2 NPs (10 and 50 mg/L). When compared to the LB-EPS, the functional groups in the TB-EPS were more tolerant to the presence of CeO2 NPs, owing to the protective role of the outer layers. As the concentration of CeO2 NPs increased to 10 mg/L, the –OH and –NH2 of the hydroxyl and amine groups, as well as the C–H and C–N peptide bonds (amide III) in the protein, were involved in a reaction between the CeO2 NPs and the TB-EPS. The differences between

the LB-EPS and TB-EPS FT-IR spectra suggest that the different functional groups of each EPS fraction were affected by the addition of CeO2 NPs.

3.4. Effect of CeO2 NPs on flocculating efficiency and zeta potential of EPS In wastewater treatment systems, flocculation is a common and effective method for the removal of suspended solids and metal ions (Wu and Ye, 2007), and the bioflocculating of EPS is a main topic of interest in terms of the adsorption of suspended particles and toxic matters in biofilms (Sheng and Liu, 2011). In this study, the effects of CeO2 NPs on the flocculating of EPS are shown in Fig. 3. The flocculating efficiency of EPS (TB-EPS and LB-EPS) did not show any variation after exposure to 1 mg/L of CeO2 NPs, compared to the control sample. However, when the concentration of CeO2 NPs increased from 1 to 50 mg/L, the flocculating efficiency of the LB-EPS declined (p < 0.05) from 23.66% to 11.41%, indicating the vulnerability of LB-EPS to high doses of CeO2 NPs. The decrease in flocculating efficiency of the LB-EPS may be related to the increase in the LB-EPS content, since an excess of LB-EPS might weaken the structure of a biofilm (Li and Yang, 2007). In particular, as CeO2 NP concentration was increased, large numbers of binding sites were occupied by CeO2 NPs, and hence it was hard for the suspended kaolin clay particles to adsorb onto the LB-EPS. It was reported that the TB-EPS are the most active fractions responsible for high flocculating efficiency (Yu et al., 2009). The flocculating efficiency of TB-EPS is more than 10% higher than that of LB-EPS. The higher flocculating efficiency of the TB-EPS fraction can probably be attributed to the fact that it contains more PRO, compared to the LB-EPS. (Yu et al., 2009). PRO contains multivalent cations that are thought to be the key factor in bioflocculation, and hence, is more involved in electrostatic bonding than PS (Laspidou and Rittmann, 2002; Yu et al., 2009). When the concentration of CeO2 NPs increased from 1 to 50 mg/L, the high flocculating efficiency of the TB-EPS did not change significantly (p > 0.05), and the average flocculating efficiency declined only slightly from 34.66% to 28.72%. These results indicate that the CeO2 NPs had a mild effect on the bioflocculation of the TB-EPS that existed in the inner layer

Fig. 3. Effect of CeO2 NP concentration on the flocculating efficiency and zeta potential of EPS exacted from a biofilm (FE-T, flocculating efficiency of TB-EPS; FE-L, flocculating efficiency of LB-EPS; ZP-T, zeta potential of the TB-EPS; ZP-L, zeta potential of the LB-EPS). Asterisks indicate statistical differences compared to the control (p < 0.05). Error bars represent standard deviations determined from triplicate measurements.

G. You et al. / Bioresource Technology 194 (2015) 91–98

of the biofilm, while the bioflocculation of the LB-EPS on the outer layer of biofilm was greatly decreased, due to binding with the CeO2 NPs. In addition, the cell death and cell lysis in the biofilm may weaken the EPS. The enhanced content of the EPS was not conducive to flocculating efficiency, instead reducing the flocculating capacity of each EPS fraction. This could imply that flocculating efficiency might be dominated by the characteristics, rather than the amount, of the EPS. The zeta potentials of each EPS fraction (1 mg/L) before and after exposure to CeO2 NPs were measured in order to explore the possible mechanism of suppressed flocculation. The zeta potentials of the different EPS fractions were negative, and increased with the lessening of the flocculating efficiency, as shown in Fig. 3. The results for the zeta potentials positively correlated with the trends of the flocculating efficiencies (R2 = 0.94235 in LB-EPS, R2 = 0.94636 in TB-EPS, respectively) (Fig. S6). This agrees with a previous study, which reported that electrostatic interactions could be predominant in bioflocculation (Zhang et al., 2014). The less negative charges of the EPS make it easier to adsorb the negatively charged suspended kaolin clay particles, in order to achieve a high flocculating efficiency (Yu et al., 2009). In addition, the electrostatic double-layer repulsion plays a vital role in the structure of the biofilm (Renner and Weibel, 2011). The less negative charges of the EPS lead to a weaker electrostatic repulsion, which contributes to the aggregation of the cells and the dense structure of the TB-EPS.

4. Conclusions The effects of CeO2 NPs on the production and physicochemical properties of EPS fractions in a SBBR were studied. After exposure to 50 mg/L CeO2 NPs, the production of EPS in the biofilms was obviously increased, owing to the increase of PRO and slight increase of PS. However, the HS content decreased, compared to that of the control test (without CeO2 NPs). 3D-EEM fluorescence and FT-IR spectra results indicated that CeO2 NPs, especially at higher concentrations (10 and 50 mg/L), have a significant effect on fluorescence substances and functional groups, showing that the main chemical composition and structures of LB-EPS and TB-EPS varied after exposure to the CeO2 NPs. The flocculating capacity of each EPS fraction, especially the LB-EPS, decreased in the presence of higher doses of CeO2 NPs (10 and 50 mg/L), a result which is consistent with the obtained zeta potential.

Acknowledgements We are grateful for the grants from Project supported by National Science Funds for Creative Research Groups of China (No. 51421006), National Natural Science Foundation of China (Nos. 51479047, 51479065, 51209069), National Science Funds for Distinguished Young Scholars (No. 51225901), Program for Changjiang Scholars and Innovative Research Team in University (No. IRT13061), the Key Program of National Natural Science Foundation of China (No. 41430751), Jiangsu Province Ordinary University Graduate Student Scientific Research Innovation Plan (No. KYZZ14_0157) and PAPD.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2015.07. 006.

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