Physiological responses of Microcystis aeruginosa against the algicidal bacterium Pseudomonas aeruginosa

Ecotoxicology and Environmental Safety 127 (2016) 214–221

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Physiological responses of Microcystis aeruginosa against the algicidal bacterium Pseudomonas aeruginosa Su Zhou a, Hua Yin a,n, Shaoyu Tang a, Hui Peng b, Donggao Yin a, Yixuan Yang a, Zehua Liu a, Zhi Dang a a Key Laboratory of Ministry of Education on Pollution Control and Ecosystem Restoration in Industry Clusters, School of Environment and Energy, South China University of Technology, Guangzhou 510006,Guangdong, China b Department of Chemistry, Jinan University, Guangzhou 510632, Guangdong, China

art ic l e i nf o

a b s t r a c t

Article history: Received 2 November 2015 Received in revised form 2 February 2016 Accepted 2 February 2016 Available online 8 February 2016

Proliferation of cyanobacteria in aquatic ecosystems has caused water security problems throughout the world. Our preliminary study has showed that Pseudomonas aeruginosa can inhibit the growth of cyanobacterium, Microcystis aeruginosa. In order to explore the inhibitory mechanism of P. aeruginosa on the cell growth and synthesis of intracellular substances of M. aeruginosa, concentrations of Chlorophyll-a, intracellular protein, carbohydrate, enzyme activities and ion metabolism of M. aeruginosa, were investigated. The results indicated that 83.84% algicidal efficiency of P. aeruginosa was achieved after treatment for 7 days. The strain inhibited the reproduction of M. aeruginosa by impeding the synthesis of intracellular protein and carbohydrate of cyanobacterium, and only a very small part of intracellular protein and carbohydrate was detected after exposure to P. aeruginosa for 5 days. P. aeruginosa caused the alteration of intracellular antioxidant enzyme activity of M. aeruginosa, such as catalase, peroxidase. The accumulation of malondialdehyde aggravated membrane injury after treatment for 3 days. P. aeruginosa also affected the ion metabolism of cyanobacteria. The release of Na þ and Cl  was significantly enhanced while the uptake of K þ , Ca2 þ , Mg2 þ , NO3  and SO42  decreased. Surface morphology and intracellular structure of cyanobacteria and bacterial cells changed dramatically over time as evidenced by electron microscope (SEM) and transmission electron microscope (TEM) analysis. These results revealed that the algicidal activity of P. aeruginosa was primarily due to the fermentation liquid of P. aeruginosa that impeded the synthesis of intracellular protein and carbohydrate, and damaged the cell membrane through membrane lipid peroxidation. & 2016 Elsevier Inc. All rights reserved.

Keywords: Algicidal mechanism Microcystis aeruginosa Pseudomonas aeruginosa Physiological responses Ion metabolism Microstructure

1. Introduction The occurrence of cyanobacterial blooms in many large freshwater lakes has been considered as a universal problem threatening the ecosystem and human health (Anderson, 2009; McLean, 2013). In particular, Microcystis aeruginosa, a preponderant alga in cyanobacterial blooms, has been considered to be a serious threat to human health and biological safety due to microcystin, a kind of poisonous metabolite secreted by M. aeruginosa (Pivokonsky et al., 2014; Zakaria et al., 2014). Extensive studies have revealed that microcystin was closely related to allergies, irritation reactions, gastroenteritis, liver diseases, and tumors (Dawson et al., 1998; Papageorgiou et al., 2004). In order to curb the bloom of M. aeruginosa and reduce the release of n

Corresponding author. E-mail address: [email protected] (H. Yin).

http://dx.doi.org/10.1016/j.ecoenv.2016.02.001 0147-6513/& 2016 Elsevier Inc. All rights reserved.

microcystin from the source, some control methods such as UV irradiation (Ou et al., 2012), ultrasonication (Rajasekhar et al., 2012), hydrogen peroxide (Matthijs et al., 2012) and allelochemicals (Shao et al., 2011) have been used, nevertheless, these techniques were not applauded for their drawbacks such as high price and potential risk of secondary pollution (Mohamed and Alamri, 2012). Microbial technology has been regarded as an effective and environmentally-friendly approach in alleviating the harmful impact of cyanobacterial blooms (Zakaria et al., 2014). To date, a number of algicidal bacteria have been isolated and their algicidal characteristics for controlling cyanobacterial blooms have been investigated (Manage et al., 2001; Feng et al., 2013). Algicidal bacteria inhibited the growth of cyanobacteria through direct and indirect attack (Berger et al., 2003; Wang et al., 2013). Direct attack refers to that algicidal bacteria adhere to or invade into the cyanobacterial cells directly. The cyanobacterial cells are decomposed further to death owing to the destruction of cellular

S. Zhou et al. / Ecotoxicology and Environmental Safety 127 (2016) 214–221

structure and some metabolic functions. Some studies have found that certain bacteria exhibited algicidal activity through excretion of extracellular algicidal compounds, such as protein (Wang et al., 2012), peptides (Park et al., 2011), amino acids (Chena et al., 2011), antibiotics, surfactant (Gustafsson et al., 2009), pigments, etc. (Mitsutani et al., 2001; Sakata et al., 2011), rather than direct contact. These materials intrude into the cells, stopping the growth and reproduction of cyanobacterial cells by intervening in their normal metabolism. During the inhibition process of cyanobacterial cells by algicidal bacteria, cyanobacteria showed a series of responses against the adverse condition, such as morphological change, migration, cyst formation, and production of bioactive compounds (Van Donk et al., 2011). Certain cyanobacteria have potential ability of forming cysts to defense algicidal bacteria (Mayali et al., 2008; Lin et al., 2014). As is well-known, some typical representatives of antioxidant enzymes existed in cells, such as catalase (CAT), an important intracellular enzyme which can decompose H2O2 into O2 and H2O, and peroxidase (POD), one of the endogenic scavengers for reactive oxygen (Shao et al., 2012). During the inhibition process, these enzyme activities of cyanobacterial cells increased markedly to alleviate the damage from external stress. Membrane lipid peroxidation of cyanobacterial cells indicated the degree of cell injury and the malondialdehyde (MDA), one of the most important by-product of the inhibition process, aggravated membrane injury of the cells (Hong et al., 2008). In addition, algicidal bacteria destroyed the cyanobacterial cells by interfering with their normal metabolism, such as suppressing the synthesis of intracellular protein and carbohydrate and disturbing ions metabolism pathways, etc. Although cyanobacteria degradation by algicidal bacteria has been generally investigated, few studies were available on the physiological responses of cyanobacteria in the presence of algicidal bacteria, and cyanobacteria suppression mechanism has not yet been thoroughly analyzed so far. Therefore, in this study, we chose Pseudomonas aeruginosa, a potential cyanobacterium degrading strain, as the algicidal bacterium, and M. aeruginosa as the model of harmful cyanobacteria to explore the cyanobacteria inhibitory mechanism. The physiological responses of M. aeruginosa, such as intracellular protein and carbohydrate, intracellular enzyme activity and ion metabolism were investigated. Also, surface morphology and intracellular structure of cyanobacteria and bacterial cells were observed by SEM and TEM analysis. We hope it is helpful to provide a better understanding of cyanobacteria inhibitory mechanism by algicidal cells.

2. Materials and methods 2.1. Strain and cultivation P. aeruginosa, a strain isolated from the contaminated sediment of Guiyu in Guangdong Province, China, was preserved in our laboratory (Shi et al., 2013), and was identified as having excellent algicidal activity on M. aeruginosa. It was inoculated into nutrient medium (g L  1): beef extracts (3), peptone (10), NaCl (5), pH 7.0– 7.2, at 30 °C with a shaking speed of 130 rpm for 24 h. Before adding into the degradation system, the strain was separated at 8000g for 5 min, and then was washed three times with sterile distilled water (Ye et al., 2014). The fermentation liquid was prepared by incubating the strain at 30 °C with a shaking speed of 130 rpm for 48 h, and was treated as follows before usage: the mixture was centrifuged at 8000g for 10 min, the supernatant thus obtained was filtered through a 0.22 μm cellulose acetate membrane to acquire the fermentation liquid. M. aeruginosa FACHB-905 was purchased from the Freshwater

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Algae Culture Collection at the Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China. Prior to using as inoculant, it was cultured in the sterilized BG11 medium for 15 days to reach the log phase (Zhou et al., 2014). The culture was incubated at 257 1 °C under constant 2000 lx white light: dark light ¼ 14 h: 10 h. 2.2. Test of algicidal activity of P. aeruginosa on M. aeruginosa Fermentation liquid with different dosages (0, 2%, 5% and 10%, v/v) was respectively added into 180 mL M. aeruginosa containing solution with OD680 of 0.6 (70.02), making sure that the total volume of the solution was 200 mL, and the insufficient section was supplied with corresponding amount of nutrient broth. The chlorophyll-a was extracted by grinding in dark with 100% acetone after centrifuging at 8000g for 5 min. Then the supernate was measured with ultraviolet–visible spectrophotometer at 750 nm, 663 nm, 645 nm and 630 nm, respectively. The concentration of chlorophyll-a was calculated as follows:

[11.64 × (D663 − D750) − 2.16 × (D645 − D750) Ca =

+ 0.1 × (D630 − D750)] × V1 V×δ

(1)

where Ca represents the concentration of chlorophyll-a (mg/L); V and V1 refer to the volume of sample and chlorophyll-a extracting solution (L), respectively; δ stands for the optical path of cuvette (cm); D750, D663, D645 and D630 are different light absorption values. Algicidal efficiency of P. aeruginosa was calculated by following equation:

Ae =

(C0 − C ) × 100% C0

(2)

where Ae represents the algicidal efficiency of P. aeruginosa on M. aeruginosa; C0 and C stand for the chlorophyll-a concentration of control and treatment groups, respectively. 2.3. Determination of intracellular protein and carbohydrate Cell disruption liquid preparation: 40 mL of M. aeruginosa solution was regularly sampled each day, then it was centrifuged at 6000g for 10 min at 4 °C. Deposits were washed twice with 0.1 M phosphate buffer saline (PBS, pH 7.0). The cells were disrupted by means of Ultrasonic Cell Disruption System (NingBo Scientz Biotechnology Co., Ltd., China) at 500 W for 10 min with interval time 5 s: 5 s to extract cell disruption liquid (Qian et al., 2012). Glucose (AR grade) was chosen to prepare glucose standard solution and bovine serum albumin was adopted as standard protein. Carbohydrates were detected by phenol–sulfuric acid colorimetry (Liu et al., 2011). Intracellular protein was determined by the following method: 3 mL coomassie brilliant blue chromogenic agent was injected into each tube, then 0.05 mL disruption liquid was transferred into the experimental group and 0.05 mL of 0.563 g L  1 standard solution was dropt to the standard group, and then corresponding volume of sterile distilled water was added into the control group. All the groups kept still for 10 min after shaking, then were determined at 595 nm by using ultraviolet– visible spectrophotometer. Protein concentration can be calculated by Eq. (3).

protein concentration (g L−1) = 0.563 (eOD595 − cOD595) /(sOD595 − cOD595)

(3)

where, e OD595, c OD595 and s OD595 represent the OD595 value in

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experiment, control and standard group, respectively.

2.4. Analyses of antioxidant enzyme system 2.4.1. Catalase (CAT) activity measurement In the reaction system, 1 mL 0.3% H2O2 (AR grade) and 1.95 mL H2O were contained, and 0.05 mL enzyme solution was added into the system. The decrease rate of OD240 was measured through spectrophotometer. Decreasing 0.01 per min at OD240 was defined as a unit of energy. CAT activity was represented by U mg  1 protein (Qian et al. 2009). 2.4.2. Peroxidase (POD) activity measurement In the reaction system, 1 mL 0.3% H2O2 (AR grade), 0.95 mL 0.2% guaiacol (AR grade) and 1 mL PBS (pH 7.0) were contained, and 0.05 mL enzyme solution was added into the system. The increase rate of OD470 was measured by using ultraviolet–visible spectrophotometer, and increasing 0.01 per min at OD470 was defined as a unit of energy. POD activity was expressed by U mg  1 protein. 2.4.3. Malondialdehyde content measurement MDA was measured by means of thiobarbituric acid method (Dogru et al. 2008).

2.5. Ion metabolism analysis The cyanobacterial samples were harvested periodically by centrifugating at 6000g for 10 min. Ions (K þ , Na þ , Ca2 þ , Mg2 þ , Cl  , NO3  and SO42  ) in the solution were analyzed by ion chromatograph (ICS-900 America). Analytical column for anion was Dionex IonPacs AS15 (dimensions 2 mm  250 mm), using 30 mmol L  1 of NaOH (GR grade) as moving phase with a flow rate of 0.25 mL min-1, the injection volume was 25 μL. Analytical column for cation was Dionex IonPas CS12A (dimensions 4 mm  250 mm), using 1.6 mmol L  1 of H2SO4 (GR grade) as moving phase with a flow rate of 1.0 mL min  1, the injection volume was 20 μL.

Fig. 1. Changes and reduction rate of chlorophyll-a in M. aeruginosa containing system treated with different constituent of P. aeruginosa. (a) Changes of Chlorophyll-a in M. aeruginosa containing system over time; (b) reduction rate of chlorophyll-a in M. aeruginosa containing system over time. * Represents a statistically significant difference of p o 0.05 when compared to the control and ** stands for a statistically significant difference of po 0.01.

3. Results and discussion 3.1. Inhibitory effect of P. aeruginosa on the growth of M. aeruginosa

2.6. SEM and TEM observation The cells were harvested by centrifugation at 6000g for 10 min, then were dehydrated by different concentration of alcohol successively after fixing with 2.5% glutaraldehyde for 24 h. Isoamyl acetate was used to drench into the cells for at least 30 min. After drying naturally, they were treated by spray gold under vacuum condition and then were observed through SEM (MERLIN Compact, Germany). The cells were pre-fixed at 4 °C refrigerator overnight by using 4% glutaraldehyde, then the cells were post-fixed in 1% osmium tetroxide for 1–2 h. After that, they were dehydrated by means of ethanol, and were covered using embedding medium. Finally the samples were cut into ultra-thin slices for transmission electron observation (TEM, FEI Tecnai 12, Netherlands).

2.7. Statistical analysis SPSS 17.0 was used for statistical analysis of the results. All of the experiments were performed in triplicate, and the mean values were used in the calculations. The standard deviations for all measurements ranged from 0.5% to 8.0%.

As shown in Fig. 1, the inhibition effect of fermentation liquid (5%, 10%, v/v) on the growth of M. aeruginosa was comparable to that of intact P. aeruginosa cells, indicating that the inhibition effect of P. aeruginosa was mainly through secretion of extracellular active substances. The concentration of chlorophyll-a in control increased from 0.437 0.021 mg L  1 to 0.96 70.024 mg L  1 after treatment for 7 days. The growth of M. aeruginosa in sample group was notably inferior to that in control after treatment for 3 days. Fig. 1a showed that the concentration of chlorophyll-a in M. aeruginosa containing system decreased to 0.497 0.012, 0.35 70.029 and 0.34 70.015 mg L  1, respectively on the 3rd day when adding different dosage of fermentation liquid (2%, 5%, 10%, v/v), and the value decreased to 0.30 70.015 mg L  1 when adding intact P. aeruginosa cells (1 g L  1). The algicidal effect of both fermentation liquid and P. aeruginosa cells reached stable level after the 5th day. Fig. 1b showed that algicidal efficiency with different dosages of fermentation liquid achieved 41.27% (2%, v/v), 81.21% (5%, v/v) and 83.84% (10%, v/v), respectively after 7 days. These results inferred that P. aeruginosa had great potential for cyanobacteria degradation, and it was clear that some dose-effect relation existed when P. aeruginosa attacked M. aeruginosa. The algicidal efficiency with higher amount (5% and 10%, v/v) of sterile fermentation liquid and

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Fig. 2. Effect of sterile fermentation liquid of P. aeruginosa on intracellular protein and carbohydrate with reaction time. (a) The changing curve of intracellular protein concentration with reaction time; (b) the changing curve of intracellular carbohydrate content with reaction time. * Represents a statistically significant difference of p o0.05 when compared to the control and ** stands for a statistically significant difference of p o0.01.

P. aeruginosa cells (1 g L  1) were significantly superior to that with lower dosage (2%, v/v). 3.2. Effect of P. aeruginosa on the intracellular protein and carbohydrate content of M. aeruginosa As shown in Fig. 2a and b, the intracellular protein and carbohydrate content did not change much at the beginning, however, they dropped dramatically after treatment for 3 days, and tended to be stable after 5 days. This phenomenon might be attributed to the physical and chemical disruption of cyanobacterial function by a certain kind of component in the fermentation liquid (Henderson et al., 2008). On one hand, the fermentation liquid inhibited the synthesis of intracellular protein and carbohydrate, on the other hand, it also led to the outflow of intracellular protein and carbohydrate through cell membrane destruction. Due to the significant degradation of cyanobacteria after 5 days, only a very small part of intracellular protein and carbohydrate could be detected. 3.3. Effect of P. aeruginosa on antioxidant enzyme activity and MDA content of M. aeruginosa The changes of cyanobacterial antioxidant enzyme activity under the stress of P. aeruginosa were shown in Fig. 3. Antioxidant

Fig. 3. Effect of fermentation liquid of P. aeruginosa on antioxidant enzyme activity of M. aeruginosa. (a) Changes of catalase activity; (b) Changes of peroxidase activity; (c) changes of malondialdehyde content. * Represents a statistically significant difference of p o 0.05 when compared to the control and ** stands for a statistically significant difference of p o 0.01.

enzyme activity of the control group kept a lower level all the time, while that in the treatment groups changed significantly over time. As illustrated in Fig. 3a and b, both CAT and POD activity rose quickly on the 1st day, and up to the highest on the 3rd day in the case of adding 5% and 10% (v/v) of fermentation liquid. CAT increased by 4.7 times and 4.6 times on the 3rd day respectively with 5% and 10% (v/v) fermentation liquid addition, compared to the control. Then the CAT activity started to decrease and was even lower than the control group on the 7th day. As for POD activity, it showed a same trend as CAT activity. Fig. 3c demonstrated that when cyanobacterial cells were exposed to the fermentation liquid

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a3 2

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Fig. 4. Effect of fermentation liquid of P. aeruginosa on intracellular ion metabolism of M. aeruginosa. (a) K þ concentration; (b) Na þ concentration; (c) Ca2 þ concentration; (d) Mg2 þ concentration; (e) Cl  concentration; (f) SO42  concentration; (g) NO3  concentration.

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of P. aeruginosa, significant increase of intracellular MDA content was observed during the first five days, then MDA content tended to be stable after the 5th day. Meanwhile, there was an obvious dose-response relationship between fermented liquid and MDA content. As is well-known, the rapid increase of MDA content indicates the aggravation of cyanobacterial membrane lipid peroxidation and reflects the severity of cell membrane damage (Wang et al., 2011). These results revealed that the activity of cyanobacterial antioxidant enzymes strengthened immediately to defense the damage from P. aeruginosa. The antioxidant enzyme activity decreased after the 3rd day due to the toxicant accumulation, such as the accumulation of MDA, which has already proven to be a kind of toxic substance to destroy the cytomembrane system and pigment synthesis of cells (Qian et al., 2008). 3.4. Effect of P. aeruginosa on the intracellular ion metabolism of cyanobacterial cells Cell membrane of cyanobacteria can selectively transfer extracellular K þ , Ca2 þ , Mg2 þ , SO42  and NO3  into cell and release Na þ and Cl  outside due to its ion permselectivity. Studies have showed that algicidal bacteria can affect the ion metabolism of normal cyanobacterial cells through changing the structure and permeability of cyanobacterial membrane (Li and Hu, 2005). Fig. 4 showed that cyanobacterial cells absorbed K þ , Ca2 þ , Mg2 þ , SO42  and NO3  as it released Na þ and Cl  at the same time. Relatively speaking, low level of fermentation liquid (2%, v/v) had less effect on ion metabolism in comparison with high dosage. The release of Na þ and Cl  was significantly enhanced with increasing fermentation liquid dosage (5% and 10%), especially when the system was treated for 5 days, it was mainly due to the fact that intracellular

a

219

Na þ and Cl  outflowed seriously along with the destruction and death of cyanobacterial cells. Also, the uptake of K þ , Ca2 þ , Mg2 þ , NO3  and SO42  decreased gradually, among which the uptake of SO42  turned to release after 4 day treatment, and that of K þ and Ca2 þ changed to release after treatment for 5 days. As a whole, the changes of these typical ions metabolism revealed that P. aeruginosa altered the cell membrane permeability or disturbed ions metabolic pathways of M. aeruginosa cells, thus affecting intracellular ion metabolism. After 5 days, intracellular ions leaked seriously as a result of cell wall and membrane rupture. It was also found that there existed a dose-effect relationship between fermentation liquid and the ion metabolism, and the degree of the effect depended on the ions species. 3.5. Observation of cellular surface morphology and intracellular structure The surface morphology and intracellular structure of microbial cells were prone to change or even devastation when exposed to the toxic substances (Bruins et al., 2000). In order to further study the physiological defensive responses of cyanobacteria against P. aeruginosa, SEM and TEM analyses were used and the results were exhibited in Figs. 5 and 6. It was demonstrated in Fig. 5a that the surface of cyanobacterial cells was elliptical, full and smooth. The morphology characterized by TEM was in line with that imaged by SEM. The TEM observation exhibited that M. aeruginosa cells were intact; cell wall and membrane combined closely; and important organelle like thylakoids, ribosome and polyphosphates particles were distributed homogeneously in cytoplasm (Fig. 6a). As shown in Fig. 5b, there was no significant change on the cell shape after treatment for 1 day, except a number of P. aeruginosa cells adhered

b Algal cell

Bacteria

c

d

Fig. 5. SEM images of M. aeruginosa and P. aeruginosa in the symbiotic system. (a) Control; (b) co-incubation for 1 day; (c) co-incubation for 3 days; (d) co-incubation for 5 days.

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a

b

THY RIB

PP CM CW

c

d

PlA

Fig. 6. TEM images of M. aeruginosa and P. aeruginosa in the symbiotic system. (a) control; (b) co-incubation for 1 day; (c) co-incubation for 3 days; (d) co-incubation for 5 days; CW, cell wall; CM, cell membrane; THY, thylakoid; RIB, ribosome; PP, polyphosphates particle; PIA, plasmolysis.

to the cyanobacterial cells. Some studies have indicated that certain kind of algicidal bacteria was prepared for degradation by secreting mucus to cyanobacterial cells to build the bridge between cyanobacteria and algicidal bacteria (Costerton and Irwin, 1981; Panova and Ivanova, 2000). Algicidal active substances might have the function of inducing cyanobacterial cells to move close to algicidal bacteria (Bowman, 2007). Fig. 6b indicated that plasmolysis began to appear in M. aeruginosa cells under the stress of P. aeruginosa (Liao et al., 2015). At the same time, internal structure started to damage. Thylakoids turned sparsely and irregularly, but the shape of cyanobacterial cells did not change significantly (Tan et al., 2015). Simultaneously, there was an increase in population of P. aeruginosa cells as it utilized the cyanobacterial cells or certain component of cyanobacteria as carbon source for growth. After treatment for 3 days, remarkable changes for both cyanobacteria and algicidal bacteria cells could be observed (Figs. 5c and 6c). Obvious wrinkle occurred in cyanobacterial cells, and cell membrane of cyanobacteria began to be broken down. The internal structure of cyanobacterial cells was also seriously affected. The photosynthetic pigments which attached to thylakoids were disrupted with the impairment of thylakoids.

Meanwhile, P. aeruginosa cells also shrank, and produced numerous vacuoles within the cytoplasm. The most evident variation of the cells was obtained on the 5th day. Figs. 5d and 6d suggested that after 5 day treatment, thylakoids and polyphosphates particles were fully biodegraded, and the cyanobacterial cells were thoroughly decomposed, leading to the release of intracellular substances, such as ions, proteins and carbohydrates, which agreed with the previous demonstration in this paper. As for P. aeruginosa cells, it was noteworthy that they gathered into a mass along with surface corrugation and cell invagination. After a prolonged treatment time, the cell membrane of P. aeruginosa was broken, as a result, the intracellular substances severely run off (Figs. 5d and 6d).

4. Conclusions The most obvious damage of M. aeruginosa under the stress of P. aeruginosa was obtained at the dosage of fermentation liquid exceeded 5% (v/v). Exposure to high dosage of fermentation liquid led to abnormal metabolism of the cyanobacteria, including the

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suppression of synthesis of intracellular protein and carbohydrate and the disturbances of ion metabolism. Though intracellular CAT and POD increased immediately to relieve the damage from fermentation liquid of P. aeruginosa, cell membrane was still decomposed with over accumulation of MDA. These results, along with the cell morphology and intracellular structure observation, inferred that oxidative damage and membrane destruction might be the primary algicidal mechanism of P. aeruginosa on M. aeruginosa cells.

Acknowledgments The authors would like to thank the National Natural Science Foundation of China (Nos. U1501234, U0933002 and 41330639), the Natural Science Foundation of Guangdong Province, China (S2013020012808) and the Fundamental Research Funds for the Central Universities (No. D2153610) for the financial support of this study.

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