The first evidence of deinoxanthin from Deinococcus sp. Y35 with strong algicidal effect on the toxic dinoflagellate Alexandrium tamarense

Journal of Hazardous Materials 290 (2015) 87–95

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The first evidence of deinoxanthin from Deinococcus sp. Y35 with strong algicidal effect on the toxic dinoflagellate Alexandrium tamarense Yi Li a,1 , Hong Zhu a,1 , Xueqian Lei a , Huajun Zhang a , Chengwei Guan a , Zhangran Chen a , Wei Zheng a , Hong Xu a , Yun Tian a , Zhiming Yu b,∗ , Tianling Zheng a,∗ a State Key Laboratory of Marine Environmental Science and Key Laboratory of MOE for Coast and Wetland Ecosystems, School of Life Sciences, Xiamen University, Xiamen 361005, China b Key Laboratory of Marine Ecology and Environmental Science, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China

h i g h l i g h t s • • • • •

A potential active bioresource was isolated and purified from Deinococcus. Deinoxanthin was firstly determined as novel algicide to A. tamarense. Unique structure and characteristics of deinoxanthin induced ROS production. Overproduction of ROS destroyed cell integrity and damaged algal cells. Efficient and eco-environmental algicidal compound was used to HABs-control.

a r t i c l e

i n f o

Article history: Received 6 September 2014 Received in revised form 24 February 2015 Accepted 25 February 2015 Available online 27 February 2015 Keywords: Algicidal bacteria Novel algicide Deinoxanthin Alexandrium tamarense Reactive oxygen species

a b s t r a c t Harmful algal blooms (HABs) could be deemed hazardous materials in aquatic environment. Alexandrium tamarense is a toxic HAB causing alga, which causes serious economic losses and health problems. In this study, the bacterium Deinococcus xianganensis Y35 produced a new algicide, showing a high algicidal effect on A. tamarense. The algicidal compound was identified as deinoxanthin, a red pigment, based on high resolution mass spectrometry and NMR after the active compound was isolated and purified. Deinoxanthin exhibited an obvious inhibitory effect on algal growth, and showed algicidal activity against A. tamarense with an EC50 of 5.636 ␮g/mL with 12 h treatment time. Based on the unique structure and characteristics of deinoxanthin, the content of reactive oxygen species (ROS) increased after 0.5 h exposure, the structure of organelles including chloroplasts and mitochondria were seriously damaged. All these results firstly confirmed that deinoxanthin as the efficient and eco-environmental algicidal compound has potential to be used for controlling harmful algal blooms through overproduction of ROS. © 2015 Elsevier B.V. All rights reserved.

1. Introduction

∗ Corresponding authors at: Room 316, Huang Chaoyang Building, Xiang’an Campus, Xiamen University, Xiamen, Fujian, China. Tel.: +86 592 2183217; fax: +86 592 2184528. E-mail addresses: [email protected] (Y. Li), [email protected] (H. Zhu), [email protected] (X. Lei), [email protected] (H. Zhang), [email protected] (C. Guan), [email protected] (Z. Chen), [email protected] (W. Zheng), [email protected] (H. Xu), [email protected] (Y. Tian), [email protected] (Z. Yu), [email protected] (T. Zheng). 1 These two authors contributed equally to the work. http://dx.doi.org/10.1016/j.jhazmat.2015.02.070 0304-3894/© 2015 Elsevier B.V. All rights reserved.

Marine algae are the main component of marine ecosystems and, as an important primary producer, they supply food and energy for other marine organisms [1]. However, overgrowths of algae is harmful to the stability of marine ecosystems and the food chain cycle [2], and we call this phenomenon “harmful algal blooms (HABs)” [3]. HABs have become more severe and outbreak more frequently in recent years following global climate change [4] and increased marine pollution [5,6] . A particular problem is the HABs caused by blooms of toxic algae, where the toxin passes to other marine organisms, and brings about a large number of death, and even threatens human security [7,8]. Alexandrium tamarense is a

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notorious bloom-forming dinoflagellate [9], and can produce paralytic shellfish poison toxin during its growth [10], which can pass through the food chain, creating great economic losses and health risks [11,12]. In order to solve the problems of HABs, many methods have been implemented, such as aminoclay [13], chemicals [14], UV irradiation [15], and microwaves [16]. Although these methods can take effect on HABs, their high cost and the secondary damage make them impractical and infeasible for HABs control [17,18]. Therefore, biological agents, including plants [19], protozoan [20], viruses [21] and bacteria [22,23] are considered as effective and biosecure ways to mitigate HABs, and the relationship between bacteria and algae has received particular interest in recent years [24–26]. Some bacteria supply cobalamin for algal growth [27], and get nutrients from algae, other bacteria compete with algae for nutrients [28], and some bacteria even produce secondary metabolites with a diverse range of algicidal activities to inhibit or kill algal cells [29]. Therefore, we need to isolate the bacteria with algicidal effect on specific algae, in order to achieve the purpose of effectively solving the problems of HABs. Most of the algicidal bacteria isolated from natural environments to date have been confirmed to show algicidal activity in an indirect way by producing an algicidal compound without contacting the algae. So far, many algicidal bacteria have been isolated from the environment and identified [30–32], but little is known concerning the algicidal compounds which are produced. There are not enough reports about the isolation and identification of these bioactive compounds, and the known algicidal compounds which could be used to control HABs are still very scarce. In this study, a bacterial strain with algicidal effects on A. tamarense was isolated from Xiang’an Lake, Fujian Province of China. This bacterium was identified as Deinococcus xianganensis, and the optimum temperature, salinity, pH and enzyme activity and carbon utilization of the strain were investigated to optimize bacterial growth. The algicidal compound was purified after extraction, and its chemical structure was further defined using high resolution mass spectrometer (Q-Exactive) and nuclear magnetic resonance (NMR). We also studied the algicidal activity of the compound, and observed the procedure involved in algal cell lysis. 2. Materials and methods 2.1. Algal cultures A. tamarense ATGD98-006 was provided by the Algal Culture Collection, Institute of Hydrobiology, Jinan University, Guangzhou, China. All cultures were maintained in f/2 medium [33] (prepared with 0.45 ␮m of filtered seawater) at 20 ± 1 ◦ C under a 12-h light/12-h dark cycle with a light intensity of 50 ␮mol photons m−2 s−1 . 2.2. Isolation of algicidal strain Water samples from 0 to 0.5 m in Xiang’an Lake were serially diluted (10-fold) using sterile distilled water, and 0.1 mL aliquots of each dilution were spread onto Luria–Bertani agar medium (LB; 10 g of Tryptone, 5 g of yeast extract, 10 g of NaCl in 1 L of 0.45 ␮m Millipore-filtered distilled water, pH 7.2–7.6) followed by incubation for 7 days at 28 ◦ C. Individual colonies of distinct morphology were further purified three times and stored at −80 ◦ C in LB medium supplemented with 10% (v/v) glycerol. In order to isolate the bacterial strains with algicidal effects on A. tamarense, a 0.4 mL aliquot of each isolate, was inoculated in triplicate into 20 mL of logarithmic-phase A. tamarense cultures, and a 0.4 mL aliquot of LB medium only was added to algal

cultures as a control. To test for algicidal activity, A. tamarense was inoculated into a 24-well cell plate, and the fluorescence intensity (RFU) of algae in each well was measured at an excitation wavelength of 440 nm and emission wavelength of 680 nm (Spectra max M2, Molecular Devices Corporation) and the result treated as algal biomass [34]. One red colored strain was isolated, which showed high algicidal activity, and this strain was designated Y35. 2.3. Identification of algicidal strain Genomic DNA was extracted based on Ausubel et al. [35], the 16S rRNA gene sequence was amplified using PCR with primers 27F and 1492R [36]. Purification of the PCR product was carried out following the protocol of the TIANquick midi purification kit (TIANGEN, China). The purified DNA was cloned into vector pMD19-T and sequenced. Sequences of related taxa were obtained from the GenBank database and EzTaxon-e server (http://www.ezbiocloud.net/eztaxon) [37]. Phylogenetic analysis was performed using MEGA version 4 [38] after multiple alignment of data by DNAMAN (version 5.1). Evolutionary distances and clustering were constructed using the neighbor-joining method [39], and were evaluated using bootstrap values based on 1000 replications. Cell morphology was observed using transmission electron microscopy (TEM, model JEM-2100HC; JEOL), with cells from LB agar medium at 37 ◦ C for 24 h. Samples were handled based on Dubey and Ben-Yehuda [40]. Colony morphology, size and color were examined from cultures on LB agar medium for 2 days. The Gram reaction was determined using the bioMérieux Gram stain kit following the manufacturer’s instructions. Anaerobic growth, hydrolysis of starch, gelatin, casein, urea and Tweens 20, 40, the optimal growth temperature and pH, tolerance to NaCl were studied as previously described [41]. Further phenotypic and enzymatic characterizations of strain Y35 were conducted using the API 20NE test kit (bioMérieux) at 28 ◦ C for 2 days, and the API ZYM test kit (bioMérieux) at 28 ◦ C for 24 h and the API 20 E test kit (bioMérieux) at 37 ◦ C for 24 h following the manufacturer’s instructions. All the commercial kits were inoculated with bacterial suspensions in 0.9% (w/v) NaCl. 2.4. Extraction of algicidal compound Before extraction of the algicidal compound from D. xianganensis strain Y35, bacterial cells were inoculated into 2 L LB medium, and cultured for 2 days at 28 ◦ C. The culture broth was centrifuged at 12,000 g for 10 min, and the pellet was resuspended with ethanol and ultrasonic oscillations for 2 h, the crude extracts were dissolved into ethanol. Then the ethanol was dried under reduced pressure in an evaporator at 30 ◦ C after centrifuged at 12,000 × g for 10 min. The crude extracts were extracted again from the concentrate by extraction three times with ethyl acetate, followed by concentration and drying under reduced pressure in an evaporator at 30 ◦ C. The crude extracts dissolved in ethyl acetate were applied to a Sephadex LH-20 (Amersham Biosciences) column with 100% methanol as eluent. Active fractions were subjected to silica gel column chromatography (170 × 30 mm in dimension and with a silica particle size of 200–300 mesh) and eluted with 50 volume ratios of dichloromethane/methanol (30:1, 20:1, and 15:1). All of the fractions were collected separately based on analysis of thin layer chromatography (TLC, GF254, pH: 6.2–6.8, Qingdao, China) and then dried and weighed. Algicidal bioassays of each fraction were determined in 24 well plates as described above. The remainder of each fraction was stored in the dark at room temperature for further analysis. A flow diagram showing the full procedure is in Fig. S4.

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The algicidal fraction was analyzed by means of high performance liquid chromatography (HPLC) and, to ensure the purity of the algicidal compound, fitted with a SunFireTm C18 (4.6 × 250 mm, 5 ␮m) column using a methanol: acetonitrile: isopropanol 5:4:1 (v/v) mixture as the eluent at a flow rate of 1 mL min−1 with 480 nm as the detection wavelength.

2.5. Identification of compounds in the active fractions The certificated purity of the algicidal fraction was analyzed by Q-Exactive in order to determine the molecular weight of the algicidal compound. The purified active fraction was dissolved in acetonitrile (HPLC grade) and carbon nanotubes were used as the assistant matrix. The mass spectra were then achieved using a Q-Exactive/Desktop FT Orbitrap mass spectrometer (Thermo Fisher Scientific, Germany). Proton nuclear magnetic resonance (NMR) spectra of the purified algicidal compound were recorded in CD3 OD using a DRX500 instrument (Bruker Biospin, Co., Karlsruhe, Germany) at 25 ◦ C, and tetramethylsilane (TMS) as the internal standard.

2.6. The algicidal activity of the algicidal compound The algicidal compound which was isolated as described above was dissolved in a non-reacting solvent, dimethyl sulfoxide (DMSO). The algicidal effect of the compound against A. tamarense was investigated using a series of concentrations (1, 5, 10, 20, 30, 50 and 70 ␮g/mL), which were inoculated into 100 mL flasks containing 20 mL A. tamarense cultures. The A. tamarense cells treated with a corresponding concentration of the DMSO were used as a control. There were three replicates for each concentration and control group. All flasks were cultivated at 20 ± 1 ◦ C under a 12-h light/12h dark cycle with a light intensity of 50 ␮mol photons m−2 s−1 . The fluorescence intensity (RFU) of algae was measured as described above.

2.7. Determination of ROS levels Intracellular ROS were detected using a fluorescent probe, 2 , 7 dichlorofluorescin diacetate (DCFH-DA), based on Choudhury et al. [42], with slight modifications. The final DCFH-DA concentration in the mixture was 10 ␮M and this was incubated with the suspended cells at 37 ◦ C in the dark for 30 min and mixed every 5 min during this time. Then the cells were immediately washed three times with sterile f/2 medium (without silicate) and finally suspended with 500 ␮L f/2 medium. The fluorescence intensity was monitored using a spectrofluorometer with excitation wavelength at 488 nm and emission wavelength at 525 nm.

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2.9. Assay of the effect of deinoxanthin on other algal species To check whether deinoxanthin take effect on other algae in aquatic environment, we tested the algicidal effect on 23 different algal species, which included typical HABs species and beneficial algae: Alexandrium catenella, Alexandrium minutum, Alexandrium tamarense, Prorocentrum donghaiense, Akashiwo sanguinea, Scrippsiella trochoidea, Phaeodactylum tricornutum, Chaetoceros compressus, Thalassiosira pseudonana, Pmphiprora alata, Thalassiosira weissflogii, Skeletonema costatum, Heterosigma akashiwo, Chattonella marina, Phaeocystis globosa, Dicrateria inornata, Isochrysis galbana, Platymonas subcordiformis, Chlorella autotrophica, Platymonas helgolandica, Prasinophyceae, (provided by College of Ocean and Earth Sciences, Xiamen University, Xiamen, China); Cyanobacteria, Dunaliella salina (provided by Professor Yahui Gao Xiamen University, Xiamen, China), adding deinoxanthin into algal culture with the concentration of 10 ␮g/mL. 2.10. Statistics All data were presented as means ± standard error of the mean and were evaluated using one-way analysis of variance followed by the least significant difference test, with p < 0.01 and p < 0.05 (Origin 8.5 for Windows). 3. Results 3.1. Characterization and identification of strain Y35 A total of 43 bacterial strains were isolated in water samples from Xiang’an Lake. Among these, strain Y35 exhibited obvious algicidal activity against A. tamarense, and the strain was deposited in the Marine Culture Collection of China, with accession number MCCC 1F01224. The bacterial cells are Gram-stain-negative, coccoid-shaped (0.6–1.0 ␮m in diameter and 0.8–1.4 ␮m in length) without flagellum (Fig. S1). Colonies on LB agar medium are shiny, red and circular with regular, smooth edges and are 1–2 mm in diameter after 48 h incubation at 37 ◦ C. Growth occurs at 4–39 ◦ C, with an optimum at 28–37 ◦ C; in NaCl concentrations of 0–3% (w/v), with optimal growth at 0–1; at pH 6–8, with optimal growth at 7. Growth does not occur under anaerobic conditions on LB. Other phenotypic characteristics are given in Table S1. A nearly full-length 16S rRNA gene sequence (1424 nt) of strain Y35 was determined. Phylogenetic analysis of the strain Y35 based on the 16S rRNA gene sequence indicated that this strain belonged to the genus Deinococcus (Fig. S2). 16S rRNA gene sequence analysis revealed that strain Y35 (GenBank accession number KJ639011) shared highest similarity (98.95%) with Deinococcus grandis DSM 3963 (Y11329). 3.2. Extraction and purification of algicidal compound

2.8. Algal cells lysis procedure after treatment with the algicidal compound A. tamarense was incubated for 24 h in the presence of the algicidal compound at a final concentration of 10 ␮g/mL, and A. tamarense with the same volume of DMSO was used as a control. Samples were fixed overnight at 4 ◦ C in 0.1 M PBS (8 g NaCl, 0.2 g KCl, 1.44 g Na2 HPO4 , 0.24 g KH2 PO4 , 1 L distilled water, 50 mM, pH 7.4) containing 2.5% glutaraldehyde (v/v) and then postfixed in 1% OsO4 in the same buffer for 2 h. After being washed twice with PBS, samples were embedded in araldite resin. Sections (60–80 nm), obtained with an ultramicrotome, were stained in 3% acetic acid uranium–citric acid and viewed using TEM (model JEM-2100HC; JEOL).

Algicidal activity against A. tamarense was almost not found in the supernatant after centrifugation, but the algicidal activity was high in the ethanol extraction from the bacterial cells (Fig. S3). After the crude extracts dissolved in ethyl acetate were applied to a Sephadex LH-20 column, three fractions were isolated based on their different colors, and the red fraction (R) was found to have algicidal effect (Fig. S4). Fraction R was then further purified using silica gel column chromatography, and four fractions (R1–R4) were found in the effluent of the chromatographic column (Fig. 1A), among of them R1, which showed an algicidal effect (data not shown). To certify the purity of R1, the R1 fraction was determined using HPLC, and HPLC separation showed the elution of the compound as a single separate peak (Fig. 1B). Separation of

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Fig. 1. TLC separation of the pure compound showing a single spot (A) using methanol/dichloromethane (1:20) as mobile phase and HPLC separation of the pure compound showing a single prominent peak indicates the purity of the compound (B). In (A), 1 represents crude extracts; 2 represents pure compound.

the compound into a single prominent peak indicated the purity and presence of a single compound in the R1 fraction.

3.3. Identification of the algicidal fraction R1 The electro-spray ionization (ESI) mass spectrum of R1 displayed a molecular ion at 583.4126 uma (M + H)+ (Fig. 2), and the molecular formula was determined to be C40 H54 O3 based on the Q-Exactive database. This showed that the molecular weight of the R1 fraction was approximately 582. The NMR data (600 MHz, ı ppm from TMS in CD3 OD) for R1 were as follows: ı = 3.84 (dd, J = 4.2, 9.0 Hz, 1H), 2.62 (dd, J = 9.0, 16.9 Hz, 1H), 2.77 (dd, J = 4.2, 16.9 Hz, 1H), 6.27 (d, J = 16.1 Hz, 1H), 6.47 (d, J = 16.1 Hz, 1H), 6.38 (m, 1H), 6.68 (m, 1H), 6.53 (d, J = 14.9 Hz, 1H), 6.36 (m, 1H), 6.77 (m, 1H), 1.21 (s, 3H), 1.27 (s, 3H), 1.87 (s, 3H), 2.00 (s, 3H), 2.00 (s, 3H), 2.33 (d, J = 7.4 Hz, 1H), 5.86 (dt, J = 7.4, 15.4 Hz, 1H), 6.21 (d, J = 15.4 Hz, 1H), 6.15 (d, J = 11.0 Hz, 1H), 6.65 (dd, J = 11.0, 14.8 Hz, 1H), 6.40 (d, J = 14.8 Hz, 1H), 6.33 (d, J = 10.3 Hz, 1H), 6.73 (m, 1H), 6.45 (d, J = 14.7 Hz, 1H), 6.35 (m, 1H), 6.77 (m, 1H), 1.22 (s, 3H), 1.21 (s, 3H), 1.95 (s, 3H), 2.02 (s, 3H), 2.05 (s, 3H). The 1 H chemical shift values of R1 were consistent with those reported by Lemee et al. [51] for deinoxanthin, and the two compounds had the same molecular weight. After comparing the UV-spectrum, chemical structure and molecular formula of the two compounds, the results showed that the R1 from D. xianganensis Y35 was the same as the deinoxanthin which was isolated from Deinococcus radiodurans DSM 20,539, and this illustrated that the algicidal compound from strain Y35 was deinoxanthin with the structure shown in Fig. 3.

3.4. The algicidal activity of deinoxanthin A. tamarense growth was significantly inhibited by deinoxanthin at all the concentrations tested, except 1 ␮g/mL, and the inhibitory effects increased with the increasing concentration of the deinoxanthin (Fig. 4). The concentration of 1 ␮g/mL promoted algal growth, but the inhibitory effects could be observed after the concentration increased to 5 ␮g/mL. The fluorescence density in the high concentrations treatment groups (30, 50 and 70 ␮g/mL) decreased obviously compared to the control group in the 2 h exposure time, however the low concentration groups (5, 10 and 20 ␮g/mL) only showed algicidal activity after the 6 h exposure time (Fig. 4). Within the 6 h exposure time, almost half the algal cells lost their fluorescence density in the high concentration treatment groups, which suggested that half of the algae were dead under the effect of deinoxanthin. Within 12 h exposure time, almost half of the algal cells lost their fluorescence density in low concentrations treatment groups and, at the same time, the algicidal rate reached 78.8%, 75.9% and 88.1% with concentrations of 30, 50 and 70 ␮g/mL. From this result, we concluded that deinoxanthin significantly inhibited the growth of A. tamarense with an EC50 of 5.636 ␮g/mL with 12 h treatment time. 3.5. ROS contents in A. tamarense Investigating the change of ROS contents was important to know the degree of damage the algicidal substance had on algal cells. The ROS content changed constantly along with the effect of deinoxanthin (10 ␮g/mL) (Fig. 5). After treatment for 0.5 h, the DCF

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Fig. 2. Mass spectrum of the R1 fraction obtained from high resolution mass spectrometry.

Fig. 3. Structure of deinoxanthin.

fluorescence was significantly increased compared to control until to 1 h exposure (p < 0.01). Within 1 h exposure, the DCF fluorescence reached its highest level (p < 0.01). However, the ROS content began to decrease to a very low level from 6 h exposure. The fluorescence intensity of ROS in the control kept low level during the whole exposure time (data not shown). 3.6. Lysis procedure of A. tamarense under the effect of deinoxanthin The TEM analysis revealed alterations in the ultrastructure of A. tamarense under the effects of deinoxanthin, with cells losing the integrity of their organelles (Fig. 6). As shown in Fig. 6a (the control group), the cell wall and plasma membrane of A. tamarense

were intact, and the intact chloroplasts and mitochondria in the cells and the cytoplasm were dense. After 6 h treatment with deinoxanthin, the structure and morphology of A. tamarense cells were changed, the fringe of organelles including chloroplasts and mitochondria became fuzzy, the distinct plasmolysis occurred in algal cells (Fig. 6b). The damage was more severe when the exposure time was prolonged to 12 h, both the cell wall and plasma membrane broke up, at this time, the cytoplasm and organelles spilled out of the cells, and a large number of vacuoles appeared (Fig. 6c). In order to study more closely the change of organelles in A. tamarense cells, their ultrastructure was examined (Fig. 6d–h). The number of lysosomes significantly increased after treatment (Fig. 6d). As shown in Fig. 6e–h, the structure and morphology of the chloroplasts and mitochondria changed obviously compared

Fig. 4. Algicidal effects of different concentrations of deinoxanthin on A. tamarense.

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Fig. 5. Effects of deinoxanthin (10 ␮g/mL) on ROS contents in A. tamarense. All error bars indicate the SE of the three biological replicates. * represents a statistically significant difference of p < 0.05 when compared to the control; ** represent a statistically significant difference of p < 0.01. Table 1 Effects of deinoxanthin on other algal species.

Pyrrophyta

Cyanophyta Diatoms

Xanthophyta Chrysophyta

Chlorophyta

Target species

Algicidal ability

Alexandrium catenella Alexandrium minutum Alexandrium tamarense Prorocentrum donghaiense Akashiwo sanguinea Scrippsiella trochoidea Cyanobacteria Phaeodactylum tricornutum Chaetoceros compressus Thalassiosira pseudonana Pmphiprora alata Thalassiosira weissflogii Skeletonema costatum Heterosigma akashiwo Chattonella marina Phaeocystis globosa Dicrateria inornata Isochrysis galbana Dunaliella salina Platymonas subcordiformis Chlorella autotrophica Platymonas helgolandica Prasinophyceae

− − + − − + − + − − − + − − + − − − − − − −

to the control. In the control group, the shape of the chloroplasts and mitochondria in the algal cells was predominantly oval, and both had a double membrane, with tightly and evenly distributed thylakoids and tubular cristae. However, from the results of Fig. 6f and h, the morphology of the chloroplasts changed to round, and chloroplast damage with thylakoid outflow was seen. Mitochondria also lost their membrane integrity; the intracellular substances had spilled out. Most of the organelles were severely damaged under the effect of deinoxanthin. 3.7. Effect of deinoxanthin on the growth of other algal species Algicidal extracts from strain LY01 showed algicidal activity against 5 of the algae tested, Alexandrium tamarense, Scrippsiella trochoidea, Phaeodactylum tricornutum, Skeletonema costatum, Phaeocystis globosa (Table 1). 4. Discussion It is well known that bacteria are considered to be one of the key biological agents in HABs control [43], and a large number of algicidal bacteria are isolated. However, the relationship between

bacteria and algae are complex 21 , and studies of the secondary metabolites produced by algicidal bacteria are especially limited and vague. The present study first confirmed that a bacterium, Deinococcus sp. Y35, and one pigment, deinoxanthin, present in the cell extracts of Deinococcus sp. Y35 had algicidal activity on A. tamarense. The genus Deinococcus, which was first proposed by Brooks and Murray [44], currently contains 50 validly named species (http://www.bacterio.net/deinococcus.html). Deinococcus is a genus of bacteria that can adapt to extreme environment [45], and it is reported many times that it has strong resistance to radiation and can also endure high temperature, desiccation, low temperature, oxidizing agents and other extreme stresses [46,47]. There are few studys on the relationship of Deinococcus and algae [48], however, up to now there are still no reports which prove that Deinococcus has an algicidal effect on A. tamarense, let alone mentioning that the metabolites from Deinococcus can show algicidal activity. Unlike the previously described algicidal methods, that of Deinococcus does not actually belong to the direct or indirect category. Thus, the algicidal substances were not released to the supernatant during bacterial growth but, at the same time, the algicidal substances were attached to unaffected bacterial cells (Fig. S3). Therefore, the supernatant did not have algicidal activity. The algicidal substances were extracted from red bacterial cells, which turned white after the red pigment was extracted. Previous studies also indicated a similar algicidal method. For example, an alkaloid, red pigment compound, prodigiosin, which shows lytic activity against Cochlodinium polykrikoides cells at very low concentrations (1 ppb), is isolated from Hahella chejuensis KCTC 2396 cells [49]; and a purple pigmented metabolite, violacein, is also extracted from the pellets after Chromobacterium violaceum culture is centrifuged [50]. Algicidal compound R1 produced by the algicidal bacterium (Deinococcus sp. Y35), was further purified and identified in this study. The compound has the same molecular weight and similar NMR data to the deinoxanthin which was isolated from D. radiodurans DSM 20,539 [51] (Fig. 2 and Table2), demonstrating their high similarity in structure. Deinococcus radiodurans DSM 20,539 showed so low similarity (92.91%) with strain Y35, therefore, they were not the same bacteria. From the molecular weight and NMR data, we concluded that the algicidal compound R1 was the same as deinoxanthin. The structure of deinoxanthin owns more conjugated double bond structures (Fig. 3). In order to determine the algicidal mechanism of the active compound, the characteristics of deinoxanthin were studied. The absorption spectrum of deinoxanthin revealed that the compound absorbed within the UV-range and, also in the blue region of visible light (Fig. S5). The cyclic structure of deinoxanthin was in agreement with the absorption observed in the UV range, and the absorption in the blue region of visible light could indicate lots of conjugated double bond structures. Shunmugam et al. report that a secondary metabolite from Nostoc XPORK14A inhibits the photosynthesis and growth of Synechocystis PCC 6803, and this algicidal mechanism maybe attributed to the special structure of the secondary metabolite M22 [19]. The absorption in the blue range of M22 makes highly possible the light-induced formation of an excited state of M22 that could interact with O2 to produce singlet oxygen (1 O2 ). Based on the above theory, we concluded that an excited state of deinoxanthin could interact with O2 to produce singlet oxygen (belonging to ROS) (Fig. 5). ROS are generated as by-products of cellular metabolism, especially when cells are under stress. Li et al. report that the ROS generated by algicidal substances attack biofilm polyunsaturated fatty acids, causing lipid peroxidation, and thus create lipid peroxides in the algal cells [52]. Zhang et al. also report that the accumulation of ROS destroys pigment synthesis and membrane integrity, and inhibits or ultimately kills the algal cells.

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Fig. 6. Transmission electron micrographs of the lysing process in A. tamarense treated with deinoxanthin (10 ␮g/mL). (a,e and g) control cells and with chloroplasts and mitochondria in A. tamarense; (b) a damaged A. tamarense cell after 6 h treatment; (c) a damaged A. tamarense cell after 12 h treatment; arrow shows where the cell wall and membrane were broken (d) lysosomes increased significantly after treatment; (f) a damaged chloroplast after 12 h treatment; (h) a damaged mitochondrion after 12 h treatment. (C: chloroplast; m: mitochondria; CM: cell membrane; CW: cell wall; L: lysosomes; V: vacuole.). Bars (a), (b) and (c) 2 ␮m; (e), (f), (g) and (h) 0.5 ␮m; (d) 0.2 ␮m.

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Table 2 Assignments of 1 H NMR Chemical Shifts of R1 (CD3 OD), 600 MHz. Proton

ı (ppm)

ı (ppm)a

Multiplicity

Coupling constants (Hz)

2 ax 3 ax 3 eq 7 8 10 11 12 14 15 16 17 18 19 20 2 3 4 6 7 8 10 11 12 14 15 16 17 18 19 20

3.84 2.62 2.77 6.27 6.47 6.38 6.68 6.53 6.36 6.77 1.21 1.27 1.87 2.00 2.00 2.33 5.86 6.21 6.15 6.65 6.40 6.33 6.73 6.45 6.35 6.77 1.22 1.21 1.95 2.02 2.05

3.89 2.61 2.80 6.22 6.38 6.30 6.64 6.44 6.28 6.66 1.21 1.25 1.89 1.98 1.99 2.31 5.76 6.21 6.14 6.60 6.36 6.24 6.65 6.38 6.28 6.66 1.24 1.24 1.93 1.99 2.00

dd dd dd d d m m d m m s s s s s d dt d d dd d d m d m m s s s s s

J2ax/3ax = 9.0; J2ax/3eq = 4.2 J3ax/3eq = 16.9; J3ax/2ax = 9.0 J3eq/3ax = 16.9; J3eq/2ax = 4.2 J7/8 = 16.1 J8/7 = 16.1

a

J12/11 = 14.9

J2 /3 = 7.4 J3 /2 = 7.4; J3 /4 = 15.4 J4 /3 = 15.4 J6 /7 = 11.0 J7 /6 = 11.0; J7 /8 = 14.8 J8 /7 = 14.8 J10 /11 = 10.3 J12 /11 = 14.7

ecological security, and could be used to bloom control after the field study. 5. Conclusions Deinoxanthin was first reported as an algicide isolated from the algicidal bacterium Deinococcus sp. Y35. The algicidal activity test showed that deinoxanthin had an EC50 value of 5.636 ␮g/mL in a 12 h treatment time against A. tamarense. The unique structure and characteristics of deinoxanthin induced ROS production, and cell membrane integrity and the structure of organelles were seriously damaged. These results prove deinoxanthin could be considered as a novel algicide for the control of HABs in future. Furthermore, further studies are needed regarding the algicidal mechanism of deinoxanthin on A. tamarense. Acknowledgements This work was financially supported by the Public Science and Technology Research Funds for Projects on the Ocean (201305016), the Joint project of National Natural Science Foundation of China (NSFC) and Shandong province: Marine ecology and environmental sciences (Grant No. U1406403), the National Natural Science Foundation of China (41376119, 40930847), and the Program for Changjiang Scholars and Innovative Research Team in University (41121091). We thank Prof. I. J. Hodgkiss of The University of Hong Kong for help with the English. Appendix A. Supplementary data

Data from Lemee et al. [51], Chemical shifts of sample (CDCl3), 500 MHz.

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhazmat. 2015.02.070. Therefore, we determined why deinoxanthin has algicidal activity [26]. Pigments are often considered as algicidal metabolites which are isolated from algicidal bacteria. A red pigment PG-L-1, which belongs to the prodigiosin members, was isolated from one marine bacterium MS-02-063, and this pigment shows potent algicidal activity against various red tide phytoplankton species in a concentration-dependent manner [53]. Kim et al. isolate red pigments from Hahella chejuensis KCTC 2396, and identify the main red-colored metabolite of the pigments as antibiotic prodigiosin [54], which shows antibacterial and algicidal activities. In our study, deinoxanthin was first proved to be an anti-algal compound, and its algicidal activity showed that deinoxanthin significantly inhibited the growth of A. tamarense with an EC50 of 5.636 ␮g/mL in a 12 h treatment time. These results showed that the inhibitory effect of deinoxanthin could cause great damage in photosynthesis and, using TEM, we also observed the destruction of the chloroplast. The cell wall and cell membrane were significantly damaged, and the structure and the shape of organelles changed under the algicidal effect. Li et al. note severe ultrastructural damage to the algae at 40 ␮g/mL concentrations of palmitoleic acid [23]. All of these results demonstrate that deinoxanthin plays an important role in controlling A. tamarense growth. The application of deinoxanthin to HABs control should promise firstly the extracts would not influence other marine algae growth. Through Table 1, we concluded that the extracts from strain Y35 only showed high algicidal effect on several dinoflagellates and diatoms (Alexandrium tamarense, Scrippsiella trochoidea, Phaeodactylum tricornutum, Skeletonema costatum, Phaeocystis globosa), and these algae always caused HABs, especially the HABs which caused by Skeletonema costatum were found in the sea area near Xiamen, China recent years. Deinoxanthin would not pose any toxicity on other harmless algae, this showed that deinoxanthin owned

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