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Microcystis aeruginosa inhibition by Zn–Fe–LDHs as photocatalyst under visible light
Journal of the Taiwan Institute of Chemical Engineers 64 (2016) 189–195
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Microcystis aeruginosa inhibition by Zn–Fe–LDHs as photocatalyst under visible light Na Gu a,c, Jinlong Gao b,c, Kuitao Wang b,c,∗, Bo Li c, Wencui Dong b,c, Yulin Ma b,c a
School of Science, Hebei University of Science and Technology, Shijiazhuang 050018, China School of Chemical and Pharmaceutical Engineering, Hebei University of Science and Technology, Shijiazhuang 050018, China c Key Laboratory of Medicinal Molecular Chemistry in Hebei Province, Shijiazhuang 050018, China b
a r t i c l e
i n f o
Article history: Received 5 August 2015 Revised 9 March 2016 Accepted 18 April 2016 Available online 4 May 2016 Keywords: Harmful algae bloom Cyanobacteria inhibition Cyanobacteria degradation Layered double hydroxide Photocatalysis under visible light
a b s t r a c t Visible light-responsive Zn–Fe layered double hydroxide (Zn–Fe LDHs) algaecide that could remove Microcystis aeruginosa when combined with visible light was prepared by the combination of Zn2+ and Fe2+ coprecipitation method and H2 O2 oxidation. A laboratory scale experiment for Microcystis aeruginosa removal was carried out under visible light to assess the effect of Zn–Fe LDHs on algae degradation by photocatalysis process. XRD, IR SEM and TEM results showed that Zn–Fe LDHs exhibited integrated crystal structure and homogeneous granularity. Zn–Fe LDHs had a strong degradation and inhibition effect on Microcystis aeruginosa under visible light and achieved a removal rate of 80%. TEM morphology of Microcystis aeruginosa and the variation of chlorophyll a level, total soluble protein content and the concentration of malondialdehyde a product of lipid peroxidation of algae cell membrane demonstrated that Zn–Fe LDHs damaged the cell membrane and degraded cell inclusions due to its visible light catalytic activity, therefore, inhibited the proliferation of Microcystis aeruginosa. Zn–Fe LDHs could be a potential algaecide used to control harmful blooms in natural water sources with solar radiation. © 2016 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
1. Introduction Harmful algae bloom arising in rivers, lakes and seas around the world have caused wide concern [1]. The excessive reproductions of harmful algae form a thick scum on the surface of the water, depleting it of dissolved oxygen and disfiguring the coastal aesthetics. Algae bloom pose a serious threat to aquaticlife, aquaculture, human health and local tourism [2]. Furthermore, some of the algae may even release toxic and malodorous substances into the water, harming the ecological environment, drinking water quality and human health [3]. Many scholars have done a lot of work in developing new technology for algae removal [4–9]. Among these, degradation of algae by photocatalysis has been an efficient method for algae removal [4]. The strong oxidizing hydroxyl radicals • OH generating in photocatalytic reaction can oxidize proteins, lipids and nucleic acids of algae cells, furthermore destroy cyst of algal cells, which cause the disintegration of algae cells [10,11].
∗ 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. Tel.: +86 311 88632236; fax: +86 311 88632236. E-mail address: [email protected] (K. Wang).
Layered double hydroxides (LDHs) are a class of host-guest layered solids with the general formula [M2+ 1-x M3+ x (OH)2 ]x+ [(An− )x/n .yH2 O], where M2+ is the divalent cation, M3+ is the trivalent cation, and An− is the interlayer anion to balance the positive charges [12]. Recently, LDHs materials used as photocatalysts are receiving more and more interests in the environmental community [13–15] due to their high anion retention capacity and simple thermal regeneration procedure. LDHs photocatalysts can be illuminated with light of energy greater than their band gap, thus producing electron–hole pairs (h+ /e− ) in the conduction and the valence bands, respectively. These charge carriers, which migrate to the LDHs surface, are capable of oxidizing [16] or reducing [17] pollutants in solution having suitable redox potentials. Parida et al. [18] demonstrated the potential applications of Zn/Fe LDH in the field of photo degradation of azo7 dyes. Xia et al. [19] proved Zn/Fe-NO3 -LDHs displayed high photocatalytic activity under visible-light irradiation for RB degradation. Therefore, LDHs which have been demonstrated photocatalytic activity under visible light can be used as promising photocatalyst for algae removal. Jin et al. [20] used Mg-Al and Zn–Al LDHs to remove biological contaminants such as MS2 and ϕ X174 viruses and Escherichia coli in waterbody. However, the literature on algae removal by LDHs as photocatalyst is very limited. In the present study, Zn–Fe LDHs intercalated by chloride and carbonate anions were prepared for effective photocatalysis degradation of Microcystis aeruginosa a
http://dx.doi.org/10.1016/j.jtice.2016.04.016 1876-1070/© 2016 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
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typical algae causing cyanobacterial bloom. Zn–Fe LDHs was prepared by the combination of Zn2+ and Fe2+ coprecipitation and H2 O2 oxidation instead of Zn2+ and Fe3+ coprecipitation, which avoided the difficulty of coprecipitation resulting from the difference of precipitation pH of Zn2+ and Fe3+ . Zn–Fe LDHs degraded Microcystis aeruginosa cells by photocatalysis under visible light therefore inhibited and removed Microcystis aeruginosa effectively. The objective of this study was to find a potential algaecide for removing and degrading algae pollutants in the field of water treatment with solar radiation. 2. Material and methods 2.1. Preparation of Zn–Fe LDHs The LDHs materials used in this study were prepared by the combination of Zn2+ and Fe2+ coprecipitation and H2 O2 oxidation. ZnCl2 8.7 g and FeCl2 •4H2 O 6.3 g with desired Zn (II)/Fe (II) molar ratio of 2:1 were dissolved in 80 mL deionized water under vigorous stirring. NaOH 0.4 g and Na2 CO3 6.8 g were dissolved in 40 mL deionized water under vigorous stirring. The two solutions were mixed rapidly achieving pH 8. After the mixture being crystallized under stirring for 2 h, 3 mL 30% H2 O2 was added into the solution in order to convert Fe2+ to Fe3+ completely. Stirring crystallization was continued for another 4 h. The resulting precipitate was filtered, washed thoroughly with deionized water, dried at 60°C for 8 h and then ground, giving the product Zn/Fe–Cl–CO3 LDHs. All chemicals used were analytical reagent grade as received. 2.2. Characterization Zn/Fe–Cl–CO3 –LDH was identified by X-ray diffractometry (D/MAX-2500, Rigaku) using Ni-filtered Cu Kα radiation ˚ with scanning speed 5°/min. The infrared spec(λ = 1.54 A) trometry of the sample was recorded on infrared spectrometer (FTS-65A1896, Bio-Rad) at room temperature using KBr pellet as the reference in the range of 40 0–40 0 0 cm−1 . The TG-DTA thermograms were recorded on thermal analyzer (STD-2960, TA) in the temperature range from 30 to 800°C at a heating rate of 10°C /min in nitrogen atmosphere. SEM images were obtained using scanning electron microscope (S-4800-I, HITACHI). TEM images were carried out by transmission electron microscope (JEM-2100, JEOL). The UV–vis DRS spectra of the samples was recorded in a SHIMADZU-2550 UV–vis spectrophotometer. 2.3. Removal of Microcystis aeruginosa Microcystis aeruginosa FACHB-942 was purchased from Institute of Hydrobiology, Chinese Academy of Sciences. Algae cells in logarithmic growth stage were harvested by centrifugation under 30 0 0 rpm. The initial cell concentration for all removal
experiments was set to 2.8 × 108 cells.L−1 . Zn–Fe LDHs was added into 30 mL of algae suspension in quartz tube and stirred vigorously for 30 min (50 r/min) in dark to establish an adsorption/desorption equilibrium. Then the algae removal reactions were conducted under visible light in photoreaction instrument equipped with a 300 W xenon lamp (Xian Puryear Precision and Scientific Instrument Corporation). The control experiment was made without Zn–Fe LDHs. After reaction finished, algae suspension was sampled for determining the physiological functions of algae cells. Chlorophyll a [21], total soluble protein content [22] and malondialdehyde content [23] were measured. Growth and metabolism activities of algae cells can 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. [24]. The algae removal assessment repeated three times and the results were expressed as a mean ± SD of three experiments. Microcystis aeruginosa after photocatalysis reaction were collected by centrifugation and the morphology of Microcystis aeruginosa cells before and after exposure to Zn–Fe LDHs were observed with transmission electron microscope (H-7650, HITACHI). 3. Results 3.1. Characterization of Zn–Fe-LDHs XRD pattern of Zn–Fe LDHs is shown in Fig. 1(a). Zn–Fe LDHs showed reflection peak around 11°, 24°, 35°, 39°, 47°, 59° and 61° corresponding to the basal reflections of planes hkl (0 03), (0 06), (009), (015), (012), (110) and (113) of layered double hydroxide, which were the typical characteristic peaks of the hydrotalcite [18].The sharp peaks with high strength and good symmetry showed that the crystals of hydrotalcite was perfect. The (003) crystal plane diffraction peaks appeared at 2θ = 11.7° indicating that the interlayer spacing of d003 was 0.749 nm calculated by Bragg equation d = nλ/2sinθ . The crystallite size of Zn–Fe LDHs was 10 nm according to the calculation of MDI JADE 7.5. Fig. 1(b) shows the IR spectra of Zn–Fe LDHs. The band at 3388 cm−1 was the stretching vibration band of a hydroxyl group in the hydrotalcite layer. The intense band around 1491 cm−1 was associated with the carbonate ion and the peak at 832 cm−1 was corresponded to chloride ion vibration [18], which showed that the Zn–Fe LDHs prepared was intercalated by chloride ions and carbonate ion. Characteristic skeleton vibration absorption band of hydrotalcite appeared in the range of 40 0–90 0 cm−1 [25]. TG/DTA curves for Zn–Fe LDHs is presented in Fig. 1(c). Decomposition of Zn–Fe LDHs occurred in three steps. The first and second steps in the range of 50–150°C and 150–200°C were due to the elimination of physical absorbed water molecules from the external surface of Zn–Fe LDHs and the removal of water intercalated respectively, which resulted in exothermic peaks at corresponding
Fig. 1. Characterization of Zn–Fe LDHs. (a) X-ray diffraction pattern. (b) IR spectrum. (c) TG and DTA curves.
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Fig. 2. SEM images of Zn–Fe LDHs.
Fig. 3. TEM images of Zn–Fe LDHs.
temperature [25]. TG curve showed that the weight loss of water was 10.36% between room temperature and 200°C. The third step, ranging from 200°C to 500°C, were mainly attributed to the dehydroxylation of the brucite-like octahedral layers in Zn–Fe LDHs and the desorption of intercalated anions, which could be corroborated to the exothermic peaks obtained. The weight loss of the third step corresponded to 16.61% of the weight of the sample. SEM and TEM images of Zn–Fe LDHs are shown in Figs. 2 and 3 respectively. It was indicated from SEM that Zn–Fe LDHs nanoparticles were agglomerated and appeared platelike morphology which were typical characteristics of hydrotalcite prepared by the coprecipitation method [25]. TEM also showed that Zn–Fe LDHs were crystallites with plate like morphology. Fig. 3(a) and (b) displays the sheet structure of Zn–Fe LDHs. From Fig. 3(c), it can be clearly observed the erect lamellar structure of hydrotalcite crystal and the interlayer spacing was 0.75 nm according with that calculated by XRD. From Fig. 4 the UV–vis DRS spectra of Zn–Fe LDHs, it is obvious that the absorbance of Zn–Fe LDHs extended to the visible light region, with the absorption edge occurring at approximately 500 nm. In addition, according to the literature [18,19], the optical absorption near the band edge followed the equation,
α hυ = K(hν − Eg )n/2
(1)
where α , h, υ , K, and Eg are the absorption coefficient, planck constant, light frequency, proportionality constant, and band gap, respectively. Assuming Zn–Fe LDHs was a direct optical transition, the n value was 1[18, 19], plotted (α hυ )2 vs. hυ and then
Fig. 4. UV–vis diffuse reflectance spectrum of Zn–Fe LDHs.
evaluated the band gap Eg by extrapolating the linear straight line to the hυ axis intercept as shown in Fig. 4. The band gap of Zn–Fe LDHs was estimated to be 2.91 eV. 3.2. Removal of Microcystis aeruginosa The concentration of chlorophyll a was proportional to the numbers of algae cell in the samples [26]; therefore, removal
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Fig. 5. Effects of catalyst dosage on Microcystis aeruginosa removal under visible light. (a) Chlorophyll a removal rate. (b) Total soluble proteins content.
Fig. 6. Effects of photocatalytic time on Microcystis aeruginosa removal under visible light. (a) Chlorophyll a removal rate and chlorophyll a content. (b) Total soluble proteins with 0.25 g/L Zn–Fe LDHs. content. without Zn–Fe LDHs;
efficiency of algae cells could be represented as the change of chlorophyll a concentration. However, the decrease of chlorophyll a in algae suspension either resulted from the degradation of algae or from the flocculation of algae by physical absorption. In addition to chlorophyll a, the protein a typical cell inclusion in algae cells can be degraded in the process of photocatalytic algae removal; therefore in this study the photocatalytic degradation effect of Zn– Fe LDHs on algae cells was also characterized by the change of total soluble protein content. Meanwhile, malondialdehyde (MDA) was the product of lipid peroxidation of algae cell membrane thus its concentration variation can indicate the damage degree of algae cell membrane. 3.2.1. Effects of Zn–Fe LDHs dosage on Microcystis aeruginosa removal Fig. 5(a) and (b) shows the chlorophyll a removal rate and total soluble protein content after adding different amounts of Zn–Fe LDHs at 2.5 h. Without Zn–Fe LDHs, the removal rate of chlorophyll a was only 9.86% and the total soluble protein changed little compared with the initial total soluble protein (131 mg/L)in algae suspension, indicating that visible light irradiation for short time had little effect on Microcystis aeruginosa. The removal rate of chlorophyll a increased and the total soluble protein content reduced
with the increase of Zn–Fe LDHs below 0.25 g/L, and the removal rate of chlorophyll a and total soluble protein content became constant over 0.25 g/L. Therefore, the optimum dosage of Zn–Fe LDHs was 0.25 g/L under trial conditions. The decrease of chlorophyll a and total soluble protein indicated that cell inclusion were disintegrated by the photocatalysis of Zn–Fe LDHs under visible light. 3.2.2. Effects of photocatalytic time on Microcystis aeruginosa removal Fig. 6(a) and (b) shows the variation of the chlorophyll a and total soluble protein content without or with 0.25 g/L Zn–Fe LDHs at different time. After Zn–Fe LDHs and Microcystis aeruginosa had establish an adsorption-desorption equilibrium in dark(Time = 0 in Fig. 6), the removal rate of chlorophyll a was 40.6% and total soluble protein changed little compared with initial total soluble protein before photocatalytic reaction. The decrease of chlorophyll a after Zn–Fe LDHs and Microcystis aeruginosa had established an adsorption-desorption equilibrium in dark was mainly due to the absorption flocculation of Microcystis aeruginosa cells by Zn–Fe LDHs. The total soluble protein content changed little indicated the inclusions of algae cells had not been degraded in the dark which meant that Zn–Fe LDHs could not degrade Microcystis aeruginosa without illumination. After Zn–Fe LDHs was illuminated by
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and intact structure with clear-cut gas vesicles and thylakoid, which was regular, plump, elliptical, or globular[11]. The surface was intact which distinguished from the solution and the mean size of algae cells was 3.91 μm. Fig. 8(c) shown that Microcystis aeruginosa treated by photocatalysis of Zn–Fe LDHs under visible light still had round-shaped, however, there were some white part in algae cells indicating the decomposition of cell inclusions, furthermore, the average size of algae cells was 2.57 μm which was smaller than initial algae cells implying the growth inhibition of Microcystis aeruginosa. From Fig. 8(d), it can be seen that the cell boundary of Microcystis aeruginosa blurred obviously and the cell membrane ruptured, which resulted in the leakage of cell inclusions. 4. Discussion
Fig. 7. MDA concentration variation with time. Table 1 Photosynthetic oxygen and oxygen uptake rate. Samples
Control
Zn–Fe LDHs
Photosynthetic oxygen rate [μmol(O2 ).h−1 mg−1 (Cha)] Oxygen uptake rate [μmol(O2 ).h−1 mg−1 (Cha)]
35.65 ± 0.68
9.57 ± 0.15
30.16 ± 0.51
7.79 ± 0.13
visible light, chlorophyll a and total soluble protein of Microcystis aeruginosa were degraded by photocatalysis of Zn–Fe LDHs, therefore the chlorophyll a removal rate increased and total soluble protein content decreased with prolonged illumination time. And the chlorophyll a and total soluble protein content tended to be constant over 2.5 h, therefore, the optimum time for Microcystis aeruginosa removal by Zn–Fe LDHs was 2.5 h under trial conditions. Fig. 6 also demonstrated that chlorophyll a and total soluble protein content increased slightly without Zn–Fe LDHs as illumination time extended, which may result from the growth of Microcystis aeruginosa under visible light. 3.2.3. Lipid peroxidation of algae cell membrane Fig. 7 shows MDA concentration at 0.25 g/L Zn–Fe LDHs in various photocatalysis time. It is clear that MDA concentration increased with photocatalysis time and reached maximum at 1.5 h, whereas, MDA concentration decreased when photocatalysis time exceeding 1.5 h. The increase of MDA concentration indicated the disruption of membrane of Microcystis aeruginosa cell. The decrease of MDA concentration after 1.5 h was attributed to the further oxidation of MDA released from disrupted cell membrane by active radical generated by photocatalysis of Zn–Fe LDHs. 3.2.4. Photosynthetic oxygen rate and oxygen uptake rate Table 1 shows the photosynthetic oxygen rate and oxygen uptake rate of Microcystis aeruginosa. Control experiment was carried out under visible light in absence of Zn–Fe LDHs. The photosynthetic oxygen rate and oxygen uptake rate of Microcystis aeruginosa treated by Zn–Fe LDHs were far below the control value, indicating the photosynthesis and normal metabolism of Microcystis aeruginosa were inhibited. 3.2.5. Morphology of Microcystis aeruginosa Fig. 8 shows the morphology of Microcystis aeruginosa cells before and after photocatalysis by Zn–Fe LDHs. The morphology of Microcystis aeruginosa cells showed significant changes after photocatalysis by Zn–Fe LDHs under visible light. As shows in Fig. 8(a) and (b), the initial Microcystis aeruginosa appeared round-shaped
4.1. Mechanism on Microcystis aeruginosa removal using Zn–Fe LDHs In this study, Zn–Fe LDHs was used as a photocatalyst to remove and eliminate Microcystis aeruginosa under visible light. According to the experimental results, a possible mechanism on degrading Microcystis aeruginosa by photocatalytical reaction using Zn–Fe LDHs was proposed. The photocatalytic process started with the light absorption by the materials and the migration of the light-induced electrons and holes [19]. Zn–Fe LDHs acted as doped semiconductor and Fe+3 in Zn–Fe LDHs existed as dopant which facilitated the shifting of light absorbance towards visible range [27]. Furthermore, hydroxide groups present in LDHs surface captured the photo produced holes h+ , consequently preventing the recombination of the hole and electron as a result improving photocatalytic activity [28]. As shown in Fig. 4, the absorbance of Zn–Fe LDHs extended to the visible light region, therefore, the excited electrons of d5 Fe: 3d ions in FeO6 octahedron were promoted from the valence band to the conduction band under visible light, yielding holes h+ and photo generated electrons e− [18] (see Eq. (2)). The holes (h+ ) were transferred to H2 O initiating its photoxidation, yielding hydroxyal radical (•OH) as shown in Eq. (3), on the other hand, the electrons (e− ) reacted with dissolved oxygen to produce superoxide ions (O2 − •) as illustrated in Eq. (4). Furthermore, O2 − •could be transformed into H2 O2 , which could be reacted with e− to give OH• [19] (see Eqs. (5)–(7)). Both hydroxyl radicals (OH•), superoxide ions(O2 − •) and H2 O2 generated in the photocatalytic reaction could cause damage to living algae cells [29,30]. However, H2 O2 had weaker oxidative activity compared with OH• and O2 − •and could lead to the production of OH•. Furthermore the destructive properties of H2 O2 to algae cells resulted primarily from their role in hydroxyl radical production rather than from direct damage. [30]
ZnFe − LDHs + hγ → h+ + e− +
h + H2 O → •OH + H
+
(3)
e− + O2 → O2 •− −
(4)
+
O2 • + H → HOO• −
(5)
+
e + HOO • +H → H2 O2 −
H2 O2 + e → OH • +OH
(2)
−
(6) (7)
Fig. 9 is the schematic illustration of Microcystis aeruginosa degradation by Zn–Fe LDHs. The degradation of Microcystis aeruginosa began with photocatalytic action by the Zn–Fe LDHs on the protective cell structures of algae such as the cell wall and the cell membrane. The protective wall and membrane of the cell undergone radical-induced changes first, and then chlorophyll a and other inclusions in algae cells were oxidatively transformed by the action of photocatalyst [31]. MDA concentration and TEM image of
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Fig. 8. TEM images of Microcystis aeruginosa. (a) and (b) initial algae cells; (c) and (d) with Zn–Fe LDHs under visible light after 2.5 h.
Fig. 9. The schematic illustration of Microcystis aeruginosa degradation by Zn–Fe LDHs.
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Microcystis aeruginosa indicated the damage of the protective wall and membrane of Microcystis aeruginosa was obvious under action of the hydroxyl radicals (OH•) and superoxide ions(O2− •) generated by Zn–Fe LDHs photocatalysis, which resulted in the leakage of inclusions such as chlorophyll a and protein and then the further degradation of cell inclusion by the oxidation of active radicals. In a word, Zn–Fe LDHs irradiated by visible light caused clearly discernible effects on Microcystis aeruginosa behavior and cell structure. The green color of the cells had faded, the cellular surface structure was damaged and the intracellular components were degraded. 5. Conclusions Zn–Fe LDHs was synthesized by the combination of Zn2+ and Fe2+ coprecipitation and H2 O2 oxidation instead of Zn2+ and Fe3+ coprecipitation, which avoided the difficulty of coprecipitation resulting from the difference of precipitation pH of Zn2+ and Fe3+ . Zn–Fe LDHs synthesized was visible light-responsive and high photocatalytic activity. The photocatalytic activity of Zn–Fe LDHs under visible light contributed to the degradation of Microcystis aeruginosa. The cell membrane of Microcystis aeruginosa was damaged, leading to the leakage of cell inclusions and then the deep degradation of cell inclusions such as chlorophyll a and total soluble protein, which eventually resulted in the growth inhibition of Microcystis aeruginosa. The chlorophyll a removal rate reached 80.6% in 2.5 h using 0.25 g L−1 Zn–Fe LDHs under visible light. Zn–Fe LDHs is a potential algaecide that can be used to control harmful algae blooms in natural water sources with solar radiation. Acknowledgments This work was supported by Hebei province Science and Technology support program (15272615D) and Scientific Research Foundation for doctor, Hebei University of Science and Technology (010 048). References [1] Yan QY, Yu YH, Feng W, Pan G, Chen H, Chen JA, et al. Plankton community succession in artificial systems subjected to cyanobacterial blooms removal using chitosan-modified soils. Microb Ecol 2009;58:47–55. doi:10.1007/ s0 0248-0 08-9434-3. [2] Beaulieu SE, Sengco MR, Anderson DM. Using clay to control harmful algal blooms: deposition and resuspension of clay/algal flocs. Harmful Algae 2005;4:123–38. doi:10.1016/j.hal.2003.12.008. [3] Nagayama K, Shibata T, Fujimoto K, Honjo T, Nakamura T. Algicidal effect of phlorotannins from the brown alga Ecklonia kurome on red tide microalgae. Aquaculture 2003;218:601–11. doi:10.1016/S0 044-8486(02)0 0255-7. [4] Hong JM, Otaki M. Association of photosynthesis and photocatalytic inhibition of algal growth by TiO2 . J Biosci Bioeng 2006;101:185–9. doi:10.1263/jbb.101. 185. [5] Pan G, Zhang MM, Chen H, Zou H, Yan H. Removal of cyanobacterial blooms in Taihu Lake using local soils. I. Equilibrium and kinetic screening on the flocculation of Microcystis aeruginosa using commercially available clays and minerals. Environ Pollut 2006;141:195–200. doi:10.1016/j.envpol.2005.08.041. [6] Gustafsson S, Hultberg M, Figueroa RI, Rengefors K. On the control of HAB species using low biosurfactant concentrations. Harmful Algae 2009;8:857–63. doi:10.1016/j.hal.20 09.04.0 02. [7] Wang Q, Su M, Zhu W, Li X, Jia Y, Guo P, et al. Growth inhibition of Microcystis aeruginosa by white-rot fungus Lopharia spadicea. Water Sci Technol 2010;62:317–23. doi:10.2166/wst.2010.214. [8] Tang Y, Zhang H, Liu XA, Cai DQ, Feng HY, Miao CG, et al. Flocculation of harmful algal blooms by modified attapulgite and its safety evaluation. Water Res 2011;45:2855–62. doi:10.1016/j.watres.2011.03.003.
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Microcystis aeruginosa inhibition by Zn–Fe–LDHs as photocatalyst under visible light Na Gu a,c, Jinlong Gao b,c, Kuitao Wang b,c,∗, Bo Li c, Wencui Dong b,c, Yulin Ma b,c a
School of Science, Hebei University of Science and Technology, Shijiazhuang 050018, China School of Chemical and Pharmaceutical Engineering, Hebei University of Science and Technology, Shijiazhuang 050018, China c Key Laboratory of Medicinal Molecular Chemistry in Hebei Province, Shijiazhuang 050018, China b
a r t i c l e
i n f o
Article history: Received 5 August 2015 Revised 9 March 2016 Accepted 18 April 2016 Available online 4 May 2016 Keywords: Harmful algae bloom Cyanobacteria inhibition Cyanobacteria degradation Layered double hydroxide Photocatalysis under visible light
a b s t r a c t Visible light-responsive Zn–Fe layered double hydroxide (Zn–Fe LDHs) algaecide that could remove Microcystis aeruginosa when combined with visible light was prepared by the combination of Zn2+ and Fe2+ coprecipitation method and H2 O2 oxidation. A laboratory scale experiment for Microcystis aeruginosa removal was carried out under visible light to assess the effect of Zn–Fe LDHs on algae degradation by photocatalysis process. XRD, IR SEM and TEM results showed that Zn–Fe LDHs exhibited integrated crystal structure and homogeneous granularity. Zn–Fe LDHs had a strong degradation and inhibition effect on Microcystis aeruginosa under visible light and achieved a removal rate of 80%. TEM morphology of Microcystis aeruginosa and the variation of chlorophyll a level, total soluble protein content and the concentration of malondialdehyde a product of lipid peroxidation of algae cell membrane demonstrated that Zn–Fe LDHs damaged the cell membrane and degraded cell inclusions due to its visible light catalytic activity, therefore, inhibited the proliferation of Microcystis aeruginosa. Zn–Fe LDHs could be a potential algaecide used to control harmful blooms in natural water sources with solar radiation. © 2016 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
1. Introduction Harmful algae bloom arising in rivers, lakes and seas around the world have caused wide concern [1]. The excessive reproductions of harmful algae form a thick scum on the surface of the water, depleting it of dissolved oxygen and disfiguring the coastal aesthetics. Algae bloom pose a serious threat to aquaticlife, aquaculture, human health and local tourism [2]. Furthermore, some of the algae may even release toxic and malodorous substances into the water, harming the ecological environment, drinking water quality and human health [3]. Many scholars have done a lot of work in developing new technology for algae removal [4–9]. Among these, degradation of algae by photocatalysis has been an efficient method for algae removal [4]. The strong oxidizing hydroxyl radicals • OH generating in photocatalytic reaction can oxidize proteins, lipids and nucleic acids of algae cells, furthermore destroy cyst of algal cells, which cause the disintegration of algae cells [10,11].
∗ 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. Tel.: +86 311 88632236; fax: +86 311 88632236. E-mail address: [email protected] (K. Wang).
Layered double hydroxides (LDHs) are a class of host-guest layered solids with the general formula [M2+ 1-x M3+ x (OH)2 ]x+ [(An− )x/n .yH2 O], where M2+ is the divalent cation, M3+ is the trivalent cation, and An− is the interlayer anion to balance the positive charges [12]. Recently, LDHs materials used as photocatalysts are receiving more and more interests in the environmental community [13–15] due to their high anion retention capacity and simple thermal regeneration procedure. LDHs photocatalysts can be illuminated with light of energy greater than their band gap, thus producing electron–hole pairs (h+ /e− ) in the conduction and the valence bands, respectively. These charge carriers, which migrate to the LDHs surface, are capable of oxidizing [16] or reducing [17] pollutants in solution having suitable redox potentials. Parida et al. [18] demonstrated the potential applications of Zn/Fe LDH in the field of photo degradation of azo7 dyes. Xia et al. [19] proved Zn/Fe-NO3 -LDHs displayed high photocatalytic activity under visible-light irradiation for RB degradation. Therefore, LDHs which have been demonstrated photocatalytic activity under visible light can be used as promising photocatalyst for algae removal. Jin et al. [20] used Mg-Al and Zn–Al LDHs to remove biological contaminants such as MS2 and ϕ X174 viruses and Escherichia coli in waterbody. However, the literature on algae removal by LDHs as photocatalyst is very limited. In the present study, Zn–Fe LDHs intercalated by chloride and carbonate anions were prepared for effective photocatalysis degradation of Microcystis aeruginosa a
http://dx.doi.org/10.1016/j.jtice.2016.04.016 1876-1070/© 2016 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
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typical algae causing cyanobacterial bloom. Zn–Fe LDHs was prepared by the combination of Zn2+ and Fe2+ coprecipitation and H2 O2 oxidation instead of Zn2+ and Fe3+ coprecipitation, which avoided the difficulty of coprecipitation resulting from the difference of precipitation pH of Zn2+ and Fe3+ . Zn–Fe LDHs degraded Microcystis aeruginosa cells by photocatalysis under visible light therefore inhibited and removed Microcystis aeruginosa effectively. The objective of this study was to find a potential algaecide for removing and degrading algae pollutants in the field of water treatment with solar radiation. 2. Material and methods 2.1. Preparation of Zn–Fe LDHs The LDHs materials used in this study were prepared by the combination of Zn2+ and Fe2+ coprecipitation and H2 O2 oxidation. ZnCl2 8.7 g and FeCl2 •4H2 O 6.3 g with desired Zn (II)/Fe (II) molar ratio of 2:1 were dissolved in 80 mL deionized water under vigorous stirring. NaOH 0.4 g and Na2 CO3 6.8 g were dissolved in 40 mL deionized water under vigorous stirring. The two solutions were mixed rapidly achieving pH 8. After the mixture being crystallized under stirring for 2 h, 3 mL 30% H2 O2 was added into the solution in order to convert Fe2+ to Fe3+ completely. Stirring crystallization was continued for another 4 h. The resulting precipitate was filtered, washed thoroughly with deionized water, dried at 60°C for 8 h and then ground, giving the product Zn/Fe–Cl–CO3 LDHs. All chemicals used were analytical reagent grade as received. 2.2. Characterization Zn/Fe–Cl–CO3 –LDH was identified by X-ray diffractometry (D/MAX-2500, Rigaku) using Ni-filtered Cu Kα radiation ˚ with scanning speed 5°/min. The infrared spec(λ = 1.54 A) trometry of the sample was recorded on infrared spectrometer (FTS-65A1896, Bio-Rad) at room temperature using KBr pellet as the reference in the range of 40 0–40 0 0 cm−1 . The TG-DTA thermograms were recorded on thermal analyzer (STD-2960, TA) in the temperature range from 30 to 800°C at a heating rate of 10°C /min in nitrogen atmosphere. SEM images were obtained using scanning electron microscope (S-4800-I, HITACHI). TEM images were carried out by transmission electron microscope (JEM-2100, JEOL). The UV–vis DRS spectra of the samples was recorded in a SHIMADZU-2550 UV–vis spectrophotometer. 2.3. Removal of Microcystis aeruginosa Microcystis aeruginosa FACHB-942 was purchased from Institute of Hydrobiology, Chinese Academy of Sciences. Algae cells in logarithmic growth stage were harvested by centrifugation under 30 0 0 rpm. The initial cell concentration for all removal
experiments was set to 2.8 × 108 cells.L−1 . Zn–Fe LDHs was added into 30 mL of algae suspension in quartz tube and stirred vigorously for 30 min (50 r/min) in dark to establish an adsorption/desorption equilibrium. Then the algae removal reactions were conducted under visible light in photoreaction instrument equipped with a 300 W xenon lamp (Xian Puryear Precision and Scientific Instrument Corporation). The control experiment was made without Zn–Fe LDHs. After reaction finished, algae suspension was sampled for determining the physiological functions of algae cells. Chlorophyll a [21], total soluble protein content [22] and malondialdehyde content [23] were measured. Growth and metabolism activities of algae cells can 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. [24]. The algae removal assessment repeated three times and the results were expressed as a mean ± SD of three experiments. Microcystis aeruginosa after photocatalysis reaction were collected by centrifugation and the morphology of Microcystis aeruginosa cells before and after exposure to Zn–Fe LDHs were observed with transmission electron microscope (H-7650, HITACHI). 3. Results 3.1. Characterization of Zn–Fe-LDHs XRD pattern of Zn–Fe LDHs is shown in Fig. 1(a). Zn–Fe LDHs showed reflection peak around 11°, 24°, 35°, 39°, 47°, 59° and 61° corresponding to the basal reflections of planes hkl (0 03), (0 06), (009), (015), (012), (110) and (113) of layered double hydroxide, which were the typical characteristic peaks of the hydrotalcite [18].The sharp peaks with high strength and good symmetry showed that the crystals of hydrotalcite was perfect. The (003) crystal plane diffraction peaks appeared at 2θ = 11.7° indicating that the interlayer spacing of d003 was 0.749 nm calculated by Bragg equation d = nλ/2sinθ . The crystallite size of Zn–Fe LDHs was 10 nm according to the calculation of MDI JADE 7.5. Fig. 1(b) shows the IR spectra of Zn–Fe LDHs. The band at 3388 cm−1 was the stretching vibration band of a hydroxyl group in the hydrotalcite layer. The intense band around 1491 cm−1 was associated with the carbonate ion and the peak at 832 cm−1 was corresponded to chloride ion vibration [18], which showed that the Zn–Fe LDHs prepared was intercalated by chloride ions and carbonate ion. Characteristic skeleton vibration absorption band of hydrotalcite appeared in the range of 40 0–90 0 cm−1 [25]. TG/DTA curves for Zn–Fe LDHs is presented in Fig. 1(c). Decomposition of Zn–Fe LDHs occurred in three steps. The first and second steps in the range of 50–150°C and 150–200°C were due to the elimination of physical absorbed water molecules from the external surface of Zn–Fe LDHs and the removal of water intercalated respectively, which resulted in exothermic peaks at corresponding
Fig. 1. Characterization of Zn–Fe LDHs. (a) X-ray diffraction pattern. (b) IR spectrum. (c) TG and DTA curves.
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Fig. 2. SEM images of Zn–Fe LDHs.
Fig. 3. TEM images of Zn–Fe LDHs.
temperature [25]. TG curve showed that the weight loss of water was 10.36% between room temperature and 200°C. The third step, ranging from 200°C to 500°C, were mainly attributed to the dehydroxylation of the brucite-like octahedral layers in Zn–Fe LDHs and the desorption of intercalated anions, which could be corroborated to the exothermic peaks obtained. The weight loss of the third step corresponded to 16.61% of the weight of the sample. SEM and TEM images of Zn–Fe LDHs are shown in Figs. 2 and 3 respectively. It was indicated from SEM that Zn–Fe LDHs nanoparticles were agglomerated and appeared platelike morphology which were typical characteristics of hydrotalcite prepared by the coprecipitation method [25]. TEM also showed that Zn–Fe LDHs were crystallites with plate like morphology. Fig. 3(a) and (b) displays the sheet structure of Zn–Fe LDHs. From Fig. 3(c), it can be clearly observed the erect lamellar structure of hydrotalcite crystal and the interlayer spacing was 0.75 nm according with that calculated by XRD. From Fig. 4 the UV–vis DRS spectra of Zn–Fe LDHs, it is obvious that the absorbance of Zn–Fe LDHs extended to the visible light region, with the absorption edge occurring at approximately 500 nm. In addition, according to the literature [18,19], the optical absorption near the band edge followed the equation,
α hυ = K(hν − Eg )n/2
(1)
where α , h, υ , K, and Eg are the absorption coefficient, planck constant, light frequency, proportionality constant, and band gap, respectively. Assuming Zn–Fe LDHs was a direct optical transition, the n value was 1[18, 19], plotted (α hυ )2 vs. hυ and then
Fig. 4. UV–vis diffuse reflectance spectrum of Zn–Fe LDHs.
evaluated the band gap Eg by extrapolating the linear straight line to the hυ axis intercept as shown in Fig. 4. The band gap of Zn–Fe LDHs was estimated to be 2.91 eV. 3.2. Removal of Microcystis aeruginosa The concentration of chlorophyll a was proportional to the numbers of algae cell in the samples [26]; therefore, removal
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Fig. 5. Effects of catalyst dosage on Microcystis aeruginosa removal under visible light. (a) Chlorophyll a removal rate. (b) Total soluble proteins content.
Fig. 6. Effects of photocatalytic time on Microcystis aeruginosa removal under visible light. (a) Chlorophyll a removal rate and chlorophyll a content. (b) Total soluble proteins with 0.25 g/L Zn–Fe LDHs. content. without Zn–Fe LDHs;
efficiency of algae cells could be represented as the change of chlorophyll a concentration. However, the decrease of chlorophyll a in algae suspension either resulted from the degradation of algae or from the flocculation of algae by physical absorption. In addition to chlorophyll a, the protein a typical cell inclusion in algae cells can be degraded in the process of photocatalytic algae removal; therefore in this study the photocatalytic degradation effect of Zn– Fe LDHs on algae cells was also characterized by the change of total soluble protein content. Meanwhile, malondialdehyde (MDA) was the product of lipid peroxidation of algae cell membrane thus its concentration variation can indicate the damage degree of algae cell membrane. 3.2.1. Effects of Zn–Fe LDHs dosage on Microcystis aeruginosa removal Fig. 5(a) and (b) shows the chlorophyll a removal rate and total soluble protein content after adding different amounts of Zn–Fe LDHs at 2.5 h. Without Zn–Fe LDHs, the removal rate of chlorophyll a was only 9.86% and the total soluble protein changed little compared with the initial total soluble protein (131 mg/L)in algae suspension, indicating that visible light irradiation for short time had little effect on Microcystis aeruginosa. The removal rate of chlorophyll a increased and the total soluble protein content reduced
with the increase of Zn–Fe LDHs below 0.25 g/L, and the removal rate of chlorophyll a and total soluble protein content became constant over 0.25 g/L. Therefore, the optimum dosage of Zn–Fe LDHs was 0.25 g/L under trial conditions. The decrease of chlorophyll a and total soluble protein indicated that cell inclusion were disintegrated by the photocatalysis of Zn–Fe LDHs under visible light. 3.2.2. Effects of photocatalytic time on Microcystis aeruginosa removal Fig. 6(a) and (b) shows the variation of the chlorophyll a and total soluble protein content without or with 0.25 g/L Zn–Fe LDHs at different time. After Zn–Fe LDHs and Microcystis aeruginosa had establish an adsorption-desorption equilibrium in dark(Time = 0 in Fig. 6), the removal rate of chlorophyll a was 40.6% and total soluble protein changed little compared with initial total soluble protein before photocatalytic reaction. The decrease of chlorophyll a after Zn–Fe LDHs and Microcystis aeruginosa had established an adsorption-desorption equilibrium in dark was mainly due to the absorption flocculation of Microcystis aeruginosa cells by Zn–Fe LDHs. The total soluble protein content changed little indicated the inclusions of algae cells had not been degraded in the dark which meant that Zn–Fe LDHs could not degrade Microcystis aeruginosa without illumination. After Zn–Fe LDHs was illuminated by
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and intact structure with clear-cut gas vesicles and thylakoid, which was regular, plump, elliptical, or globular[11]. The surface was intact which distinguished from the solution and the mean size of algae cells was 3.91 μm. Fig. 8(c) shown that Microcystis aeruginosa treated by photocatalysis of Zn–Fe LDHs under visible light still had round-shaped, however, there were some white part in algae cells indicating the decomposition of cell inclusions, furthermore, the average size of algae cells was 2.57 μm which was smaller than initial algae cells implying the growth inhibition of Microcystis aeruginosa. From Fig. 8(d), it can be seen that the cell boundary of Microcystis aeruginosa blurred obviously and the cell membrane ruptured, which resulted in the leakage of cell inclusions. 4. Discussion
Fig. 7. MDA concentration variation with time. Table 1 Photosynthetic oxygen and oxygen uptake rate. Samples
Control
Zn–Fe LDHs
Photosynthetic oxygen rate [μmol(O2 ).h−1 mg−1 (Cha)] Oxygen uptake rate [μmol(O2 ).h−1 mg−1 (Cha)]
35.65 ± 0.68
9.57 ± 0.15
30.16 ± 0.51
7.79 ± 0.13
visible light, chlorophyll a and total soluble protein of Microcystis aeruginosa were degraded by photocatalysis of Zn–Fe LDHs, therefore the chlorophyll a removal rate increased and total soluble protein content decreased with prolonged illumination time. And the chlorophyll a and total soluble protein content tended to be constant over 2.5 h, therefore, the optimum time for Microcystis aeruginosa removal by Zn–Fe LDHs was 2.5 h under trial conditions. Fig. 6 also demonstrated that chlorophyll a and total soluble protein content increased slightly without Zn–Fe LDHs as illumination time extended, which may result from the growth of Microcystis aeruginosa under visible light. 3.2.3. Lipid peroxidation of algae cell membrane Fig. 7 shows MDA concentration at 0.25 g/L Zn–Fe LDHs in various photocatalysis time. It is clear that MDA concentration increased with photocatalysis time and reached maximum at 1.5 h, whereas, MDA concentration decreased when photocatalysis time exceeding 1.5 h. The increase of MDA concentration indicated the disruption of membrane of Microcystis aeruginosa cell. The decrease of MDA concentration after 1.5 h was attributed to the further oxidation of MDA released from disrupted cell membrane by active radical generated by photocatalysis of Zn–Fe LDHs. 3.2.4. Photosynthetic oxygen rate and oxygen uptake rate Table 1 shows the photosynthetic oxygen rate and oxygen uptake rate of Microcystis aeruginosa. Control experiment was carried out under visible light in absence of Zn–Fe LDHs. The photosynthetic oxygen rate and oxygen uptake rate of Microcystis aeruginosa treated by Zn–Fe LDHs were far below the control value, indicating the photosynthesis and normal metabolism of Microcystis aeruginosa were inhibited. 3.2.5. Morphology of Microcystis aeruginosa Fig. 8 shows the morphology of Microcystis aeruginosa cells before and after photocatalysis by Zn–Fe LDHs. The morphology of Microcystis aeruginosa cells showed significant changes after photocatalysis by Zn–Fe LDHs under visible light. As shows in Fig. 8(a) and (b), the initial Microcystis aeruginosa appeared round-shaped
4.1. Mechanism on Microcystis aeruginosa removal using Zn–Fe LDHs In this study, Zn–Fe LDHs was used as a photocatalyst to remove and eliminate Microcystis aeruginosa under visible light. According to the experimental results, a possible mechanism on degrading Microcystis aeruginosa by photocatalytical reaction using Zn–Fe LDHs was proposed. The photocatalytic process started with the light absorption by the materials and the migration of the light-induced electrons and holes [19]. Zn–Fe LDHs acted as doped semiconductor and Fe+3 in Zn–Fe LDHs existed as dopant which facilitated the shifting of light absorbance towards visible range [27]. Furthermore, hydroxide groups present in LDHs surface captured the photo produced holes h+ , consequently preventing the recombination of the hole and electron as a result improving photocatalytic activity [28]. As shown in Fig. 4, the absorbance of Zn–Fe LDHs extended to the visible light region, therefore, the excited electrons of d5 Fe: 3d ions in FeO6 octahedron were promoted from the valence band to the conduction band under visible light, yielding holes h+ and photo generated electrons e− [18] (see Eq. (2)). The holes (h+ ) were transferred to H2 O initiating its photoxidation, yielding hydroxyal radical (•OH) as shown in Eq. (3), on the other hand, the electrons (e− ) reacted with dissolved oxygen to produce superoxide ions (O2 − •) as illustrated in Eq. (4). Furthermore, O2 − •could be transformed into H2 O2 , which could be reacted with e− to give OH• [19] (see Eqs. (5)–(7)). Both hydroxyl radicals (OH•), superoxide ions(O2 − •) and H2 O2 generated in the photocatalytic reaction could cause damage to living algae cells [29,30]. However, H2 O2 had weaker oxidative activity compared with OH• and O2 − •and could lead to the production of OH•. Furthermore the destructive properties of H2 O2 to algae cells resulted primarily from their role in hydroxyl radical production rather than from direct damage. [30]
ZnFe − LDHs + hγ → h+ + e− +
h + H2 O → •OH + H
+
(3)
e− + O2 → O2 •− −
(4)
+
O2 • + H → HOO• −
(5)
+
e + HOO • +H → H2 O2 −
H2 O2 + e → OH • +OH
(2)
−
(6) (7)
Fig. 9 is the schematic illustration of Microcystis aeruginosa degradation by Zn–Fe LDHs. The degradation of Microcystis aeruginosa began with photocatalytic action by the Zn–Fe LDHs on the protective cell structures of algae such as the cell wall and the cell membrane. The protective wall and membrane of the cell undergone radical-induced changes first, and then chlorophyll a and other inclusions in algae cells were oxidatively transformed by the action of photocatalyst [31]. MDA concentration and TEM image of
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Fig. 8. TEM images of Microcystis aeruginosa. (a) and (b) initial algae cells; (c) and (d) with Zn–Fe LDHs under visible light after 2.5 h.
Fig. 9. The schematic illustration of Microcystis aeruginosa degradation by Zn–Fe LDHs.
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Microcystis aeruginosa indicated the damage of the protective wall and membrane of Microcystis aeruginosa was obvious under action of the hydroxyl radicals (OH•) and superoxide ions(O2− •) generated by Zn–Fe LDHs photocatalysis, which resulted in the leakage of inclusions such as chlorophyll a and protein and then the further degradation of cell inclusion by the oxidation of active radicals. In a word, Zn–Fe LDHs irradiated by visible light caused clearly discernible effects on Microcystis aeruginosa behavior and cell structure. The green color of the cells had faded, the cellular surface structure was damaged and the intracellular components were degraded. 5. Conclusions Zn–Fe LDHs was synthesized by the combination of Zn2+ and Fe2+ coprecipitation and H2 O2 oxidation instead of Zn2+ and Fe3+ coprecipitation, which avoided the difficulty of coprecipitation resulting from the difference of precipitation pH of Zn2+ and Fe3+ . Zn–Fe LDHs synthesized was visible light-responsive and high photocatalytic activity. The photocatalytic activity of Zn–Fe LDHs under visible light contributed to the degradation of Microcystis aeruginosa. The cell membrane of Microcystis aeruginosa was damaged, leading to the leakage of cell inclusions and then the deep degradation of cell inclusions such as chlorophyll a and total soluble protein, which eventually resulted in the growth inhibition of Microcystis aeruginosa. The chlorophyll a removal rate reached 80.6% in 2.5 h using 0.25 g L−1 Zn–Fe LDHs under visible light. Zn–Fe LDHs is a potential algaecide that can be used to control harmful algae blooms in natural water sources with solar radiation. Acknowledgments This work was supported by Hebei province Science and Technology support program (15272615D) and Scientific Research Foundation for doctor, Hebei University of Science and Technology (010 048). References [1] Yan QY, Yu YH, Feng W, Pan G, Chen H, Chen JA, et al. Plankton community succession in artificial systems subjected to cyanobacterial blooms removal using chitosan-modified soils. Microb Ecol 2009;58:47–55. doi:10.1007/ s0 0248-0 08-9434-3. [2] Beaulieu SE, Sengco MR, Anderson DM. Using clay to control harmful algal blooms: deposition and resuspension of clay/algal flocs. Harmful Algae 2005;4:123–38. doi:10.1016/j.hal.2003.12.008. [3] Nagayama K, Shibata T, Fujimoto K, Honjo T, Nakamura T. Algicidal effect of phlorotannins from the brown alga Ecklonia kurome on red tide microalgae. Aquaculture 2003;218:601–11. doi:10.1016/S0 044-8486(02)0 0255-7. [4] Hong JM, Otaki M. Association of photosynthesis and photocatalytic inhibition of algal growth by TiO2 . J Biosci Bioeng 2006;101:185–9. doi:10.1263/jbb.101. 185. [5] Pan G, Zhang MM, Chen H, Zou H, Yan H. Removal of cyanobacterial blooms in Taihu Lake using local soils. I. Equilibrium and kinetic screening on the flocculation of Microcystis aeruginosa using commercially available clays and minerals. Environ Pollut 2006;141:195–200. doi:10.1016/j.envpol.2005.08.041. [6] Gustafsson S, Hultberg M, Figueroa RI, Rengefors K. On the control of HAB species using low biosurfactant concentrations. Harmful Algae 2009;8:857–63. doi:10.1016/j.hal.20 09.04.0 02. [7] Wang Q, Su M, Zhu W, Li X, Jia Y, Guo P, et al. Growth inhibition of Microcystis aeruginosa by white-rot fungus Lopharia spadicea. Water Sci Technol 2010;62:317–23. doi:10.2166/wst.2010.214. [8] Tang Y, Zhang H, Liu XA, Cai DQ, Feng HY, Miao CG, et al. Flocculation of harmful algal blooms by modified attapulgite and its safety evaluation. Water Res 2011;45:2855–62. doi:10.1016/j.watres.2011.03.003.
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