Bioresource Technology 176 (2015) 65–70
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Effect of CuO nanoparticles on the production and composition of extracellular polymeric substances and physicochemical stability of activated sludge flocs Jun Hou, Lingzhan Miao ⇑, Chao Wang, Peifang Wang, Yanhui Ao, 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 Effect of CuO-NPs on EPS and physicochemical stability of sludge flocs was studied. The production of LB-EPS (polysaccharides) was enhanced to resist the nanotoxicity. CAOAC and carboxyl groups in the EPS changed in the presence of nano-CuO. Exposure to 50 mg/L CuO NPs caused a decrease in flocculation and dewaterability.
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Article history: Received 30 September 2014 Received in revised form 4 November 2014 Accepted 5 November 2014 Available online 13 November 2014 Keywords: Nano-CuO Extracellular polymeric substances (EPS) Activated sludge floc Bioflocculation Dewaterability
a b s t r a c t The effects of CuO nanoparticles (NPs) on the production and composition of extracellular polymeric substances (EPS) and the physicochemical stability of activated sludge were investigated. The results showed enhanced production of loosely bound extracellular polymeric substances (LB-EPS), protecting against nanotoxicity. Specifically, polysaccharide production increased by 89.7% compared to control upon exposure to CuO NPs (50 mg/L). Fourier transform-infrared spectroscopy analysis revealed changes in the polysaccharide CAOAC group and the carboxyl group of proteins in the EPS in the presence of CuO NPs. The sludge flocs were unstable after exposure to CuO NPs (50 mg/L) because of excess LB-EPS. This also corresponded with decreased cell viability of the sludge flocs, as determined by the production of reactive oxygen species and the release of lactate dehydrogenase. These results are key to assessing the adverse effects of the CuO NPs on activated sludge in wastewater treatment plants. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction With the rapid development and application of nanotechnology, a large number of nanomaterials are used in consumer and industrial products such as semiconductors, cosmetics, textiles, and pig-
Abbreviations: CLSM, confocal laser scanning microscopy; COD, chemical oxygen demand; CST, capillary suction time; DCF, dichlorofluorescein; EPS, extracellular polymeric substance; ESS, effluent suspended solid; FT-IR, Fourier transforminfrared spectroscopy; LB-EPS, loosely bound EPS; LDH, lactate dehydrogenase; MLVSS, mixed liquor volatile suspended solid; NP, nanoparticle; PRO, protein; PS, polysaccharide; ROS, reactive oxygen species; SBR, sequencing batch reactor; SEM, scanning electron microscope; SVI, sludge volume index; TB-EPS, tightly bound EPS; TOC, total organic carbon; WWTP, wastewater treatment plant. ⇑ 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] (L. Miao). http://dx.doi.org/10.1016/j.biortech.2014.11.020 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
ments (Gottschalk et al., 2009). Some reports have suggested that the wide use of nanoparticles (NPs) has inevitably caused their release into the environment, such as into wastewater treatment plants (WWTPs), natural water bodies, and the soil (Nowack and Bucheli, 2007). At the same time, there are increasing concerns over the risks posed by NPs to the health of humans and the ecosystem (Sharifi et al., 2012). Therefore, it is imperative to understand the environmental impact of NPs. WWTPs are important for preventing NPs from entering the natural environment (Nowack and Bucheli, 2007). While a significant amount of NPs can be removed from the wastewater treatment systems through biosorption using activated sludge (Kiser et al., 2010), the adsorbed NPs may induce adverse effects on microbial growth (Zhang et al., 2014). Their toxicity to some microorganisms within the biological systems of the WWTPs (Brar et al., 2010) is of particular concern. Recent studies have shown that
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some types of NPs such as TiO2, ZnO, CeO2, and Ag could decrease the population of the microbial community and disturb the microbial diversity in activated sludge systems (García et al., 2012; Zheng et al., 2011). Most of the studies so far have focused on the effect of NPs on the microbial growth activity (Choi et al., 2008; Liang et al., 2010; Zhang et al., 2014), change in the bacterial community structure (Sun et al., 2013; Yang et al., 2014), and decrease in the chemical oxygen demand (COD) and nitrogen/ phosphorus contents (Hou et al., 2012; Li et al., 2013). However, the effect of NPs on the physicochemical stability of activated sludge has been seldom reported (Yang et al., 2013). The physicochemical stability of activated sludge flocs plays an important role in the performance of wastewater treatment systems (Li and Yang, 2007; Ye et al., 2011). Activated sludge generally exists as flocs, which are suspended microbial aggregates containing microorganisms and organic/inorganic compounds (Biggs and Lant, 2000). Flocculating ability, sludge settling, and sludge dewatering are the regularly monitored physicochemical properties of activated sludge (Jin et al., 2003; Wilén et al., 2003). In particular, extracellular polymeric substances (EPS), which are the major components of the activated sludge, act as a gel-like matrix that binds the cells together to form sludge flocs (Sheng et al., 2010). There are two types of EPS, namely the loosely bound EPS (LB-EPS) and the tightly bound EPS (TB-EPS) (Li et al., 2014). The production and composition of the EPS can significantly affect biomass granulation (Caudan et al., 2012), flocculation (Li and Yang, 2007), and the structure of the bioaggregates (Seviour et al., 2012). It is reasonable to assume that the EPS play a leading role in flocculation, settling, and dewatering properties of the flocs. In addition, EPS are usually thought to protect the inner microorganisms from the harsh external environmental conditions such as exposure to heavy metals or chemicals (Ma et al., 2013; Sheng et al., 2013). Thus, the EPS may determine the physicochemical properties and give the floc its structural and functional integrity (Niu et al., 2013; Sheng et al., 2010). Therefore, it is very important to explore the nature and composition of the EPS in activated sludge in the presence of NPs. In previous studies, CuO NPs were chosen as model NPs, since they are widely used in antibactericide coatings, biomedicines, and toothpastes owing to their unique physicochemical properties such as enhanced magnetic, electrical, and optical features (Applerot et al., 2012; Zhao et al., 2013). However, the effects of CuO NPs on the physicochemical properties of the sludge and the production and composition of the EPS have never been reported. For these reasons, the effect of CuO NPs on the physicochemical properties (such as flocculating ability, settleability, and dewaterability) of activated sludge, and in particular, on the EPS production and composition, were investigated. In addition, possible mechanisms explaining the changes in the physicochemical characteristics of the sludge induced by different concentrations of CuO NPs were explored.
2. Methods 2.1. CuO nanoparticles and activated sludge Powdered nanosized CuO NPs (with a mean diameter of 92 ± 12 nm; see Supplementary material, Table S1 for detailed information on the properties) were purchased from Sigma– Aldrich (St. Louis, MO). A scanning electron microscope (SEM) image of the CuO NPs was obtained using a Hitachi S-4800 SEM to get a visual idea of their shape (Fig. S1, Supplementary material). In this study, a stock suspension of the CuO NPs was prepared by adding 0.3 g of the CuO NPs to 1 L of Milli-Q deionized water (pH 6.9 ± 0.1). Subsequently, the stock suspension was ultrasonicated
(20 °C, 250 W, 40 kHz) for 30 min before diluting it to the desired exposure concentration (Zhao et al., 2013). The particle size distribution and the zeta potential of the CuO NPs were measured using a Malvern Zetasizer Nano ZS90 (Malvern Instruments, UK). The flocculent sludge samples were collected from the sedimentation tank of the Jiangning Municipal Wastewater Treatment Plant (Nanjing, China). The flocculent sludge (5 g of mixed liquor volatile suspended solids (MLVSS)/L) was first acclimatized to the synthetic wastewater in a parent sequencing batch reactor (SBR) for about two months until stable performance was achieved (details are provided in the Supplementary material). The synthetic wastewater contained 1060 mg/L of glucose, 90 mg/L of NH4Cl, 400 mg/L of K2HPO43H2O, 360 mg/L of KH2PO42H2O, 12 mg/L of CaCl22H2O, 50 mg/L of MgSO4, MgSO4 50 mg/L of MgSO4, 8 mg/L of MnSO4H2O, 2 mg/L of ZnSO47H2O, 1 mg/L of CuSO45H2O, and 1 mg/L of (NH4)6MoO244H2O. The temperature was maintained at about 20–22 °C and the pH was controlled at 7.0–7.5. 2.2. Experimental design In order to investigate the effect of the CuO NPs on the stability of the activated sludge, three different concentrations of CuO NPs were examined in batch experiments, which were conducted in a series of reactors, each having a working volume of 250 mL. The stock suspension containing the CuO NPs was diluted to concentrations of 5 mg/L, 20 mg/L, and 50 mg/L and added into the reactors. The activated sludge, withdrawn from the parent SBR reactor, was inoculated into each reactor along with approximately 3 g of MLVSS/L. The mixtures were then equilibrated in a rotary shaker, with a rotation speed of 160 rpm (20 °C) for 12 h. Control tests in the absence of the CuO NPs were also conducted for comparison and all the experiments were carried out in triplicate. After exposure to the CuO NPs, the sludge samples were withdrawn and the amount and the composition of the EPS in the activated sludge were measured. 2.3. EPS extraction and analysis Extraction of the EPS was carried out using a process involving centrifugation, sonication, and thermal extraction (Ye et al., 2011). At the end of the batch experiments, 2000g of the activated sludge was harvested by centrifugation for 10 min. The sludge pellet was re-suspended in a 0.05% (w/w) NaCl solution, and sonicated at 20 kHz for 2 min. The suspension was then centrifuged at 8000g for 20 min and the liquid was collected carefully for measuring the LB-EPS. The residual sludge pellet left in the centrifuge tube was re-suspended in a 0.05% (w/w) NaCl solution and heated at 70 °C for 30 min. These conditions have been previously shown to provide a relatively high extraction efficiency and low cell lysis (D’Abzac et al., 2010). The suspension was centrifuged at 12,000g for 20 min, and the supernatant was the TB-EPS fraction. Finally, all the EPS fractions were filtered through 0.45 lm acetate cellulose membranes. Both the LB-EPS and TB-EPS extractions were analyzed for total organic carbon (TOC), proteins (PRO), and polysaccharides (PS). The TOC was measured using a TOC analyzer (Liqui TOC II, Elementar, Germany), using the combustion-infrared method. The PRO and PS were analyzed using the modified Lowry method and the anthrone–sulfuric acid method respectively, according to reports in the literature (Li and Yang, 2007). 2.4. Fourier transform-infrared spectroscopy (FT-IR) FT-IR analysis was used to obtain information on the major functional groups in EPS and their interaction with Cu2+/CuO NPs (Sheng et al., 2013). Prior to the analysis, the wet samples were
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freeze-dried. All the infrared spectra were recorded over the frequency range of 4000–400 cm 1 and the averaged spectra were obtained at a resolution of 4 cm 1. The FT-IR spectra (Tensor 27, Bruker) were measured using KBr pellets prepared by pressing mixtures containing 1 mg of the dry powdered sample and 100 mg of spectrometry grade KBr under vacuum, to avoid moisture uptake.
tistically significant. Significant data points are denoted with an asterisk.
2.5. Characterization of sludge flocculation and settleability
In activated sludge wastewater treatment systems, adsorption onto the biomass is believed to be one of the primary mechanisms for the removal of pollutants, and this adsorption process causes the accumulation of these contaminants in activated sludge (Kiser et al., 2010). In the present study, the SEM images showed that large amounts of the CuO NPs were adsorbed onto the activated sludge after their addition (Fig. S2). Such observations agree with previous studies concerning the behavior of Ag NPs in wastewater treatment systems (Gu et al., 2014). In addition, CuO aggregates with diameters larger than 500 nm were found on the surfaces of the activated sludge, which was confirmed by the EDS examination of the activated sludge. It has been reported that some types of NPs such as Ag and CeO2 NPs are able to depress cell viability (Choi et al., 2008; Ma et al., 2013). Analysis of ROS production and LDH release assays have been widely used to evaluate the influences of such toxicants on cell growth and viability (Gu et al., 2014; Ma et al., 2013). While the level of extracellular LDH is an indicator of the cell membrane integrity (Hou et al., 2014), ROS is an important indicator of oxidative stress (Applerot et al., 2012). Therefore, in this study, these two parameters were measured to reveal the possible toxicological mechanisms of CuO NPs towards activated sludge. As shown in Fig. 1, an increase in the intracellular ROS production was observed with an increase in the concentration of CuO NPs. However, a significant increase in LDH was only observed at a CuO NPs dosage of 50 mg/L (p < 0.05), indicating that cell leakage occurred. Previous studies have addressed the higher antibacterial activity of CuO NPs in comparison to the NPs of other metal oxides such as ZnO, NiO, and Sb2O3 (Zhao et al., 2013). According to the results of the present study, the exposure to high concentrations of CuO NPs (50 mg/L) decreased the cell viability of activated sludge. The CLSM technique was conducted to further investigate the live and dead cells in the activated sludge cultures, in the absence and presence of 50 mg/L of CuO NPs. As seen from Fig. S3, the density of the dead cells significantly increased after
Bioflocculation was performed by slowly mixing a sludge suspension at 50 rpm for 3 min in a beaker (Ye et al., 2011). The size of the flocs was measured after 3 min of mixing. Subsequently, the mixture was allowed to settle for 30 min and the supernatant fraction was collected as the effluent. The turbidity of the effluent and the effluent suspended solids (ESS) were measured, which provided an indication of the sludge flocculation and effluent clarification. The sludge volume index (SVI) was used to evaluate the settleability of the sludge mixture. The ESS content and the SVI were determined according to the standard methods (APHA et al., 1998). The mixed liquor was diluted to about 2 g/L before the SVI test. 2.6. Reactive oxygen species (ROS) accumulation and lactate dehydrogenase (LDH) release In order to explore the possible toxicity mechanism of the CuO NPs towards the sludge, the production of ROS and the LDH activity after exposure of the sludge to the CuO NPs were measured. The intracellular ROS in the sludge was measured by the dichlorodihydrofluorescein (DCF) assay method according to the procedure reported in the literature (Gu et al., 2014). Briefly, the mixed sludge liquor was first centrifuged at 10,000g for 10 min and was washed with a 0.85% (w/w) NaCl solution. The collected pellets were then re-suspended in a 0.85% (w/w) NaCl solution and incubated with 20 lmol/L H2DCF-DA (Molecular Probes, Invitrogen) for 30 min. The mixed liquor was transferred into 96-well microtiter plates (Molecular Devices, USA) for fluorescence spectroscopy at excitation/emission wavelengths of 495/525 nm. The LDH level was determined using a LDH kit and a cell counting kit-8 (Jiancheng Bioengineering Co. Ltd., Nanjing, China), according to the protocol specified by the manufacturer. The sludge samples with and without the CuO NPs were centrifuged at 10,000g for 10 min (Zheng et al., 2011). A supernatant volume of 100 lL was used to provide sufficient volume for LDH detection. The absorbance of the mixture at a wavelength of 340 nm was monitored using UV–vis spectroscopy (UV-3600, Shimadzu, Japan).
3. Results and discussion 3.1. Adsorption of CuO NPs onto activated sludge and its toxic effect on sludge flocs
2.7. Other analytical methods and statistical analysis The surface morphology of the bioflocs after exposure to the CuO NPs was observed using SEM following the procedures reported by Yang et al. (2013). The details are provided in the Supplementary material. Multiple fluorescence labeling and confocal laser scanning microscopy (CLSM) observations were performed according to the analytical procedures described by Ma et al. (2013) (details are provided in the Supplementary material). In order to determine the sludge dewatering ability for sludges with and without CuO NPs exposure, the capillary suction time (CST) was measured according to the standard methods (APHA et al., 1998). All the tests were performed in triplicate and the mean and the standard deviation of the results are reported. The analysis of variance (ANOVA) method was used to test the statistical significance of the results, and p values less than 0.05 was considered to be sta-
Fig. 1. Relative ROS production and LDH release for activated sludge at different concentrations of CuO NPs. Asterisks indicate statistical differences (p < 0.05) from the controls. Error bars represent the standard deviations determined from triplicate measurements.
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the exposure of the activated sludge to 50 mg/L of CuO NPs, indicating that there was a great loss in the cell viability. The increase in the amount of dead cells at higher CuO NPs dosages was in agreement with the observed increase in the ROS production and LDH release. When exposed to CuO NPs, the EPS secreted by the sludge bacteria can serve as a protective barrier for the microbes inside the activated sludge against the toxicity of the added CuO NPs. However, owing to the loose structure, the CuO NPs may diffuse to deeper positions in the sludge flocs (Ma et al., 2013). Thus, the protective capability of the EPS to impede the access of the CuO NPs to the activated sludge, become limited under high toxicity conditions (50 mg/L CuO NPs in this study). 3.2. Effect of CuO NPs on EPS production and components EPS, which is as an important component of the activated sludge, play a key role in protecting inner microorganisms against environmental stress (Sheng et al., 2010). In view of this, the effect of the CuO NPs on the secretion of the EPS in the activated sludge was investigated. As seen in Fig. 2, the TB-EPS content of the biomass did not change significantly upon exposure to different concentrations of CuO NPs, when compared to the control test (p > 0.05). However, the amount of LB-EPS noticeably increased with an increase in the concentration of the CuO NPs. For example, as the concentration of the CuO NPs increased from 0 mg/L to 50 mg/L, the average LB-EPS in the sludge increased from 12.7 mg TOC/g VSS (control) to 23.4 mg TOC/g VSS (50 mg/L CuO NPs). According to previous studies (Seviour et al., 2012; Ye et al., 2011), proteins and polysaccharides were the primary and secondary components in the EPS matrix in the activated sludge system, respectively. Therefore, in this study, only the amount of proteins and polysaccharides in the EPS were analyzed. As shown in Table 1, for both the LB-EPS and TB-EPS in the control samples, the proteins were the predominant component at quantities of 68.8% and 70.7%, respectively. The presence of 5 mg/L CuO NPs did not significantly affect the chemical constituents of the LBEPS. However, with an increase in the dosage of the CuO NPs to 20 mg/L and 50 mg/L, the production of polysaccharides in the LB-EPS increased by 45.7% and 89.7%, respectively, compared to the control. A significant increase in the protein content was observed only at a CuO NP dosage of 50 mg/L (p < 0.05). Compared to the variations in the LB-EPS with increasing concentrations of
Fig. 2. Effect of CuO NPs on the amount of LB-EPS and TB-EPS, expressed in terms of TOC content. Asterisks indicate statistical differences (p < 0.05) from the controls. Error bars represent standard deviations determined from triplicate measurements.
Table 1 Effect of the concentration of CuO NPs on the composition of EPSa (LB-EPS and TBEPS) in activated sludge. CuO NPs (mg/ L)
LB-EPS Protein
Polysaccharides
TB-EPS Protein
Polysaccharides
0 5 20 50
40.8 ± 2.5 41.0 ± 3.2 43.5 ± 1.0 53.6 ± 1.8
18.4 ± 2.1 19.2 ± 1.4 26.8 ± 3.0 34.9 ± 2.5
127.4 ± 2.8 129.2 ± 3.7 127.6 ± 5.7 131.5 ± 4.9
52.7 ± 4.8 50.6 ± 2.4 54.3 ± 1.2 55.8 ± 2.6
a The data reported are the averages and their standard deviations determined from triplicate measurements, and the unit is mg/SS.
the CuO NPs, the polysaccharides and protein contents in the TBEPS did not vary as much on increasing the concentration of the CuO NPs. The response of the activated sludge upon exposure to the CuO NPs can be explained as follows. The LB-EPS diffused from the TBEPS may function as the primary surface for the contact and interaction with the NPs. The sludge would accumulate more EPS under toxic conditions as a protective response to toxicants (Sheng et al., 2010). Recently, several studies have reported that the production of EPS can be enhanced by some metal ions or metal nanoparticles. As a result, the toxic resistance of the activated sludge would increase by retarding the contact of the metals with the bacteria inside the sludge (Ma et al., 2013; Wang et al., 2010). In addition, EPS (especially polysaccharides) could increase the hydrodynamic diameter of the nanoparticles and promote their aggregation (Ma et al., 2013), which may be another important reason for the enhanced resistance to the toxicity of the CuO NPs with an increase in the concentration of the polysaccharides, since larger CuO NPs displayed less toxicity than smaller ones and can hardly move into sludge flocs. Studies have demonstrated the potential toxicity of CuO NPs to bacteria. The toxicity has been attributed to both the NPs and the dissolved Cu ions (Applerot et al., 2012; Zhao et al., 2013). In this study, the addition of CuO NP concentrations of 5 mg/L, 20 mg/L, and 50 mg/L could result in 0.24 mg/L, 0.92 mg/L, and 1.85 mg/L of Cu2+ cations in synthetic wastewater (see the results of the experiments on CuO NPs dissolution in synthetic wastewater, Supporting information). The dissolved Cu2+ ions and CuO NPs would bind on the anionic functional groups on the sludge surface, since the EPS could strongly interact with the polymer matrix to impede the access of these pollutants to the bacterial cells (Sheng et al., 2013). Thus, FT-IR analysis was performed to identify the major functional groups in EPS and to confirm their possible participation in the interaction with metal ions or nanoparticles. The FT-IR spectrum of EPS exhibited a number of bands, indicating the complex nature of the EPS (Fig. S4, Supporting information). The bands in the FT-IR spectrum were assigned to various groups according to the wavenumbers at which they occurred (Table S2, Supporting information). As illustrated in Fig. S4, the strong band at 3407.43 cm 1 originated from the OH and NH2 stretching vibrations of the hydroxyl and amine groups in the EPS. The distinct band at 1638.65 cm 1 was a result of the stretching vibrations of C@O and CAN (amide I) peptidic bonds in the protein. The bands at 1458.52 cm 1 and 1405.39 cm 1 corresponding to the CAH bending from the proteins may also have a contribution from the amine III. The band at 1081.28 cm 1 could be attributed to the stretching vibration of CAOAC in the polysaccharides (Zheng et al., 2011). Further, the finger region demonstrated the existence of sulfur or phosphate groups. After exposure to the CuO NPs, the extracted EPS from the sludge flocs showed a slightly different spectrum. Some of the peaks were shifted and became weak, while other peaks disappeared. The peak at 3407.43 cm 1 in the FT-IR spectrum of the
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EPS prior to exposure to the CuO NPs was shifted to 3396.56 cm 1 in the sample obtained after exposure and intensity of the peak was reduced. In addition, the band at 1405.39 cm 1 in the sample prior to exposure shifted to 1385.48 cm 1, whereas the band at 1458.52 cm 1 disappeared after the exposure of the activated sludge to the CuO NPs. These changes in the wave numbers provided evidence for the involvement of the amine in the carboxyl groups in the reaction. While the band at 1081.28 cm 1 shifted towards a higher wave number (1103.24 cm 1), its intensity increased slightly. These differences suggest that different functional groups in the EPS were involved in the interaction between the CuO NPs/Cu2+ with the activated sludge (Sheng et al., 2013). 3.3. Effects of CuO NPs on sludge flocculation, settleability, and dewaterability Bioflocculation is a process in which microbial cells (predominantly bacteria) and particles aggregate in the wastewater (Li et al., 2014). The sludge with a large size and a compact structure has good flocculating ability, resulting in low ESS and superior settleability. Therefore, effective bioflocculation is key to achieving stability of the activated sludge, and may have a significant influence on both the settleability and the dewaterability of sludge flocs (Ye et al., 2011). The exposure to the CuO NPs resulted in marked variations in the flocculating ability of the sludge flocs. As shown in Fig. 3A, when the concentration of the CuO NPs increased from 0 mg/L to 20 mg/L, the turbidity was not significantly affected (p > 0.05). However, the ESS content increased from 0.12 g/L to 0.25 g/L. The results also implied that the turbidity might be not a good indicator for the flocculation of the sludge. With the concentration of the CuO NPs increasing to 50 mg/L, the turbidity and the ESS content increased greatly (p < 0.05), indicating severely deteriorated flocculation of the sludge. It was reported that an excess of LB-EPS might deteriorate the cell attachment and weaken the floc structure, resulting in poor sludge-water separation (Li and Yang, 2007). The decrease in the flocculation ability at higher CuO NP dosages (50 mg/L) corresponded with the observed increase in the LB-EPS content of the activated sludge. In addition, the toxicity of the CuO NPs, as determined from the damaged cell membranes and the death of the cells (Fig. 1), may be another reason for the declined bioflocculation. Owing to the declined flocculating ability, the sludge flocs exist as dispersive small particles, providing a larger area to interact with the CuO NPs, which in turn may increase the toxicity of the CuO NPs towards the bacteria. Furthermore, the decreasing amount of the C@O, CAN, and O@CAOH functional groups in the EPS observed in the present study (Fig. S4) may also contribute to a deterioration in the flocculation of the sludge (Badireddy et al., 2010). Besides, the cell hydrophobicity and the surface charge have been reported to be related to the flocculation ability of the sludge (Jin et al., 2003). However, these potential mechanisms need to be further investigated. Many investigations have been carried out with regards to the relationship between the bioflocculation and settleability/dewaterability of the activated sludge. However, the results of the studies in the literature are often inconsistent, either showing that the bioflocculating ability and settleability/dewaterability are positively correlated or that there is no correlation at all (Li and Yang, 2007; Jin et al., 2003; Wilén et al., 2003). In the present study, as the concentration of the CuO NPs increased from 5 mg/L to 50 mg/L, the SVI values were almost unchanged compared to the control sample (p > 0.05) (Fig. 3B), indicating that the exposure of the activated sludge to different concentrations of the CuO NPs had no effect on the settleability. However, the results of the dewatering tests showed that by increasing the CuO NP concentration, the dewatering capacity of the activated sludge was significantly
Fig. 3. Effect of CuO NPs on the physiochemical properties of the sludge. (A) flocculation as measured by the turbidity and the ESS content; (B) settleability and dewaterability as measured by the SVI and CST. Asterisks indicate statistical differences (p < 0.05) from the controls. Error bars represent standard deviations of triplicate measurements.
reduced, as a result of the increase in the CST. A 50% increase in the dewatering time was observed for a CuO NP concentration of 50 mg/L, compared to the control sample (Fig. 3B). A possible cause for this phenomenon could be a combination of the chemical reactivity and the antimicrobial characteristics of the CuO NPs, and this needs to be further investigated. In this study, the influences of nano-CuO on the biological stability of activated sludge and the mechanism of its interaction with the EPS of sludge bioflocs have been studied. However, future studies are needed to better understand the effects of CuO NPs accumulation in the activated sludge flocs as well as the potential chronic toxicity of this nanomaterial to the microbial community. 4. Conclusions The effect of CuO NPs on the production and chemical composition of EPS and the flocculation, settleability, and dewaterability of the activated sludge was investigated. The production of the ROS and release of LDH show that exposing the activated sludge to CuO NPs caused oxidative stress and damaged cell membranes in the sludge flocs. The production of LB-EPS, particularly polysaccharides, was enhanced by 89.7% upon exposure to 50 mg/L CuO NPs, compared to the control. The sludge flocs became less stable
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after exposure to 50 mg/L CuO NPs, as seen from the decrease in the flocculation ability and dewaterability. 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, 51109058, 51209069), National Science Funds for Distinguished Young Scholars (No. 51225901), Program for Changjiang Scholars and Innovative Research Team in University (No. IRT13061) and Jiangsu Province Ordinary University Graduate Student Scientific Research Innovation Plan (No. KYZZ14_0157). 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.2014.11.020. References APHA, AWWA, WEF, 1998. Standard Methods for the Examinations of Water and Wastewater. American Public Health Association, Washington, DC, USA. Applerot, G., Lellouche, J., Lipovsky, A., Nitzan, Y., Lubart, R., Gedanken, A., Banin, E., 2012. Understanding the antibacterial mechanism of CuO nanoparticles: revealing the route of induced oxidative stress. Small 8, 3326–3337. Badireddy, A.R., Chellam, S., Gassman, P.L., Engelhard, M.H., Lea, A.S., Rosso, K.M., 2010. Role of extracellular polymeric substances in bioflocculation of activated sludge microorganisms under glucose-controlled conditions. Water Res. 44, 4505–4516. Biggs, C.A., Lant, P.A., 2000. Activated sludge flocculation: on-line determination of floc size and the effect of shear. Water Res. 34, 2542–2550. Brar, S.K., Verma, M., Tyagi, R.D., Surampalli, R.Y., 2010. Engineered nanoparticles in wastewater and wastewater sludge – evidence and impacts. Waste Manage. 30, 504–520. Caudan, C., Filali, A., Lefebvre, D., Spérandio, M., Girbal-Neuhauser, E., 2012. Extracellular polymeric substances (EPS) from aerobic granular sludges: extraction, fractionation, and anionic properties. Appl. Biochem. Biotechnol. 166, 1685–1702. Choi, O., Deng, K.K., Kim, N.J., Ross Jr, L., Surampalli, R.Y., Hu, Z., 2008. The inhibitory effects of silver nanoparticles, silver ions, and silver chloride colloids on microbial growth. Water Res. 42, 3066–3074. D’Abzac, P., Bordas, F., Van Hullebusch, E., Lens, P.N., Guibaud, G., 2010. Extraction of extracellular polymeric substances (EPS) from anaerobic granular sludges: comparison of chemical and physical extraction protocols. Appl. Microbiol. Biotechnol. 85, 1589–1599. García, A., Delgado, L., Torà, J.A., Casals, E., González, E., Puntes, V., Sánchez, A., 2012. Effect of cerium dioxide, titanium dioxide, silver, and gold nanoparticles on the activity of microbial communities intended in wastewater treatment. J. Hazard. Mater. 199, 64–72. Gottschalk, F., Sonderer, T., Scholz, R.W., Nowack, B., 2009. Modeled environmental concentrations of engineered nanomaterials (TiO2, ZnO, Ag, CNT, fullerenes) for different regions. Environ. Sci. Technol. 43, 9216–9222. Gu, L., Li, Q., Quan, X., Cen, Y., Jiang, X., 2014. Comparison of nanosilver removal by flocculent and granular sludge and short-and long-term inhibition impacts. Water Res. 58, 62–70. Hou, J., Miao, L.Z., Wang, C., Wang, P.F., Ao, Y.H., Qian, J., Dai, S.S., 2014. Inhibitory effects of ZnO nanoparticles on aerobic wastewater biofilms from oxygen
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Effect of CuO nanoparticles on the production and composition of extracellular polymeric substances and physicochemical stability of activated sludge flocs Jun Hou, Lingzhan Miao ⇑, Chao Wang, Peifang Wang, Yanhui Ao, 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 Effect of CuO-NPs on EPS and physicochemical stability of sludge flocs was studied. The production of LB-EPS (polysaccharides) was enhanced to resist the nanotoxicity. CAOAC and carboxyl groups in the EPS changed in the presence of nano-CuO. Exposure to 50 mg/L CuO NPs caused a decrease in flocculation and dewaterability.
a r t i c l e
i n f o
Article history: Received 30 September 2014 Received in revised form 4 November 2014 Accepted 5 November 2014 Available online 13 November 2014 Keywords: Nano-CuO Extracellular polymeric substances (EPS) Activated sludge floc Bioflocculation Dewaterability
a b s t r a c t The effects of CuO nanoparticles (NPs) on the production and composition of extracellular polymeric substances (EPS) and the physicochemical stability of activated sludge were investigated. The results showed enhanced production of loosely bound extracellular polymeric substances (LB-EPS), protecting against nanotoxicity. Specifically, polysaccharide production increased by 89.7% compared to control upon exposure to CuO NPs (50 mg/L). Fourier transform-infrared spectroscopy analysis revealed changes in the polysaccharide CAOAC group and the carboxyl group of proteins in the EPS in the presence of CuO NPs. The sludge flocs were unstable after exposure to CuO NPs (50 mg/L) because of excess LB-EPS. This also corresponded with decreased cell viability of the sludge flocs, as determined by the production of reactive oxygen species and the release of lactate dehydrogenase. These results are key to assessing the adverse effects of the CuO NPs on activated sludge in wastewater treatment plants. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction With the rapid development and application of nanotechnology, a large number of nanomaterials are used in consumer and industrial products such as semiconductors, cosmetics, textiles, and pig-
Abbreviations: CLSM, confocal laser scanning microscopy; COD, chemical oxygen demand; CST, capillary suction time; DCF, dichlorofluorescein; EPS, extracellular polymeric substance; ESS, effluent suspended solid; FT-IR, Fourier transforminfrared spectroscopy; LB-EPS, loosely bound EPS; LDH, lactate dehydrogenase; MLVSS, mixed liquor volatile suspended solid; NP, nanoparticle; PRO, protein; PS, polysaccharide; ROS, reactive oxygen species; SBR, sequencing batch reactor; SEM, scanning electron microscope; SVI, sludge volume index; TB-EPS, tightly bound EPS; TOC, total organic carbon; WWTP, wastewater treatment plant. ⇑ 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] (L. Miao). http://dx.doi.org/10.1016/j.biortech.2014.11.020 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
ments (Gottschalk et al., 2009). Some reports have suggested that the wide use of nanoparticles (NPs) has inevitably caused their release into the environment, such as into wastewater treatment plants (WWTPs), natural water bodies, and the soil (Nowack and Bucheli, 2007). At the same time, there are increasing concerns over the risks posed by NPs to the health of humans and the ecosystem (Sharifi et al., 2012). Therefore, it is imperative to understand the environmental impact of NPs. WWTPs are important for preventing NPs from entering the natural environment (Nowack and Bucheli, 2007). While a significant amount of NPs can be removed from the wastewater treatment systems through biosorption using activated sludge (Kiser et al., 2010), the adsorbed NPs may induce adverse effects on microbial growth (Zhang et al., 2014). Their toxicity to some microorganisms within the biological systems of the WWTPs (Brar et al., 2010) is of particular concern. Recent studies have shown that
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some types of NPs such as TiO2, ZnO, CeO2, and Ag could decrease the population of the microbial community and disturb the microbial diversity in activated sludge systems (García et al., 2012; Zheng et al., 2011). Most of the studies so far have focused on the effect of NPs on the microbial growth activity (Choi et al., 2008; Liang et al., 2010; Zhang et al., 2014), change in the bacterial community structure (Sun et al., 2013; Yang et al., 2014), and decrease in the chemical oxygen demand (COD) and nitrogen/ phosphorus contents (Hou et al., 2012; Li et al., 2013). However, the effect of NPs on the physicochemical stability of activated sludge has been seldom reported (Yang et al., 2013). The physicochemical stability of activated sludge flocs plays an important role in the performance of wastewater treatment systems (Li and Yang, 2007; Ye et al., 2011). Activated sludge generally exists as flocs, which are suspended microbial aggregates containing microorganisms and organic/inorganic compounds (Biggs and Lant, 2000). Flocculating ability, sludge settling, and sludge dewatering are the regularly monitored physicochemical properties of activated sludge (Jin et al., 2003; Wilén et al., 2003). In particular, extracellular polymeric substances (EPS), which are the major components of the activated sludge, act as a gel-like matrix that binds the cells together to form sludge flocs (Sheng et al., 2010). There are two types of EPS, namely the loosely bound EPS (LB-EPS) and the tightly bound EPS (TB-EPS) (Li et al., 2014). The production and composition of the EPS can significantly affect biomass granulation (Caudan et al., 2012), flocculation (Li and Yang, 2007), and the structure of the bioaggregates (Seviour et al., 2012). It is reasonable to assume that the EPS play a leading role in flocculation, settling, and dewatering properties of the flocs. In addition, EPS are usually thought to protect the inner microorganisms from the harsh external environmental conditions such as exposure to heavy metals or chemicals (Ma et al., 2013; Sheng et al., 2013). Thus, the EPS may determine the physicochemical properties and give the floc its structural and functional integrity (Niu et al., 2013; Sheng et al., 2010). Therefore, it is very important to explore the nature and composition of the EPS in activated sludge in the presence of NPs. In previous studies, CuO NPs were chosen as model NPs, since they are widely used in antibactericide coatings, biomedicines, and toothpastes owing to their unique physicochemical properties such as enhanced magnetic, electrical, and optical features (Applerot et al., 2012; Zhao et al., 2013). However, the effects of CuO NPs on the physicochemical properties of the sludge and the production and composition of the EPS have never been reported. For these reasons, the effect of CuO NPs on the physicochemical properties (such as flocculating ability, settleability, and dewaterability) of activated sludge, and in particular, on the EPS production and composition, were investigated. In addition, possible mechanisms explaining the changes in the physicochemical characteristics of the sludge induced by different concentrations of CuO NPs were explored.
2. Methods 2.1. CuO nanoparticles and activated sludge Powdered nanosized CuO NPs (with a mean diameter of 92 ± 12 nm; see Supplementary material, Table S1 for detailed information on the properties) were purchased from Sigma– Aldrich (St. Louis, MO). A scanning electron microscope (SEM) image of the CuO NPs was obtained using a Hitachi S-4800 SEM to get a visual idea of their shape (Fig. S1, Supplementary material). In this study, a stock suspension of the CuO NPs was prepared by adding 0.3 g of the CuO NPs to 1 L of Milli-Q deionized water (pH 6.9 ± 0.1). Subsequently, the stock suspension was ultrasonicated
(20 °C, 250 W, 40 kHz) for 30 min before diluting it to the desired exposure concentration (Zhao et al., 2013). The particle size distribution and the zeta potential of the CuO NPs were measured using a Malvern Zetasizer Nano ZS90 (Malvern Instruments, UK). The flocculent sludge samples were collected from the sedimentation tank of the Jiangning Municipal Wastewater Treatment Plant (Nanjing, China). The flocculent sludge (5 g of mixed liquor volatile suspended solids (MLVSS)/L) was first acclimatized to the synthetic wastewater in a parent sequencing batch reactor (SBR) for about two months until stable performance was achieved (details are provided in the Supplementary material). The synthetic wastewater contained 1060 mg/L of glucose, 90 mg/L of NH4Cl, 400 mg/L of K2HPO43H2O, 360 mg/L of KH2PO42H2O, 12 mg/L of CaCl22H2O, 50 mg/L of MgSO4, MgSO4 50 mg/L of MgSO4, 8 mg/L of MnSO4H2O, 2 mg/L of ZnSO47H2O, 1 mg/L of CuSO45H2O, and 1 mg/L of (NH4)6MoO244H2O. The temperature was maintained at about 20–22 °C and the pH was controlled at 7.0–7.5. 2.2. Experimental design In order to investigate the effect of the CuO NPs on the stability of the activated sludge, three different concentrations of CuO NPs were examined in batch experiments, which were conducted in a series of reactors, each having a working volume of 250 mL. The stock suspension containing the CuO NPs was diluted to concentrations of 5 mg/L, 20 mg/L, and 50 mg/L and added into the reactors. The activated sludge, withdrawn from the parent SBR reactor, was inoculated into each reactor along with approximately 3 g of MLVSS/L. The mixtures were then equilibrated in a rotary shaker, with a rotation speed of 160 rpm (20 °C) for 12 h. Control tests in the absence of the CuO NPs were also conducted for comparison and all the experiments were carried out in triplicate. After exposure to the CuO NPs, the sludge samples were withdrawn and the amount and the composition of the EPS in the activated sludge were measured. 2.3. EPS extraction and analysis Extraction of the EPS was carried out using a process involving centrifugation, sonication, and thermal extraction (Ye et al., 2011). At the end of the batch experiments, 2000g of the activated sludge was harvested by centrifugation for 10 min. The sludge pellet was re-suspended in a 0.05% (w/w) NaCl solution, and sonicated at 20 kHz for 2 min. The suspension was then centrifuged at 8000g for 20 min and the liquid was collected carefully for measuring the LB-EPS. The residual sludge pellet left in the centrifuge tube was re-suspended in a 0.05% (w/w) NaCl solution and heated at 70 °C for 30 min. These conditions have been previously shown to provide a relatively high extraction efficiency and low cell lysis (D’Abzac et al., 2010). The suspension was centrifuged at 12,000g for 20 min, and the supernatant was the TB-EPS fraction. Finally, all the EPS fractions were filtered through 0.45 lm acetate cellulose membranes. Both the LB-EPS and TB-EPS extractions were analyzed for total organic carbon (TOC), proteins (PRO), and polysaccharides (PS). The TOC was measured using a TOC analyzer (Liqui TOC II, Elementar, Germany), using the combustion-infrared method. The PRO and PS were analyzed using the modified Lowry method and the anthrone–sulfuric acid method respectively, according to reports in the literature (Li and Yang, 2007). 2.4. Fourier transform-infrared spectroscopy (FT-IR) FT-IR analysis was used to obtain information on the major functional groups in EPS and their interaction with Cu2+/CuO NPs (Sheng et al., 2013). Prior to the analysis, the wet samples were
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freeze-dried. All the infrared spectra were recorded over the frequency range of 4000–400 cm 1 and the averaged spectra were obtained at a resolution of 4 cm 1. The FT-IR spectra (Tensor 27, Bruker) were measured using KBr pellets prepared by pressing mixtures containing 1 mg of the dry powdered sample and 100 mg of spectrometry grade KBr under vacuum, to avoid moisture uptake.
tistically significant. Significant data points are denoted with an asterisk.
2.5. Characterization of sludge flocculation and settleability
In activated sludge wastewater treatment systems, adsorption onto the biomass is believed to be one of the primary mechanisms for the removal of pollutants, and this adsorption process causes the accumulation of these contaminants in activated sludge (Kiser et al., 2010). In the present study, the SEM images showed that large amounts of the CuO NPs were adsorbed onto the activated sludge after their addition (Fig. S2). Such observations agree with previous studies concerning the behavior of Ag NPs in wastewater treatment systems (Gu et al., 2014). In addition, CuO aggregates with diameters larger than 500 nm were found on the surfaces of the activated sludge, which was confirmed by the EDS examination of the activated sludge. It has been reported that some types of NPs such as Ag and CeO2 NPs are able to depress cell viability (Choi et al., 2008; Ma et al., 2013). Analysis of ROS production and LDH release assays have been widely used to evaluate the influences of such toxicants on cell growth and viability (Gu et al., 2014; Ma et al., 2013). While the level of extracellular LDH is an indicator of the cell membrane integrity (Hou et al., 2014), ROS is an important indicator of oxidative stress (Applerot et al., 2012). Therefore, in this study, these two parameters were measured to reveal the possible toxicological mechanisms of CuO NPs towards activated sludge. As shown in Fig. 1, an increase in the intracellular ROS production was observed with an increase in the concentration of CuO NPs. However, a significant increase in LDH was only observed at a CuO NPs dosage of 50 mg/L (p < 0.05), indicating that cell leakage occurred. Previous studies have addressed the higher antibacterial activity of CuO NPs in comparison to the NPs of other metal oxides such as ZnO, NiO, and Sb2O3 (Zhao et al., 2013). According to the results of the present study, the exposure to high concentrations of CuO NPs (50 mg/L) decreased the cell viability of activated sludge. The CLSM technique was conducted to further investigate the live and dead cells in the activated sludge cultures, in the absence and presence of 50 mg/L of CuO NPs. As seen from Fig. S3, the density of the dead cells significantly increased after
Bioflocculation was performed by slowly mixing a sludge suspension at 50 rpm for 3 min in a beaker (Ye et al., 2011). The size of the flocs was measured after 3 min of mixing. Subsequently, the mixture was allowed to settle for 30 min and the supernatant fraction was collected as the effluent. The turbidity of the effluent and the effluent suspended solids (ESS) were measured, which provided an indication of the sludge flocculation and effluent clarification. The sludge volume index (SVI) was used to evaluate the settleability of the sludge mixture. The ESS content and the SVI were determined according to the standard methods (APHA et al., 1998). The mixed liquor was diluted to about 2 g/L before the SVI test. 2.6. Reactive oxygen species (ROS) accumulation and lactate dehydrogenase (LDH) release In order to explore the possible toxicity mechanism of the CuO NPs towards the sludge, the production of ROS and the LDH activity after exposure of the sludge to the CuO NPs were measured. The intracellular ROS in the sludge was measured by the dichlorodihydrofluorescein (DCF) assay method according to the procedure reported in the literature (Gu et al., 2014). Briefly, the mixed sludge liquor was first centrifuged at 10,000g for 10 min and was washed with a 0.85% (w/w) NaCl solution. The collected pellets were then re-suspended in a 0.85% (w/w) NaCl solution and incubated with 20 lmol/L H2DCF-DA (Molecular Probes, Invitrogen) for 30 min. The mixed liquor was transferred into 96-well microtiter plates (Molecular Devices, USA) for fluorescence spectroscopy at excitation/emission wavelengths of 495/525 nm. The LDH level was determined using a LDH kit and a cell counting kit-8 (Jiancheng Bioengineering Co. Ltd., Nanjing, China), according to the protocol specified by the manufacturer. The sludge samples with and without the CuO NPs were centrifuged at 10,000g for 10 min (Zheng et al., 2011). A supernatant volume of 100 lL was used to provide sufficient volume for LDH detection. The absorbance of the mixture at a wavelength of 340 nm was monitored using UV–vis spectroscopy (UV-3600, Shimadzu, Japan).
3. Results and discussion 3.1. Adsorption of CuO NPs onto activated sludge and its toxic effect on sludge flocs
2.7. Other analytical methods and statistical analysis The surface morphology of the bioflocs after exposure to the CuO NPs was observed using SEM following the procedures reported by Yang et al. (2013). The details are provided in the Supplementary material. Multiple fluorescence labeling and confocal laser scanning microscopy (CLSM) observations were performed according to the analytical procedures described by Ma et al. (2013) (details are provided in the Supplementary material). In order to determine the sludge dewatering ability for sludges with and without CuO NPs exposure, the capillary suction time (CST) was measured according to the standard methods (APHA et al., 1998). All the tests were performed in triplicate and the mean and the standard deviation of the results are reported. The analysis of variance (ANOVA) method was used to test the statistical significance of the results, and p values less than 0.05 was considered to be sta-
Fig. 1. Relative ROS production and LDH release for activated sludge at different concentrations of CuO NPs. Asterisks indicate statistical differences (p < 0.05) from the controls. Error bars represent the standard deviations determined from triplicate measurements.
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the exposure of the activated sludge to 50 mg/L of CuO NPs, indicating that there was a great loss in the cell viability. The increase in the amount of dead cells at higher CuO NPs dosages was in agreement with the observed increase in the ROS production and LDH release. When exposed to CuO NPs, the EPS secreted by the sludge bacteria can serve as a protective barrier for the microbes inside the activated sludge against the toxicity of the added CuO NPs. However, owing to the loose structure, the CuO NPs may diffuse to deeper positions in the sludge flocs (Ma et al., 2013). Thus, the protective capability of the EPS to impede the access of the CuO NPs to the activated sludge, become limited under high toxicity conditions (50 mg/L CuO NPs in this study). 3.2. Effect of CuO NPs on EPS production and components EPS, which is as an important component of the activated sludge, play a key role in protecting inner microorganisms against environmental stress (Sheng et al., 2010). In view of this, the effect of the CuO NPs on the secretion of the EPS in the activated sludge was investigated. As seen in Fig. 2, the TB-EPS content of the biomass did not change significantly upon exposure to different concentrations of CuO NPs, when compared to the control test (p > 0.05). However, the amount of LB-EPS noticeably increased with an increase in the concentration of the CuO NPs. For example, as the concentration of the CuO NPs increased from 0 mg/L to 50 mg/L, the average LB-EPS in the sludge increased from 12.7 mg TOC/g VSS (control) to 23.4 mg TOC/g VSS (50 mg/L CuO NPs). According to previous studies (Seviour et al., 2012; Ye et al., 2011), proteins and polysaccharides were the primary and secondary components in the EPS matrix in the activated sludge system, respectively. Therefore, in this study, only the amount of proteins and polysaccharides in the EPS were analyzed. As shown in Table 1, for both the LB-EPS and TB-EPS in the control samples, the proteins were the predominant component at quantities of 68.8% and 70.7%, respectively. The presence of 5 mg/L CuO NPs did not significantly affect the chemical constituents of the LBEPS. However, with an increase in the dosage of the CuO NPs to 20 mg/L and 50 mg/L, the production of polysaccharides in the LB-EPS increased by 45.7% and 89.7%, respectively, compared to the control. A significant increase in the protein content was observed only at a CuO NP dosage of 50 mg/L (p < 0.05). Compared to the variations in the LB-EPS with increasing concentrations of
Fig. 2. Effect of CuO NPs on the amount of LB-EPS and TB-EPS, expressed in terms of TOC content. Asterisks indicate statistical differences (p < 0.05) from the controls. Error bars represent standard deviations determined from triplicate measurements.
Table 1 Effect of the concentration of CuO NPs on the composition of EPSa (LB-EPS and TBEPS) in activated sludge. CuO NPs (mg/ L)
LB-EPS Protein
Polysaccharides
TB-EPS Protein
Polysaccharides
0 5 20 50
40.8 ± 2.5 41.0 ± 3.2 43.5 ± 1.0 53.6 ± 1.8
18.4 ± 2.1 19.2 ± 1.4 26.8 ± 3.0 34.9 ± 2.5
127.4 ± 2.8 129.2 ± 3.7 127.6 ± 5.7 131.5 ± 4.9
52.7 ± 4.8 50.6 ± 2.4 54.3 ± 1.2 55.8 ± 2.6
a The data reported are the averages and their standard deviations determined from triplicate measurements, and the unit is mg/SS.
the CuO NPs, the polysaccharides and protein contents in the TBEPS did not vary as much on increasing the concentration of the CuO NPs. The response of the activated sludge upon exposure to the CuO NPs can be explained as follows. The LB-EPS diffused from the TBEPS may function as the primary surface for the contact and interaction with the NPs. The sludge would accumulate more EPS under toxic conditions as a protective response to toxicants (Sheng et al., 2010). Recently, several studies have reported that the production of EPS can be enhanced by some metal ions or metal nanoparticles. As a result, the toxic resistance of the activated sludge would increase by retarding the contact of the metals with the bacteria inside the sludge (Ma et al., 2013; Wang et al., 2010). In addition, EPS (especially polysaccharides) could increase the hydrodynamic diameter of the nanoparticles and promote their aggregation (Ma et al., 2013), which may be another important reason for the enhanced resistance to the toxicity of the CuO NPs with an increase in the concentration of the polysaccharides, since larger CuO NPs displayed less toxicity than smaller ones and can hardly move into sludge flocs. Studies have demonstrated the potential toxicity of CuO NPs to bacteria. The toxicity has been attributed to both the NPs and the dissolved Cu ions (Applerot et al., 2012; Zhao et al., 2013). In this study, the addition of CuO NP concentrations of 5 mg/L, 20 mg/L, and 50 mg/L could result in 0.24 mg/L, 0.92 mg/L, and 1.85 mg/L of Cu2+ cations in synthetic wastewater (see the results of the experiments on CuO NPs dissolution in synthetic wastewater, Supporting information). The dissolved Cu2+ ions and CuO NPs would bind on the anionic functional groups on the sludge surface, since the EPS could strongly interact with the polymer matrix to impede the access of these pollutants to the bacterial cells (Sheng et al., 2013). Thus, FT-IR analysis was performed to identify the major functional groups in EPS and to confirm their possible participation in the interaction with metal ions or nanoparticles. The FT-IR spectrum of EPS exhibited a number of bands, indicating the complex nature of the EPS (Fig. S4, Supporting information). The bands in the FT-IR spectrum were assigned to various groups according to the wavenumbers at which they occurred (Table S2, Supporting information). As illustrated in Fig. S4, the strong band at 3407.43 cm 1 originated from the OH and NH2 stretching vibrations of the hydroxyl and amine groups in the EPS. The distinct band at 1638.65 cm 1 was a result of the stretching vibrations of C@O and CAN (amide I) peptidic bonds in the protein. The bands at 1458.52 cm 1 and 1405.39 cm 1 corresponding to the CAH bending from the proteins may also have a contribution from the amine III. The band at 1081.28 cm 1 could be attributed to the stretching vibration of CAOAC in the polysaccharides (Zheng et al., 2011). Further, the finger region demonstrated the existence of sulfur or phosphate groups. After exposure to the CuO NPs, the extracted EPS from the sludge flocs showed a slightly different spectrum. Some of the peaks were shifted and became weak, while other peaks disappeared. The peak at 3407.43 cm 1 in the FT-IR spectrum of the
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EPS prior to exposure to the CuO NPs was shifted to 3396.56 cm 1 in the sample obtained after exposure and intensity of the peak was reduced. In addition, the band at 1405.39 cm 1 in the sample prior to exposure shifted to 1385.48 cm 1, whereas the band at 1458.52 cm 1 disappeared after the exposure of the activated sludge to the CuO NPs. These changes in the wave numbers provided evidence for the involvement of the amine in the carboxyl groups in the reaction. While the band at 1081.28 cm 1 shifted towards a higher wave number (1103.24 cm 1), its intensity increased slightly. These differences suggest that different functional groups in the EPS were involved in the interaction between the CuO NPs/Cu2+ with the activated sludge (Sheng et al., 2013). 3.3. Effects of CuO NPs on sludge flocculation, settleability, and dewaterability Bioflocculation is a process in which microbial cells (predominantly bacteria) and particles aggregate in the wastewater (Li et al., 2014). The sludge with a large size and a compact structure has good flocculating ability, resulting in low ESS and superior settleability. Therefore, effective bioflocculation is key to achieving stability of the activated sludge, and may have a significant influence on both the settleability and the dewaterability of sludge flocs (Ye et al., 2011). The exposure to the CuO NPs resulted in marked variations in the flocculating ability of the sludge flocs. As shown in Fig. 3A, when the concentration of the CuO NPs increased from 0 mg/L to 20 mg/L, the turbidity was not significantly affected (p > 0.05). However, the ESS content increased from 0.12 g/L to 0.25 g/L. The results also implied that the turbidity might be not a good indicator for the flocculation of the sludge. With the concentration of the CuO NPs increasing to 50 mg/L, the turbidity and the ESS content increased greatly (p < 0.05), indicating severely deteriorated flocculation of the sludge. It was reported that an excess of LB-EPS might deteriorate the cell attachment and weaken the floc structure, resulting in poor sludge-water separation (Li and Yang, 2007). The decrease in the flocculation ability at higher CuO NP dosages (50 mg/L) corresponded with the observed increase in the LB-EPS content of the activated sludge. In addition, the toxicity of the CuO NPs, as determined from the damaged cell membranes and the death of the cells (Fig. 1), may be another reason for the declined bioflocculation. Owing to the declined flocculating ability, the sludge flocs exist as dispersive small particles, providing a larger area to interact with the CuO NPs, which in turn may increase the toxicity of the CuO NPs towards the bacteria. Furthermore, the decreasing amount of the C@O, CAN, and O@CAOH functional groups in the EPS observed in the present study (Fig. S4) may also contribute to a deterioration in the flocculation of the sludge (Badireddy et al., 2010). Besides, the cell hydrophobicity and the surface charge have been reported to be related to the flocculation ability of the sludge (Jin et al., 2003). However, these potential mechanisms need to be further investigated. Many investigations have been carried out with regards to the relationship between the bioflocculation and settleability/dewaterability of the activated sludge. However, the results of the studies in the literature are often inconsistent, either showing that the bioflocculating ability and settleability/dewaterability are positively correlated or that there is no correlation at all (Li and Yang, 2007; Jin et al., 2003; Wilén et al., 2003). In the present study, as the concentration of the CuO NPs increased from 5 mg/L to 50 mg/L, the SVI values were almost unchanged compared to the control sample (p > 0.05) (Fig. 3B), indicating that the exposure of the activated sludge to different concentrations of the CuO NPs had no effect on the settleability. However, the results of the dewatering tests showed that by increasing the CuO NP concentration, the dewatering capacity of the activated sludge was significantly
Fig. 3. Effect of CuO NPs on the physiochemical properties of the sludge. (A) flocculation as measured by the turbidity and the ESS content; (B) settleability and dewaterability as measured by the SVI and CST. Asterisks indicate statistical differences (p < 0.05) from the controls. Error bars represent standard deviations of triplicate measurements.
reduced, as a result of the increase in the CST. A 50% increase in the dewatering time was observed for a CuO NP concentration of 50 mg/L, compared to the control sample (Fig. 3B). A possible cause for this phenomenon could be a combination of the chemical reactivity and the antimicrobial characteristics of the CuO NPs, and this needs to be further investigated. In this study, the influences of nano-CuO on the biological stability of activated sludge and the mechanism of its interaction with the EPS of sludge bioflocs have been studied. However, future studies are needed to better understand the effects of CuO NPs accumulation in the activated sludge flocs as well as the potential chronic toxicity of this nanomaterial to the microbial community. 4. Conclusions The effect of CuO NPs on the production and chemical composition of EPS and the flocculation, settleability, and dewaterability of the activated sludge was investigated. The production of the ROS and release of LDH show that exposing the activated sludge to CuO NPs caused oxidative stress and damaged cell membranes in the sludge flocs. The production of LB-EPS, particularly polysaccharides, was enhanced by 89.7% upon exposure to 50 mg/L CuO NPs, compared to the control. The sludge flocs became less stable
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