Montmorillonite-supported with Cu2O nanoparticles for damage and removal of Microcystis aeruginosa under visible light

CLAY-03867; No of Pages 11 Applied Clay Science xxx (2016) xxx–xxx

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Research paper

Montmorillonite-supported with Cu2O nanoparticles for damage and removal of Microcystis aeruginosa under visible light Na Gu a,b, Jinlong Gao b,c, Heng Li a,b, Yunxia Wu b,c, Yulin Ma b,c, Kuitao Wang b,c,⁎ a b c

School of Science, Hebei University of Science and Technology, Shijiazhuang 050018,China Key Laboratory of Medicinal Molecular Chemistry in Hebei Province, Shijiazhuang 050018, China School of Chemical and Pharmaceutical Engineering, Hebei University of Science and Technology, Shijiazhuang 050018,China

a r t i c l e

i n f o

Article history: Received 7 September 2015 Received in revised form 17 May 2016 Accepted 18 May 2016 Available online xxxx Keywords: Montmorillonite Cu2O Microcystis aeruginosa Cyanobacteria bloom Photocatalysis Flocculation

a b s t r a c t The montmorillonite supported with Cu2O nanoparticles was prepared by reduction of Cu2+ absorbed by montmorillonite using glucose and ethylene glycol as reductant. X-ray diffraction and IR spectrum depicted the maintenance of the host structure of montmorillonite and the presence of crystallite Cu2O nanoparticles. X-ray diffraction and transmission electron microscopy demonstrated the Cu2O nanoparticles were either intercalated into the interlayer space of montmorillonite or dispersed homogeneously on the surface of montmorillonite, and the size of Cu2O nanoparticles were varying from 5 to 10 nm. Energy dispersive X-ray showed the loading amount of Cu2O in montmorillonite interlayer was around 26.31%. Cu2O-montmorillonite was used for adsorbent, flocculant and photocatalyst to remove Microcystis aeruginosa, removing 90.4% of M. aeruginosa in 3 h under visible light. The synergy of adsorption-flocculation and photocatalysis of Cu2O-montmorillonite promoted the aggregation of M. aeruginosa and then the cell damage mainly associated with cell membrane attack and inclusion degradation by photocatalysis of Cu2O, leading to the inhibition of physiological activity of M. aeruginosa. Cu2O-montmorillonite was an effective algae removal material for the emergency control of cyanobacteria bloom. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Occurrence of cyanobacterial blooms in eutrophic water bodies has become more frequent in many countries of the world and they are predicted to be a rapidly expanding global problem (Mohamed et al., 2014; Pei et al., 2014; Shao et al., 2014). The excessive growth of cyanobacteria color the water, produce odor and toxins which are harmful to humans, animals and aquatic biota and disfigure the coastal aesthetics (Laughinghouse et al., 2012). An in situ control of the overgrown algae is in high demand (Chang et al., 2014). Some approaches, such as chemical algaecides (Gustafsson et al., 2009), clay flocculation (Tang et al., 2011), ultrasonic irradiation (Wu et al., 2012), aquatic macrophytes (Chen et al., 2012), biological techniques (Schmack et al., 2012) and ultraviolet irradiation (Tao et al., 2010) on the removal of algae blooms species were conducted. Among these, clay flocculation, an economical and environmentally acceptable method of controlling algae bloom, was used in several countries as a mean to remove harmful algae from the water bodies

⁎ Corresponding author at: Room 114, Key Laboratory of Medicinal Molecular Chemistry in Hebei Province, Hebei University of Science and Technology, No. 70, Yuhua Road, Yuhua District, Shijiazhuang City, Hebei 050018, China. E-mail address: [email protected] (K. Wang).

(Hagstrom and Graneli, 2005; Sengco et al., 2005, Jiang and Kim, 2008). The removal ability of clay could be enhanced with chemical flocculants or surfactants, such as polyaluminum chloride (PAC) (Pan et al., 2011), chitosan (Pan et al., 2006) and hexadecyltrimethylammonium bromide (CTAB) (Liu et al., 2010) to increase their adhesiveness. However, the motility of the algae cells in aggregates or flocs formed by clay particles and algae cells caused the re-suspension of aggregates or flocs, which influenced the effect of algae removal (Beaulieu et al., 2005). Currently, some research indicated that active radical generated by photocatalytic semiconducting metal oxides could oxidize the protein, lipid and nucleic acid in cyanobacterial cell, destroyed the periblastesis and caused the cytoclasis of cyanobacterial cells (Yu et al., 2010). Therefore, the combination of clay flocculation and photocatalysis will make the algae cell lose the motility when flocculated by clay, which improved the effectiveness of algae removal. Microcystis aeruginosa (M. aeruginosa) is a well-known toxin-producing cyanobacterium which caused cyanobacterial blooms in freshwater lakes and reservoirs worldwide (Wang et al., 2015a). Clay minerals have been applied as flocculants to remove M. aeruginosa cells and toxins with moderate success (Morris et al., 2000; Verspagen et al., 2006; Chang et al., 2014). Montmorillonite is layered aluminosilicate clay mineral that consists of an octahedral alumina layer fused between two tetrahedral silica layers, showing excellent absorption ability, high surface area and outstanding ability to exchange or

http://dx.doi.org/10.1016/j.clay.2016.05.017 0169-1317/© 2016 Elsevier B.V. All rights reserved.

Please cite this article as: Gu, N., et al., Montmorillonite-supported with Cu2O nanoparticles for damage and removal of Microcystis aeruginosa under visible light, Appl. Clay Sci. (2016), http://dx.doi.org/10.1016/j.clay.2016.05.017

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N. Gu et al. / Applied Clay Science xxx (2016) xxx–xxx

intercalate ions and molecules (Praveen Kumar et al., 2015). Owing to these properties, it was applied as flocculant and showed high removal abilities against harmful algae blooms (Wu et al., 2010). On the other hand, montmorillonite is an excellent support to enhance the chemical/thermal stability of nano-metal oxide catalysts (Chen et al., 2016). Montmorillonite supported with metal oxide nanoparticles displayed added physicochemical functions of the metal oxide nanoparticles and montmorillonite together. Up to now, there are little reports in literature on the removal and inhibition of algae bloom using montmorillonite supported with reactive metal oxide nanoparticles. Our previous work suggested that ZnO- montmorillonite could remove M. aeruginosa by the synergy of absorption and photocatalytic degradation under UV (Gu et al., 2015). Cu2O is p-type metal oxide semiconductor with a band gap of 2.0 eV and has been known as a visible-light responsive photocatalyst (Kakuta and Abe, 2009; Wang et al., 2015c), which may be explored to generate active free radicals to kill the microorganisms under suitable conditions (Tu et al., 2014). In the present study, we synthesized Cu2O-montmorillonite composite by the reduction of Cu2+ ion adsorbed by montmorillonite using glucose and ethylene glycol as reducing agent and studied its removal efficiency for M. aeruginosa. The Cu2O nanoparticles were found to be distributed on montmorillonite and were supported by the montmorillonite layer to avoid aggregations. Cu2O-montmorillonite could remove the algae cells by the adsorption-flocculation property of montmorillonite and the photocatalytic degradation property of Cu2O, damaging M. aeruginosa at the same time of adsorbing and flocculating M. aeruginosa cells. The combination of adsorption-flocculation and heterogeneous photocatalysis made algae removal by Cu2O-montmorillonite more effective and complete. This research aimed to develop a kind of algae removal material for lowcost cyanobacterial bloom control.

2.3. Characterizations of Cu2O-montmorillonite Powder X-ray diffraction patterns of the samples were recorded using an X-ray diffractometer (D/MAX-2500, Rigaku) using Ni-filtered Cu Kα radiation (λ = 1.54 Å) and scanning speed 5°·min−1. Infrared spectra of samples were recorded on a Bio-Rad FTS-65 A1896 spectrometer using KBr pellet method. The transmission electron micrographs (TEM) were recorded with a JEOL-2100 transmission electron microscopes, working at a 200 kV accelerating voltage. The elemental analysis was performed with EDAX (Hitachi S-4700), specimens were mounted on double-sided carbon tape and lightly platinum sputter coated for analysis.

2.4. Algal cultivation M. aeruginosa FACHB-942, obtained from the Institute of Hydrobiology, Chinese Academy of Sciences. The algae were cultured in 300 mL sterilized BG11 medium in 500 mL Erlenmeyer flask at 25 ± 1 °C under light intensity of 2000 lx with a photoperiod of 12:12 h (light: dark) in the Artificial Climate Incubator (MGC-300H, Shanghai Yiheng Technology Co. Ltd. China). The BG11 medium contained NaNO3 (1.5 g), K2HPO4 (0.04 g), MgSO4·7H2O (0.075 g), CaCl2·2H2O(0.036 g), citric acid (0.006 g), ferric ammonium citrate(0.006 g), EDTA (0.001 g), Na2CO3 (0.02 g) and 1 mL A5 solution in 1 L distilled water (A5 solution: H3BO4 (2.86 g), MnCl2·4H2O (1.81 g), ZnSO4·7H2O (0.222 g), CuSO4·5H2O(0.079 g), Na2MoO4·2H2O (0.39 g), Co(NO3)2·6H2O (0.0494 g) in 1 L distilled water). The algae cultures were shaken at least three times per day at 100 rpm. The algae were cultured for 5 days to reach the exponential phase, which were used for the assay of algae removal.

2. Experimental 2.5. Removal of algae 2.1. Materials The commercial Na+-montmorillonite used in present study, with cation exchange capacity (CEC) = 0.9 meq/g, was obtained from Fenghong Corporation (Zhejiang, China). Copper acetate, ethylene glycol, sodium hydroxide, glucose, thiobarbituric acid (TBA) were purchased from Sinopharm Chemical Reagent Co., Ltd. Coomassie brilliant blue G-250, trichloroacetic acid, ascorbic acid and bovine serum albumin were obtained from Tianjin Bodi Chemical Industry Co. Ltd., Tianjin Kaixin Chemical Industry Co. Ltd., Shanghai Yutong Bio-Technology Co. Ltd. and Sigma respectively. All the chemicals and reagents were used as received without further purification.

2.2. Synthesis of montmorillonites supported with Cu2O nanoparticles For preparing Cu2O-montmorillonite, initially 1 g montmorillonite was added to100 mL deionized water under condition of magnetic stirring for 5 h at room temperature to obtain montmorillonite slurry. Next 1.0 g of copper acetate was dissolved in a mixture of 20.0 mL of deionized water, 50.0 mL absolute alcohol and 20.0 mL ethylene glycol at room temperature to form a pillarization agent solution in a beaker. After that, pillarization agent solution was added to montmorillonite slurry and the mixed slurry were stirred for 20 h at room temperature. Later, the mixed slurry were heated to 80 °C under stirring, and then 1.4 g sodium hydroxide and 1.2 g glucose were added to the above slurry and stirred for 5 min. After the products were cooled down to room temperature, the resulting precipitate was collected by filtration, washed with distilled water and absolute ethanol, dried in vacuum at 70 °C for 5 h and then ground up and passed through 300 mesh sieve. As a reference, neat Cu2O was also prepared by the above-mentioned method without the employment of montmorillonite.

Algae cells in exponential growth stage were harvested by centrifugation under 4000 rpm for 10 min. Removal of M. aeruginosa was carried out by diluting the algae cells with deionized water to 2.8 × 107 cells·mL− 1 higher than 4.78 × 106 cells·mL− 1 in the waterbody occurred algae bloom (Zou et al., 2004), in order to see if Cu2O-montmorillonite can effectively inhibit the growth of the M. aeruginosa within a high concentration range often encountered in an extremely eutrophic water body. Prior to use in experiments, the algae solution were adjusted to pH 7. Different amounts of Cu2O-montmorillonite or montmorillonite were separately added into 50 mL of algae solution. Algae removing material were added to reach final concentrations at 0, 0.1, 0.2, 0.3, 0.4 and 0.5 g L−1. The algae removal reaction was conducted for 3 h at room temperature under visible light in photoreaction instrument equipped with a 500 W xenon lamp (80– 300 mv/cm2) from Xian Puryear Precision and Scientific Instrument Corporation. The algae removal reactions of the different materials were also conducted in the dark. The control experiment was made without any algae removal materials. After reaction finished, algae solution was sampled for determining the physiological functions of algae cells. All the physiological assays were repeated three times and the results were expressed as a mean ± SD of three experiments.

2.6. Characterization of M. aeruginosa removal effect Effects of Cu2O-montmorillonite on M. aeruginosa were investigated. Specifically, changes in chlorophyll a, total soluble protein, malondialdehyde (MDA), chemical oxygen demand (COD), growth and metabolism activities and morphology of M. aeruginosa were assessed after using Cu2O-montmorillonite, natural montmorillonite, and Cu2O.

Please cite this article as: Gu, N., et al., Montmorillonite-supported with Cu2O nanoparticles for damage and removal of Microcystis aeruginosa under visible light, Appl. Clay Sci. (2016), http://dx.doi.org/10.1016/j.clay.2016.05.017

N. Gu et al. / Applied Clay Science xxx (2016) xxx–xxx

2.6.1. Algae removal efficiency The chlorophyll a in algae was the main photosynthetic pigment, and the content of the chlorophyll a was closely related to the state of algal cell growth and photosynthesis, which could be a potential indicator of primary productivity of water bodies (Ni et al., 2015). The changes of chlorophyll a concentration was proportional to algae cell numbers in the samples (Liu et al., 2010), therefore, the removal efficiency of algae cells based on chlorophyll a was determined. The computational formula is as follows. r¼

Chla2 −Chla1  100% Chla2

ð1Þ

where Chla1 and Chla2 were the chlorophyll a after treatment and control, respectively. The amount of chlorophyll a in M. aeruginosa was assayed according to the hot-ethanol extracting method (State Environmental Protection Administration in China, 2002).

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SCIENTZ-IID, 120 W) for 10 min at 4 °C. The solution was collected and filtered through a filter with a pore diameter of 0.45 μm to remove the residual solids Wang et al., 2015b). Chemical oxygen demand (COD) of the filtrate was measured using standard methods (APHA, 1998). 2.6.5. Growth and metabolism activity of algae cells The growth and metabolism activity of algae cells could be represented by respiratory oxygen uptake rate and photosynthetic oxygen evolution rate, which were measured by dissolved oxygen analyzer (HACH) according to the method of Winkler et al. (2002). 2.6.6. Morphology of M. aeruginosa M. aeruginosa after photocatalysis reaction were collected by centrifugation and the morphology of M. aeruginosa cells before and after exposure to Cu2O-montmorillonite were observed with transmission electron microscope (H-7650, Hitachi). 3. Results and discussion

2.6.2. Concentration of total soluble protein The algae cells could be damaged and the protein in algae cells would be released and degraded in the process of algae removal through photocatalysis of Cu2O-montmorillonite under visible light, resulting the decrease of protein. Therefore, the protein content could reflect the damage degree of algae cells. Total soluble proteins are small molecular proteins which are soluble in water. The concentration of total soluble protein was determined using Bradford assay (Bradford, 1979), a widely used method to quantify the amount of non-specific proteins. 5.0 mL cyanobacteria sample were harvested by centrifugation at 9000 rpm for 15 min and the supernatant was discarded. Harvested cells were re-dispersed in 5 mL phosphate buffer solution with pH 7.2. The mixture above was disrupted for 120 s by ultrasonication in ice bath and centrifuged for 10 min under 8000 rpm after standing for 24 h at 4 °C, the supernatant cell-free extract was used for the following assays. 1 mL of supernatant was added to 3 mL of 1/5 diluted Coomassie brilliant blue G-250 and stirred for 20 min. The concentration of protein was calculated by measuring the absorbance at 595 nm using a spectrophotometer (TU-1810, Beijing Purkinje General Instrument Co. Ltd.). The standard calibration was performed using Bull Serum Albumin (Sigma). 2.6.3. Lipid peroxidation of algae cell membrane Malondialdehyde was the product of unsaturated fatty acid peroxidation and its concentration could be used as index of lipid peroxidation of algae cell membrane. The quantity of malondialdehyde was analyzed using thiobarbituric acid (TBA, Sinopharm Chemical Reagent Co., Ltd.) which formed pink-colored MDA-TBA adduct and exhibited an absorption maximum at 532 nm (Uchiyama and Mihara, 1978). 3 mL reaction supernatant and 3 mL of 10% (wt/vol) trichloroacetic acid were mixed and centrifuged for 30 min at 9000 rpm. Then 1 mL supernatant was added to freshly prepared 1.7 mL 0.67% (wt/vol) TBA solution and 0.20 mL 1.0 M ascorbic acid. After incubating in boiling water bath for 20 min and cooling to room temperature, the absorbance of solution at 532 nm, 600 nm and 450 nm were tested using a spectrophotometer. The concentration of MDA was calculated according to following formula (Liao, 2009).
6:45  ðA532 −A600 Þ−0:56  A450 CMDA nmol  mg−1 ðTSPÞ ¼ CTSP

ð2Þ

In the formula, CMDA represented the concentration of MDA; CTSP represented the concentration of TSP, A532, A600, A450 were the absorbance value at 532 nm, 600 nm and 450 nm respectively. 2.6.4. COD of M. aeruginosa 10.0 mL cyanobacteria sample were broken by ultrasound using ultrasonic cell disruptor (Ningbo Scientz Biotechnology Co., LTD,

3.1. Structure and morphology of Cu2O-montmorillonite The structure and characteristic of Cu2O-montmorillonite were identified in terms of XRD pattern, IR spectrum, TEM image, and EDAX analysis. Fig. 1A showed XRD patterns of Na+-montmorillonite and Cu2Omontmorillonite. Based on the resulting XRD patterns, montmorillonite showed a reflection at approximately 2θ = 7.2°, the calculated basal spacing of 1.21 nm based on Bragg's equation 2dsinθ = λ (λ is X-ray wavelength, θ is diffraction angle) was typical of predominantly Na+montmorillonite. In Cu2O-montmorillonite, reflection appeared at 2θ = 5.3° corresponding to a basal spacing of 1.63 nm as calculated by Bragg's equation. The increase of d001-value was ascribed to the intercalation of Cu2O nanoparticles via cation exchange between the Cu2+ and the interlayer Na+ associated with montmorillonite (Kakuta and Abe, 2009). In Cu2O-montmorillonite, the reflection at 2θ values of 29.7, 36.7, 42.7, 61.6 and 73.5° corresponded to the lattice plane of (110) (111) (200) (220) and (311) respectively, suggesting the presence of pure Cu2O with cubic symmetry (JCPDS No. 05-0667) (Zhang et al., 2012). From the XRD data, the average crystallite size was calculated between 5 and 10 nm for Cu2O using Scherrer's equation D = Kλ/Bcosθ (λ is X-ray wavelength, B is half-width of X-ray diffraction, θ is diffraction angle, K is Scherrer constant.). No reflection of either CuO or Cu (OH)2 phases were observed, indicating the formation of pure Cu2O in the products. Fig. 1B was the IR spectra of Na+-montmorillonite and Cu2O-montmorillonite, peak of montmorillonite at 3643 cm− 1 and 3432 cm− 1 were respectively due to the stretching vibrations of structure hydroxy Al-OH and water molecules, and band at 794 cm− 1 was due to stretching vibration of AlIV tetrahedral. The band at 1094 cm−1 was due to antisymmetric Si\\O stretching. In the spectrum of Cu2O-montmorillonite, the band shifted to lower wavenumber and appeared at around 1039 cm−1. The red-shift of antisymmetric stretching vibrations were attributed to the stretching modes of the silicate layer in Cu2Omontmorillonite (Chen et al., 2014), indicating the intercalation of Cu2O. IR spectrum of Cu2O-montmorillonite confirmed the formation of Cu2O in Cu2O-montmorillonite composite. A strong peak at 626 cm−1 which was related to the optically active lattice vibration of Cu2O (Ahmed et al., 2011; Wang et al., 2015c). Fig. 2 showed the TEM images of Na+-montmorillonite (Fig. 2A), Cu2O-montmorillonite (Fig. 2B, C and D) as well as Cu2O prepared without montmorillonite (Fig. 2E and F). Montmorillonite exhibited sheets images with 1.1 nm interlayer space (Fig. 2A). Fig. 2B indicated the existence of both Cu2O and montmorillonite. It can be seen that the small tiny particles of Cu2O were attached on the surface of montmorillonite (De et al., 2015) and the size of Cu2O particles were 5–10 nm in diameter. However the Cu2O nanoparticles intercalated into the

Please cite this article as: Gu, N., et al., Montmorillonite-supported with Cu2O nanoparticles for damage and removal of Microcystis aeruginosa under visible light, Appl. Clay Sci. (2016), http://dx.doi.org/10.1016/j.clay.2016.05.017

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N. Gu et al. / Applied Clay Science xxx (2016) xxx–xxx

Fig. 1. Structure characterization of Cu2O-montmorillonite. (A)XRD patterns of samples. (B)FT-IR spectra of samples.

interlayer of montmorillonite were not observed by TEM, because they were extremely small and non-crystalline (Kun et al., 2006). Fig. 2C showed the lattice space of nanoparticles was 0.25 nm which corresponded to the distance of (111) planes of cubic Cu2O (Zhang et al., 2012). The crystallized structure of Cu2O was also confirmed from the selected area electron diffraction pattern (Fig. 2D), where bright crystalline spots were found. Fig. 2E and F showed that Cu2O nanoparticles prepared without montmorillonite support tended to aggregate together. The results of the energy-dispersive X-ray (EDAX) analysis of Cu2Omontmorillonite were reported as numerical values in Table 1. The weight content of Cu element in Cu2O-montmorillonite was 23.37 wt% which converted to the weight content of Cu2O was 26.31 wt% according to the mass ratio of Cu element and Cu2O. The Cu element content observed was a little lower than that added from Cu (CH3COO) 2 during the preparation (which was 28.21 wt%). This was related to that a little amount of Cu2O were intercalated the interlayer of the montmorillonite which could out been observed by SEM.

3.2. Removal rate of Cu2O-montmorillonite on M. aeruginosa Fig. 3A and B showed chlorophyll a removal rates of M. aeruginosa after 3 h of treatment with various concentrations of Cu2O-montmorillonite and montmorillonite under visible light and in the dark. Under visible light and in the dark, the chlorophyll a removal rates were only 5.1% and 1.2% respectively without any algae removal material, indicating visible light irradiation or dark condition for short time had no effect on M. aeruginosa. Furthermore, the chlorophyll a removal rates were below 40% with natural Na+-montmorillonite either under visible light or in the dark. The low algae removal rates were attributed to the poor capacity of natural montmorillonite with superficial negative charge to flocculate algae cells with the same negative charge (Yu et al., 2011). In the dark, the removal efficiency of M. aeruginosa by Cu2O-montmorillonite was superior to natural montmorillonite and increased with the increase of Cu2O-montmorillonite, the superior M. aeruginosa removal rate was probably due to the better absorption coagulation capacity of Cu2O-montmorillonite or the growth inhibition

Fig. 2. TEM images of montmorillonite, Cu2O-montmorillonite and Cu2O. (A)Na+-montmorillonite. (B) Cu2O-montmorillonite. (C) Lattice space of nanoparticles in Cu2O in Cu2Omontmorillonite. (D) Electron diffraction pattern of nanoparticles in Cu2O-montmorillonite. (E) and (F) Cu2O.

Please cite this article as: Gu, N., et al., Montmorillonite-supported with Cu2O nanoparticles for damage and removal of Microcystis aeruginosa under visible light, Appl. Clay Sci. (2016), http://dx.doi.org/10.1016/j.clay.2016.05.017

N. Gu et al. / Applied Clay Science xxx (2016) xxx–xxx Table 1 Elemental analysis of Cu2O-montmorillonite. Element(w%)

O

Si

Cu

C

Al

Na

Mg

Ca

Fe

K

Cu2O-Mt

38.03

16.46

23.37

8.21

6.18

3.96

1.74

1.04

0.61

0.40

of M. aeruginosa by copper ions generated from the dissolution of Cu2O (Sunada et al., 2012). Under visible light, the removal efficiency of M. aeruginosa kept increasing with the continuing increase of Cu2O-montmorillonite dosages and reached 90.2% till the dosage was 0.4 g·L−1. By comparing Fig. 3A and B, it was clear that M. aeruginosa removal efficiency of Cu2O-montmorillonite under visible light was higher than that in the dark, indicating the photocatalysis of Cu2O-montmorillonite under visible light contributed to the removal of M. aeruginosa. In Fig. 3A, chlorophyll a removal rate decreased instead when the concentration of Cu2O-montmorillonite was greater than 0.4 g·L−1. The decrease of algae removal efficiency to some degree was probably caused by the influence of excessive dosage of Cu2O-montmorillonite on light absorption of photocatalyst, reducing the photocatalytic efficiency. Fig. 3C showed chlorophyll a removal rates with Cu2O-montmorillonite loading of 0.4 g·L−1 and Cu2O loading of 0.12 g·L−1 equal to the theoretical amount of Cu2O in Cu2O-montmorillonite, which was calculated according to the added Cu (CH3COO) 2 during the preparation of Cu2Omontmorillonite. The chlorophyll a removal rates by Cu2O-montmorillonite were all higher than Cu2O either under visible light or in the dark, which meant that Cu2O-montmorillonite was a better M. aeruginosa removal material than Cu2O. The tiny increase of chlorophyll a removal rate by Cu2O-montmorillonite comparing with Cu2O in the dark was on account of the better adsorption-flocculation capacity of Cu2O-montmorillonite on M. aeruginosa than Cu2O. The significant increase of chlorophyll a removal rate by Cu2O-montmorillonite than Cu2O under visible light arising from the superior photocatalysis capacity of Cu2O-montmorillonite. The effects of contact time on chlorophyll a concentration of M. aeruginosa using 0.4 g·L−1 Cu2O-montmorillonite in the dark and under visible light are illustrated in Fig. 3D. Cu2O

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photocatalyst could not be excited to generate active radical for degrading M. aeruginosa without light illumination, therefore, removal of chlorophyll a by Cu2O-montmorillonite in the dark condition may be attributed to the absorption and flocculation of M. aeruginosa by Cu2O-montmorillonite and the growth inhibition of M. aeruginosa by copper ions generated from the dissolution of Cu2O (Sunada et al., 2012). However, under visible light, the removal of chlorophyll a was the synergistic actions of absorption- flocculation of M. aeruginosa by Cu2O-montmorillonite, the growth inhibition of M. aeruginosa by copper ions and photocatalytic degradation of M. aeruginosa by Cu2O-montmorillonite. In the dark, chlorophyll a concentration reduced rapidly within the first 0.5 h and then decreased slowly in the subsequent 1.5 h. The removal efficiency of chlorophyll a reached 43% at 2 h and then was basically constant, indicating the achievement of adsorption flocculation equilibrium of M. aeruginosa by Cu2O-montmorillonite. Under visible light, the quick decrease of chlorophyll a concentration occurred in the first 0.5 h and then the continuous decrease of chlorophyll a concentration came up in the next 2 h. The chlorophyll a concentration under visible light was obviously lower than that in the dark, demonstrating that photocatalysis of Cu2O-montmorillonite played an important role on degradation of M. aeruginosa under visible light. 3.3. M. aeruginosa disruption by Cu2O-montmorillonite The variations of total soluble protein contents of M. aeruginosa after 3 h treatment with the dosage of Cu2O-montmorillonite and montmorillonite under visible light and in the dark were shown in Fig. 4A and B respectively. Under visible light and in the dark, the total soluble protein contents without any algae removal material didn't show much difference from initial total soluble protein content 125.1 mg·L−1, demonstrating M. aeruginosa could maintain growth without the action of any algae removal material. Furthermore, no significant decrease of total soluble protein content was found for M. aeruginosa with natural Na+-montmorillonite either under visible light or in the dark, indicating there was little impact of natural montmorillonite on the activity of M.

Fig. 3. Removal efficiency of chlorophyll a. (A) Comparison of Cu2O-montmorillonite and montmorillonite under visible light. (B) Comparison of Cu2O-montmorillonite and montmorillonite in the dark. (C) Comparison of Cu2O-montmorillonite and Cu2O. (D) Variation of chlorophyll a concentration with time.

Please cite this article as: Gu, N., et al., Montmorillonite-supported with Cu2O nanoparticles for damage and removal of Microcystis aeruginosa under visible light, Appl. Clay Sci. (2016), http://dx.doi.org/10.1016/j.clay.2016.05.017

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Fig. 4. Total soluble protein content. (A) Comparison of Cu2O-montmorillonite and montmorillonite under visible light. (B) Comparison of Cu2O-montmorillonite and montmorillonite in the dark. (C) Comparison of Cu2O-montmorillonite and Cu2O. (D) Variation of total soluble protein content with time.

aeruginosa in short time, which further proven that the decreases of chlorophyll a by natural montmorillonite in Fig. 3A and B were mainly attributed to the sedimentation of M. aeruginosa to the bottom by absorption-flocculation of montmorillonite. In Fig. 4A, under visible light, total soluble protein content of M. aeruginosa kept decreasing with the continuing increase of Cu2O-montmorillonite and achieved 70.2 mg·L− 1 when dosage was 0.4 g·L−1. It can be observed that there was dramatic reduction of total soluble protein content of M. aeruginosa by Cu2O-montmorillonite under visible light from Fig. 4A, which was due to the photocatalytic degradation of M. aeruginosa by Cu2O-montmorillonite. In Fig. 4A, total soluble protein content increased over 0.4 g·L−1 Cu2O-montmorillonite, suggesting the photocatalytic degradation of M. aeruginosa was limited at high concentration of Cu2O-montmorillonite, which was in agreement with the result of chlorophyll a in Fig. 3A. Fig. 4B showed, in the dark, the total soluble protein content of M. aeruginosa gradually decreased with the continuing increase of Cu2O-montmorillonite till the dosage was 0.3 g·L−1 and

then maintained at about 100 mg·L−1. The reducing total soluble protein content in dark condition possibly was associated with the cell lysis by copper ions generated from the dissolution of Cu2O (Sunada et al., 2012). However, it can be concluded that this impact of cell lysis by copper ions on M. aeruginosa was limited based on the result that there was only tiny decrease of total soluble protein content. The total soluble protein contents after using 0.4 g·L−1 Cu2O-montmorillonite or 0.12 g·L−1 Cu2O equal to the theoretical amount of Cu2O in Cu2Omontmorillonite, which was calculated according to the added Cu (CH3COO) 2 during the preparation of Cu2O-montmorillonite were illustrated in Fig. 4C. In the dark, there was a little decrease of total soluble protein compared to initial content of total soluble protein either using Cu2O-montmorillonite or Cu2O. The reducing total soluble protein contents in dark condition possibly were associated with the denaturation and the loss of function of M. aeruginosa by the binding of copper ions generated from the partial surface dissolution of Cu2O to M. aeruginosa (Xue and Sigg, 1990; Sunada et al., 2012). In the dark, the

Fig. 5. Malondialdehyde concentration. (A) MDA concentration of M. aeruginosa treated with 0.4 g·L−1 Cu2O-montmorillonite at different time. (B) MDA concentration of M. aeruginosa treated with 0.4 g·L−1 Cu2O-montmorillonite, 0.12 g·L−1 Cu2O and 0.4 g·L−1 montmorillonite at 2.5 h.

Please cite this article as: Gu, N., et al., Montmorillonite-supported with Cu2O nanoparticles for damage and removal of Microcystis aeruginosa under visible light, Appl. Clay Sci. (2016), http://dx.doi.org/10.1016/j.clay.2016.05.017

N. Gu et al. / Applied Clay Science xxx (2016) xxx–xxx

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Fig. 6. COD of M. aeruginosa. (A) COD of M. aeruginosa treated with 0.4 g·L−1 Cu2O-montmorillonite at different time. (B) COD of M. aeruginosa treated with 0.4 g·L−1 Cu2Omontmorillonite, 0.12 g·L−1 Cu2O, 0.4 g·L−1 montmorillonite and control.

total soluble protein content of M. aeruginosa using Cu2O was lower than that using Cu2O-montmorillonite indicating that Cu2O had stronger inhibition against M. aeruginosa than Cu2O-montmorillonite in the dark. It was speculated that Cu2O released more copper ions comparing with Cu2O-montmorillonite, therefore, damaging M. aeruginosa more powerfully. Different from dark condition, there was more significant decrease of total soluble protein content of M. aeruginosa exposed to Cu2O-montmorillonite contrast with Cu2O under visible light, revealing the superior photocatalysis capacity of Cu2O-montmorillonite than Cu2O. The relations of total soluble protein content and time using 0.4 g·L−1 Cu2O-montmorillonite in the dark and under visible light are illustrated in Fig. 4D. In the dark condition, there was little change of total soluble protein content in the first 0.5 h and the continuous decrease of total soluble protein content occurred within the next 1.5 h, suggesting that the removal of M. aeruginosa in the first 0.5 h may be attributed to the absorption and flocculation of M. aeruginosa by Cu2Omontmorillonite and in the subsequent 1.5 h may be due to the growth inhibition of M. aeruginosa by copper ions generated from the dissolution of Cu2O. Under visible light, total soluble protein content reduced slightly within the first 0.5 h and then fell continually within the next 2 h and was far lower than that in the dark condition. It was because that the photocatalytic degradation of M. aeruginosa by Cu2O-montmorillonite played an important role on M. aeruginosa removal under visible light, therefore, the total protein was degraded gradually with the extended photocatalytic time and the enhanced photocatalysis of Cu2O-montmorillonite. MDA concentration variations with extended time after treatment by 0.4 g·L−1 Cu2O-montmorillonite under visible light and in the dark were shown in Fig. 5A. Under visible light, after 2.5 h of photocatalytic reaction, a high increase of MDA concentration was observed, which was correlated to the marked damage of cell membrane of M. aeruginosa. Further photo treatment times led to the sequential degradation of MDA released by photocatalysis as a consequence the reduction of MDA concentration. Dark experiment performed showed a fast increase of MDA concentration before 2 h of the reaction and then a slow increase profile was attained, indicating that Cu2O-montmorillonite could also cause the damage of cell membrane of M. aeruginosa in the dark, which further demonstrated the speculation that Cu2O released copper ions to denature M. aeruginosa by slightly partial surface dissolution. However, the result that the MDA concentration treated by Cu2Omontmorillonite with visible light irritation was much higher than that without any light irritation manifested the photocatalysis of Cu2Omontmorillonite played a dominant role on the damage to membranes of M. aeruginosa under visible light. The experiment indicated that MDA concentration achieved the maximum value at 2.5 h using Cu2O-montmorillonite, therefore, the MDA concentrations at 2.5 h using Cu2Omontmorillonite, Cu2O and montmorillonite were compared to illustrate the damaged level of the three materials to cell membranes. Fig.

5B presented the MDA concentrations of M. aeruginosa treated with 0.4 g·L−1 montmorillonite, 0.4 g·L− 1 Cu2O-montmorillonite and 0.12 g·L−1 Cu2O equal to the theoretical amount of Cu2O in Cu2O-montmorillonite, which was calculated according to the added Cu (CH3COO) 2 during the preparation of Cu2O-montmorillonite at 2.5 h. The low MDA concentration under visible light or in the dark after using montmorillonite indicated natural montmorillonite had little effect on M. aeruginosa. Comparing with Cu2O, the higher MDA concentration of M. aeruginosa treated with Cu2O-montmorillonite under illumination indicated the more damage to membranes of M. aeruginosa, which was related with the better photocatalysis activity of Cu2O-montmorillonite than Cu2O. However, in the dark, the MDA concentration of M. aeruginosa treated with Cu2O-montmorillonite was a little lower than Cu2O, indicating that Cu2O had stronger damage to M. aeruginosa than Cu2O-montmorillonite in the dark in accordance with the result of total soluble protein in Fig. 4C. COD of M. aeruginosa at different time using 0.4 g·L−1 Cu2O-montmorillonite are illustrated in Fig. 6A. In the dark condition, there was slight decrease of COD with the extended time indicating that the degradation effect of organic matter derived from M. aeruginosa was poor in the dark. Under visible light, the COD of M. aeruginosa fell a little within the first 1 h and then decrease sharply in the text 2 h. According to the result, it can be deduced that the organic matter derived from M. aeruginosa were degraded gradually with the extension of photocatalytic action of Cu2O-montmorillonite. Fig. 6B shows COD of M. aeruginosa treated with 0.4 g·L−1 Cu2O-montmorillonite, 0.12 g·L−1 Cu2O equal to the theoretical amount of Cu2O in Cu2O-montmorillonite, 0.4 g·L−1 montmorillonite and without any material after 3 h. There were minor differences of COD after the above treatments in the dark condition. Under visible light, the COD of M. aeruginosa using montmorillonite or without any material changed little compared with that in the dark condition, the result was because there was no photocatalysis and short exposure of M. aeruginosa to light had no effect on algae. However, the COD of M. aeruginosa after using Cu2O-montmorillonite or Cu2O decreased dramatically under visible light, which was the result of photocatalytic degradation of M. aeruginosa by Cu2O. Furthermore, COD after using Cu2O-montmorillonite was lower than that using Cu2O, indicating the superior photocatalytic capacity of Cu2O-montmorillonite in comparison with Cu2O. Fig. 7 presented TEM images of M. aeruginosa morphology before and after 0.4 g·L−1 Cu2O-montmorillonite photocatalysis under visible light. TEM micrographs in Fig. 7A and B showed the ovoid or spherical shape of M. aeruginosa cells ranging from 4.0 to 7.0 μm in diameter, as well as the presence of a mucilaginous capsule (Pinho et al., 2015). From Fig. 7C it can be seen that M. aeruginosa cells tended to aggregate, which may be related with absorption-flocculation of M. aeruginosa by Cu2O-montmorillonite. Besides the aggregation, a disorganisation of the cellular content was also observed (Zhou et al., 2014), indicating

Please cite this article as: Gu, N., et al., Montmorillonite-supported with Cu2O nanoparticles for damage and removal of Microcystis aeruginosa under visible light, Appl. Clay Sci. (2016), http://dx.doi.org/10.1016/j.clay.2016.05.017

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N. Gu et al. / Applied Clay Science xxx (2016) xxx–xxx

Fig. 7. TEM images of M. aeruginosa cells. (A) and (B) initial M. aeruginosa cells. (C) and (D) M. aeruginosa cells after exposed to 0.4 g·L−1 Cu2O-montmorillonite under visible light.

the degradation of cellular inclusion of M. aeruginosa. In Fig. 7D, the fuzzy boundary of M. aeruginosa was observed after exposed to Cu2Omontmorillonite, indicating a visible damage to the membranes of M. aeruginosa. Meanwhile, there were white areas in the M. aeruginosa cell which was generated from the degradation of cell inclusions, indicating the loss of integrity of M. aeruginosa cell. The effects of Cu2O-montmorillonite on photosynthetic oxygen and oxygen uptake rate were investigated. Table 2 showed the photosynthetic oxygen and oxygen uptake rate treated by 0.4 g·L−1 Cu2O-montmorillonite after 3 h. The photosynthetic oxygen and oxygen uptake

Table 2 Photosynthetic oxygen and oxygen uptake rate. Samples

Control

Photosynthetic oxygen rate [μmol(O2)·h−1·mg−1(Cha)] Oxygen uptake rate [μmol(O2)·h−1·mg−1(Cha)]

37.62 ± 0.68 4.23 ± 0.13

Cu2O-montmorillonite

31.36 ± 0.49 3.82 ± 0.09

rate decreased under the stress of Cu2O-montmorillonite comparing to the control test, which indicated that Cu2O-montmorillonite could limit the photosynthesis of M. aeruginosa. 3.4. Regeneration of Cu2O-montmorillonite The stability and reusability of Cu2O-montmorillonite photocatalyst had significant impact on its performance in the process of photocatalytic degradation. The stability experiments of Cu2O-montmorillonite were carried out according to the method of Kıranşan et al. (2015). Cu2O-montmorillonite was separated from treated algae suspension solution, washed with distilled water and dried to use for another photocatalytic degradation cycle keeping the concentration of M. aeruginosa and dosage of photocatalyst constant. As shown in Fig. 8A, the algae removal rate of chlorophyll a decreased little after three cycles, indicating the good stability of Cu2O-montmorillonite in successive M. aeruginosa removal processes through photocatalysis. Fig. 8B presents the XRD of Cu2O-montmorillonite after three cycles, it can be seen that the lattice plane (110) (111) (200) (220) and (311) of Cu2O and the 001 plane

Please cite this article as: Gu, N., et al., Montmorillonite-supported with Cu2O nanoparticles for damage and removal of Microcystis aeruginosa under visible light, Appl. Clay Sci. (2016), http://dx.doi.org/10.1016/j.clay.2016.05.017

N. Gu et al. / Applied Clay Science xxx (2016) xxx–xxx

9

Fig. 8. Reusability behavior of Cu2O-montmorillonite in photocatalytic M. aeruginosa removal process. (A) Chlorophyll a removal rate. (B) XRD patterns.

of montmorillonite. No change of diffraction was observed compared to initial Cu2O-montmorillonite, indicating the stability of Cu2Omontmorillonite. 3.5. Mechanism of degradation of M. aeruginosa with Cu2Omontmorillonite As mentioned in Sections 3.1 and 3.2, both Cu2O-montmorillonite and Cu2O could remove and disrupt cyanobacterium M. aeruginosa either in dark condition or under visible light. It was speculated that in darkness the tiny decrease of total soluble protein and COD, the slight damage to cell membrane of M. aeruginosa and the activity inhibition to M. aeruginosa were responsible for the copper ions released by slightly partial surface dissolution (Sunada et al., 2012). In order to confirm the conjecture, the variations of copper ions concentration with time in the supernatant of M. aeruginosa solution after addition of 0.4 g·L−1 Cu2O-montmorillonite and 0.12 g·L−1 Cu2O were determined, which were shown in Fig. 9. Fig. 9 illustrated that copper ions concentration decreased with time before 10 h and maintained at steady value after 10 h. The reduction of copper ions may be related to the binding of copper ions to M. aeruginosa, which lead to the denaturation and the loss of function of M. aeruginosa (Xue and Sigg, 1990). However, Fan et al. (2013) demonstrated that 0.5 mg·L−1 copper ions caused lysis in 33% of the M. aeruginosa after 3 days treatment and the highest 1.5 mg·L−1 copper ions made all the cells lost membrane integrity. Therefore, copper ions released by surface dissolution in this study was inadequate for causing the complete injury of M. aeruginosa and the removal effect of M. aeruginosa by Cu2O-montmorillonite or Cu2O in darkness was unsatisfactory. Under visible light, photocatalysis of

Fig. 9. Variation of copper ions concentration with time.

Cu2O was the main reason causing the damage to cellular surface structure of M. aeruginosa, the degradation of intracellular components of M. aeruginosa and then the inhibition of physiological activity of M. aeruginosa. With visible light irradiation, Cu2O nanoparticles were excited and electron-hole pairs were formed as explained in Eq. (3). Hydroxyl radical (·OH) mainly arose from the oxidation reactions of holes (h+) and H2O shown in Eq. (4) and the oxidation reaction of holes (h+) and OH– shown in Eq. (5). Furthermore, ·OH partially generated by chain reaction of O2 ·− shown in Eqs. (6) to (9) (Damardji et al., 2009). On the other hand, the electrons(e−) reacted dissolved oxygen to produce superoxide ions(O–2·) as illustrated in Eq. (6).These strong oxidizing agents such as superoxide ions and hydroxyl radicals can cause irreversible damage to living algae cells (Obregón Alfaro et al., 2010). Algae removal experiments in this study showed Cu2O-montmorillonite had better effect on removal and physiological activity inhibition of M. aeruginosa than Cu2O with visible light illumination, indicating the photocatalysis of Cu2O-montmorillonite was superior to Cu2O. Montmorillonite is a good type of catalyst support that are porous, environmentally benign and resistant to harsh reactions, catalysts can be uniformly distributed on montmorillonite, and are readily protected by the clay layers to avoid aggregations even if the hybrid was exposed to harsh conditions (Chen et al., 2016). Cu2O nanoparticles prepared without montmorillonite support tended to aggregate together as shown in Fig. 2E and F, which was not convenient for the sufficient contact with M. aeruginosa. However, in Cu2O-montmorillonite, photocatalyst Cu2O nanoparticles dispersed homogenously on montmorillonite instead of aggregating as shown in Fig. 2B, which increased the direct contact between M. aeruginosa and photocatalyst Cu2O and provided more chances of Cu2O working as efficient catalysts. In addition, the catalytically active species might be immobilized through chemical bonds or weaker interactions such as hydrogen bonds or donor-acceptor interactions, besides physical adsorption. Often, the

Fig. 10. Schematic structure of Cu2O-montmorillonite and possible reaction mechanism for M. aeruginosa degradation.

Please cite this article as: Gu, N., et al., Montmorillonite-supported with Cu2O nanoparticles for damage and removal of Microcystis aeruginosa under visible light, Appl. Clay Sci. (2016), http://dx.doi.org/10.1016/j.clay.2016.05.017

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N. Gu et al. / Applied Clay Science xxx (2016) xxx–xxx

interaction between support material and catalytic active components remarkably enhanced the catalytic efficiency (Zhou, 2011). Therefore, Cu2O-montmorillonite improved the removal of M. aeruginosa compared with Cu2O. Furthermore, as shown in Fig. 9, the concentration of copper ions in Cu2O-montmorillonite system was lower than Cu2O, which demonstrated that the stability of Cu2O in Cu2O-montmorillonite was better than Cu2O without support and also could enhance the photocatalytic efficiency of Cu2O and then the removal efficiency of M. aeruginosa. Finally, as stated in Sections 3.1 and 3.2, Cu2O-montmorillonite had preferable capacity of absorbing and flocculating M. aeruginosa. In other words, Cu2O-montmorillonite could better immobilize M. aeruginosa, increasing the contact between algae cells and photocatalyst Cu2O and enhancing the efficiency of photocatalysis. In brief, the superior dispersity and stability of Cu2O in Cu2O-montmorillonite, the interaction between Cu2O and montmorillonite and the better absorption and flocculation capacity of Cu2O-montmorillonite to M. aeruginosa improved the photocatalytic efficiency of Cu2O-montmorillonite and then enhanced the removal efficiency of M. aeruginosa. þ

Cu2 O þ hy→h þ e−

ð3Þ

et al., 2010; Vilela et al., 2012; Andersen et al., 2014), so, it can be believed that Cu2O-montmorillonite will be effective for simultaneous removal of the cyanotoxin, which will be our next work. 4. Conclusions This study aimed to develop an effective algae removal material for the emergency control of cyanobacteria bloom. Cu2O-montmorillonite was prepared by reduction of Cu2+ absorbed by montmorillonite. The formation of fine particles and homogeneous dispersion of Cu2O originated in the use of montmorillonite as a photocatalyst supporter. A visible light driven Cu2O-montmorillonite photocatalytic process was successfully used for the M. aeruginosa removal and disruption. Cu2Omontmorillonite removed M. aeruginosa through absorption-flocculation and photocatalysis, which resulted in the damage of the cellular surface structure of M. aeruginosa, the degradation of the intracellular components of M. aeruginosa and then the inhibition of the physiological activity of M. aeruginosa. The good absorption ability of Cu2O-montmorillonite to M. aeruginosa cells made reactive species generated by photocatalyst form on algae cells surface promoting the photodegradation efficiency of M. aeruginosa. In turn, the photodegradation caused the physiological activity loss of M. aeruginosa improving the effect of coagulation removal of M. aeruginosa.

þ

ð4Þ

h þ OH− →  OH þ Hþ

þ

ð5Þ

e− þ O2 →O2 −

ð6Þ

Acknowledgements

O2 − þ Hþ →HO2 

ð7Þ

HO2 þ e− þ Hþ →H2 O2

ð8Þ

This work was supported by Hebei Province Science and Technology Support Program (15272615D), Scientific Research Foundation for doctor, Hebei University of Science and Technology (010048) and Natural Science Foundation of Hebei Province (B2016208054).

H2 O2 þ e− →  OH þ OH−

ð9Þ

h þ H2 O→  OH þ Hþ

The probable degradation mechanism of M. aeruginosa using Cu2Omontmorillonite under visible light was shown in Fig. 10. M. aeruginosa tended to be adsorbed onto Cu2O-montmorillonite and then transferred to adjacent photocatalyst Cu2O dispersed over montmorillonite. This direct contact between algae cells and photocatalyst Cu2O was necessary for photocatalysis of Cu2O, inducing the damages to M. aeruginosa. The algae microorganism was first immobilized then damaged by photons generated by the photocatalyst (Rodríguez-González et al., 2010). Algae degradation began with the radical-induced changes of protective cell structures of algae such as the cell wall and the cell membrane, and then chlorophyll a and other inclusions in algae cells were oxidatively transformed by the action of photocatalyst (Peller et al., 2007). In present study, Cu2O-montmorillonite was used as absorbent, flocculant and photocatalyst to remove and degrade M. aeruginosa. The synergy of absorption and photocatalytic degradation of M. aeruginosa by Cu2O-montmorillonite improved the algae removal efficiency. 3.6. Safety of Cu2O-montmorillonite Fig. 9 showed that copper ions concentration in the system using Cu2O-montmorillonite was inferior to that using Cu2O, indicating the stability of Cu2O-montmorillonite was better than neat Cu2O. Using Cu2O-montmorillonite to control cyanobacteria bloom could not cause the high concentration of copper ions. Moreover, the residual copper ions concentration was 0.08 mg·L−1, below the maximum acceptable copper ions concentration according to Environmental Quality Standards for Surface Water GB3838-2002. In the process of M. aeruginosa removal and degradation using Cu2Omontmorillonite, microcystins may be released in case of the cell lysis (Ma et al., 2012). However, many research have verified powerful reactive oxygen species generated from photocatalysis were responsible for the degradation of the cyanotoxin molecules into biodegradable compounds or the mineralization into CO2, H2O and inorganic ions (Pelaez

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Please cite this article as: Gu, N., et al., Montmorillonite-supported with Cu2O nanoparticles for damage and removal of Microcystis aeruginosa under visible light, Appl. Clay Sci. (2016), http://dx.doi.org/10.1016/j.clay.2016.05.017