Science of the Total Environment 482–483 (2014) 116–124

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Novel insights into the algicidal bacterium DH77-1 killing the toxic dinoflagellate Alexandrium tamarense Xiaoru Yang a,b,⁎,1, Xinyi Li b,c,1, Yanyan Zhou b, Wei Zheng b, Changping Yu a, Tianling Zheng b,⁎⁎ a

Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, China State Key Laboratory of Marine Environmental Science and Key Laboratory of Ministry of Education for Coast and Wetland Ecosystems, School of Life Sciences, Xiamen University, Xiamen 361005, China c Tangdu Hospital, The Fourth Military Medical University, Xi'an 710032, China b

H I G H L I G H T S • • • •

DH77-1 is the first record of a Joostella being algicidal to Alexandrium tamarense. DH77-1 killed algae by indirect attack and the active substances were stable. Algal cells responded severely after exposure to the bacterial filtrate of DH77-1. The molecular weight of the algicidal substance was 125.88

a r t i c l e

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Article history: Received 21 October 2013 Received in revised form 20 February 2014 Accepted 26 February 2014 Available online xxxx Keywords: Toxic Alexandrium tamarense Algicidal bacteria Algicidal substance Algicidal mechanism HABs control

a b s t r a c t Algicidal bacteria may play a major role in controlling harmful algal blooms (HABs) dynamics. Bacterium DH77-1 was isolated with high algicidal activity against the toxic dinoflagellate Alexandrium tamarense and identified as Joostella sp. DH77-1. The results showed that DH77-1 exhibited algicidal activity through indirect attack, which excreted active substance into the filtrate. It had a relatively wide host range and the active substance of DH77-1 was relatively stable since temperature, pH and storage condition had no obvious effect on the algicidal activity. The algicidal compound from bacterium DH77-1 was isolated based on activity-guided bioassay and the molecular weight was determined to be 125.88 by MALDI-TOF mass spectrometer, however further identification via nuclear magnetic resonance (NMR) spectra is ongoing. The physiological responses of algal cells after exposure to the DH77-1 algicidal substances were as follows: the antioxidant system of A. tamarense responded positively in self-defense; total protein content decreased significantly as did the photosynthetic pigment content; superoxide dismutase, peroxidase enzyme and malondialdehyde content increased extraordinarily and algal cell nucleic acid leaked seriously ultimately inducing cell death. Furthermore, DH77-1 is the first record of a Joostella sp. bacterium being algicidal to the harmful dinoflagellate A. tamarense, and the bacterial culture and the active compounds might be potentially used as a bio-agent for controlling harmful algal blooms. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Harmful algal blooms (HABs) are serious global marine disasters, causing a series of ecological, resource and environmental problems as well as significant economic losses (Yang et al., 2012; Zheng et al., 2013). To manage and mitigate the adverse impact of HABs, various management strategies have been developed. Physical techniques ⁎ Correspondence to: X. Yang, Institute of Urban Environment, Chinese Academy of Sciences, 1799 Jimei Road, Xiamen 361021, China. Tel.: + 86 592 6190560; fax: + 86 592 6190514. ⁎⁎ Correspondence to: T. Zheng, RoomC-301, School of Life Sciences, Xiamen University, Xiamen 361005, China. Tel.: +86 592 2183217; fax: +86 592 2184528. E-mail addresses: [email protected] (X. Yang), [email protected] (T. Zheng). 1 X. Yang and X. Li contributed equally to this work.

http://dx.doi.org/10.1016/j.scitotenv.2014.02.125 0048-9697/© 2014 Elsevier B.V. All rights reserved.

including the use of yellow loess (Choi et al., 1998) and chemical agents such as copper sulfate (Anderson, 1997) are effective in controlling blooms within a short period after application. However, their usage in aquatic ecosystems is considered to be potentially dangerous due to its potential secondary effects on bottom-dwelling organisms (Rhoads and Young, 1970; Bricelj and Malouf, 1984). Chemical agents can cause serious secondary pollution, and they can indiscriminately kill multiple organisms in the aquatic ecosystem, which may alter marine food webs and eventually cause a severe impact on natural fish communities (Jeong et al., 2000, 2008). Biological agents, including bacteria (Mayali and Azam, 2004; Su et al., 2007a, 2007b; Wang et al., 2010; Wang et al., 2010; Wang et al., 2012), actinomycete (Bai et al., 2011; Zheng et al., 2013; Zhang et al., 2013), viruses (Nagasaki et al., 2004; Cai et al., 2011), protozoa (Jeong et al., 2008), macrophytes (Nakai

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et al., 1999; Jin and Dong, 2003; Zhou et al., 2010) and microalgae (Granéli et al., 2008) are considered as potential suppressors in controlling the outbreak and control of HABs. Algal-bacterial interactions are increasingly cited as potential regulators in the sense of both decreasing and developing algal blooms (Doucette et al., 1998). Research into the relationships between algae and bacteria has resulted in the isolation of several algicidal bacteria which belong mainly to the Cytophaga/Flavobacterium/Bacteroidetes (CFB) group and the genera Cytophaga, Saprospira, Alteromonas and Pseudoalteromonas, Vibrio, Shewanella, Bacillus, Planomicrobium and Micrococcus. These bacteria show algicidal activity through either direct or indirect attack on the target algal cells (Mayali and Azam, 2004). Most reports on algicidal bacteria have dealt with a description of the alga-lysing phenomenon, the isolation and identification of the algicidal bacteria. Only a few algicidal compounds from algicidal bacteria have been purified and identified. These algicides would be ecto-proteases (Lee et al., 2002), peptides (Imamura et al., 2000), protein (Wang et al., 2012), biosurfactants (Ahn et al., 2003; Wang et al., 2005) or antibiotic-like substances (Nakashima et al., 2006). In this study, a strain of bacterium (DH77-1) was isolated with high algicidal activity against the toxic dinoflagellate Alexandrium tamarense. Algicidal mode, algicidal specificity and the basic characteristics of the bacterial supernatant of the strain against A. tamarense were studied. A study of the physiological responses of algal cells after exposure to the DH77-1 metabolites was also implemented in order to better understand its algicidal mechanism. In addition, the algicidal compound from DH77-1 was isolated based on activity-guided bioassay and its molecular weight was determined.

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in previous study (Su et al., 2011). Algicidal rate, representing the algicidal activity, was calculated using the following equation: Algicidal rateð% Þ ¼ ðNC −NT Þ=NC  100 where NC refers to the initial living algal cells concentration, NT refers to the concentration of living algal cells after incubation for 1 day. 2.3. Characterization and identification of strain DH77-1 The colony appearance of DH77-1 was described after incubation on the 2216 agar plate for about 2–3 days at 28 °C. The bacterium was dyed with 2% phosphotungstic acid and examined using a transmission electron microscope (JEM2100). Bacterial cells from DH77-1 cultures grown in 2216 liquid cultures were collected by centrifugation (5000 ×g for 20 min). Extraction of genomic DNA from DH77-1 and PCR amplification for the 16S rRNA gene were performed as described by Su (Su et al., 2007a, 2007b). The PCR products were purified from agarose gel with a GeneClean Turbo Kit (Qbiogene) and ligated with a pMD 19-T vector; the ligation products were transformed into Escherichia coli DH5α competent cells, followed by sequencing, which was performed by Shanghai Invitrogen Biotechnology Co., Ltd. The sequence for DH77-1 was compared with other 16S rRNA genes obtained from GenBank using the BLAST program. Alignments and similarity comparison were calculated by the Clustal X2 software (Thompson et al., 1997), and a phylogenetic tree was constructed using MEGA 5.0 with the neighbor-joining method. Bootstrap values were determined according to Felsenstein's method (Felsenstein, 1985).

2. Materials and methods 2.4. Characterization of the algicidal substances 2.1. A. tamarense culture A non-axenic culture of A. tamarense ATGD98-006 was provided by the Algal Culture Collection (Institute of Hydrobiology, Jinan University, Guangzhou, China). Axenic A. tamarense cultures were obtained in order to investigate the interactions between this alga and bacteria (Su et al., 2007a, 2007b). The algal cultures were maintained in f/2 medium (Guillard, 1975) prepared with natural seawater (28 practical salinity units, psu) at 20 °C ± 1 °C under a 12 h light/12 h dark cycle with a light intensity of 50 μmol photons m−2 s−1. 2.2. Algicidal activity against A. tamarense Strain DH77-1 was isolated from surface water samples of a Prorocentrum donghaiense Lu bloom (~ 108 cells L−1), accompanied with A. tamarense (~ 104 cells L− 1), in the East China Sea during the National Science Foundation of China 973 project MC2003-2 cruise on 19–21 May 2003. DH77-1 was inoculated in 4 mL of Zobell 2216 broth (5 g peptone, 1 g yeast extraction, 0.1 g ferric phosphorous acid, pH 7.6–7.8, in 1 L natural seawater) at 28 °C and 150 rpm for 7 days. The algicidal activity assay was carried out in the 24-well plates. A 40 μL aliquot of the bacterial supernatant, which was collected by centrifugation at 5000 ×g for 20 min, was inoculated in triplicate into 2 mL of logarithmic-phase (~1 × 104 cells mL−1) algal cultures, and a 40 μL aliquot of 2216 liquid medium only was added to the algal cultures as a control. The growth of A. tamarense was monitored using fluorescein diacetate (FDA, Sigma) vital stain. FDA stain was performed according to (Widholm, 1972). FDA working stock (5 mg mL− 1 in 100% acetone, preserved at 4 °C in the dark) was added into the samples of A. tamarense cultures to a final concentration of 50 μg mL−1 followed by incubation at room temperature for 3 min. The treated samples were kept in an ice bath and vital cells of A. tamarense were measured immediately by counting the green cells under an epifluorescence microscope (Olympus BX41) with blue light excitation. After co-culture for 1 d, the living algal cells were counted with FDA assay as described

2.4.1. Algicidal mode The pure DH77-1 isolate grown in 3 mL 2216 broth were inoculated as a 1% inoculum into 100 mL tryptone broth and grown to the stationary phase (28 °C at 150 rpm for 22–24 h, based on the results of the growth curve). Bacterial cells were collected using centrifugation (8000 rpm, 10 min), washed three times using sterile f/2 medium and re-suspended in sterile f/2 medium. The supernatants were filtrated through 0.22 μm Millipore membrane filters. To determine the algicidal mode of the DH77-1 bacterium, 40 μL (2%) bacterial cultures, filtrates or cell suspensions were inoculated into 2 mL exponential phase axenic A. tamarense cultures (~1 × 104 cells mL−1). Cultures with the addition of 40 μL 2216 broth but no bacteria served as a control, and a no addition control was also involved in the experiments. All treatments and controls were performed in triplicate. Algicidal mode was illustrated by the algicidal activity, calculated after co-cultured for 24 h, and microscopically observed. 2.4.2. Host range of DH77-1 The algicidal range of DH77-1 was tested on 22 different algal species, some of which are typical HABs species: Cyanobacteria, Dunaliella salina (provided by Professor Gao Yahui, Xiamen University, Xiamen, China); Chlorella autotrophica, Platymonas helgolandica, Prasinophyceae, Chlorella, Prorocentrum donghaiense, Dicrateria inornata, Isochrysis galbana, Heterosigma akashiwo, Chattonella marina, Phaeodactylum tricornutum, Chaetoceros compressus, Thalassiosira pseudonana, Amphiprora alata, Thalassiosira weissflogii, Asterionella japonica (provided by College of Ocean and Earth Sciences, Xiamen University, Xiamen, China); Alexandrium minutum TW01, Alexandrium catenella DH01, Scrippsiella trochoidea XM01 (provided by Vice Professor Zhou Lihong, Jimei University, Xiamen, China); Phaeocystis globosa (provided by the Algal Culture Collection, Institute of Hydrobiology, Jinan University, Guangzhou, China); and Nannochloropsis (provided by the Center of Marine Biotechnology, University of Maryland, Biotechnology Institute, Baltimore, USA).

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2.4.3. Effects of temperature, pH and storage condition on algicidal activity DH77-1 cultures grown in 2216 liquid medium at 28 °C for 1 day were centrifuged at 5000 ×g for 20 min to collect the supernatant. The supernatant was then incubated at 40, 60, 80, 100 and 120 °C for 1 h to test the effects of temperature on algicidal activity. The pH of the filtrate was adjusted to 3 or 11 to test the effect of pH on the algicidal activity. The treated filtrate was subsequently inoculated into A. tamarense cultures in triplicate to test the algicidal activity. In addition, the effect of storage on the algicidal activity was also determined. The sterile filtrate was lyophilized and stored at −80 °C for 6 months. A 30 mL aliquot of the sterile filtrate was lyophilized and weighed, and the weight of lyophilized powder in 1 mL of sterile filtrate was then calculated. Based on the sterile filtrate algicidal test, proportionable weights of lyophilized powder were used in the algicidal activity experiment. 2.5. Lipid peroxidation and antioxidative enzyme assays of A. tamarense Bacterial filtrates were subjected to 2 mL of the exponential phase axenic A. tamarense cultures at a range of concentrations (1%,2% and 2.5%). Axenic f/2 medium of the same volume was also added separately as a control. After co-culture for 2 h, 4 h and 24 h, algal cells were collected by centrifugation (3,500 rpm, 5 min), and resuspended in 2 mL PBS buffer (20 mM, pH 7.6). Cell disruption was conducted using Ultrasonic Cell Disruption System (Ningbo Scientific Biotechnological Co., Ltd, China (40 W, 2 s; 3 s, 40 times) below 4 °C. The crude protease solution was collected by centrifugation at 4 °C for 10 min, at 12,000 rpm. The total protein content, superoxide dismutase (SOD), peroxidase (POD) and malondialdehyde (MDA) content were determined following the kit's instructions (Nanjing Jiancheng). All treatments and controls were performed in triplicate. 2.6. Measurement of chloroplast pigments To evaluate the photosynthetic pigment of algal cells, cells were collected by centrifugation (3500 rpm, 5 min) after incubation with a time interval of 3 h during the experimental period of 24 h followed by storage at − 80 °C for 24 h to allow easy pigment release. Cells were mixed with 5 mL 95% ethanol for extraction in the dark at −20 °C for 24 h followed by centrifugation (8000 rpm, 10 min). The supernatant was used to measure absorbance values at wavelengths of 665, 649 and 470 nm. The absorbance of 95% ethanol was used as the blank. Photosynthetic pigment was calculated according to the following formula: Ca ¼ 13:95A665 −6:88A649 Cb ¼ 24:96A649 −7:32A655 CT ¼ Ca þ Cb CX:C ¼ ð1000A470 −2:05Ca −114:8 Cb Þ=245 where Ca, Cb, CT represent the concentrations of chlorophyll a, chlorophyll b and total chlorophyll concentration (mg L− 1), respectively; CX.C represents of the total carotinoid (Car) concentration (mg L−1). 2.7. Determination of cellular membrane damage The release of intracellular components is a good indicator of membrane integrity. Through the detection of absorbance at 260 nm, one can estimate the amount of the DNA and RNA released from the cytoplasm (Chen and Cooper, 2002). The experiments were conducted in triplicate. A certain amount of sterile filtrate of DH77-1 was added to each tube containing 2 mL algal culture. After co-culture for 24 h, the supernatant was obtained by centrifugation (3500 rpm, 5 min). The optical density (OD) of the supernatant was measured at 260 nm using a UV–Vis spectrophotometer (Thermo Spectronic). The OD260 ratio between experimental group and the control group was used to evaluate the releasing level of nucleotides.

2.8. Separation of the algicidal compounds DH77-1 was cultured in 30 L 2216 Zobell broth in a fermentor for 5 days (28 °C, 150 rpm). The culture filtrate was collected by centrifugation at 5000 ×g for 20 min and then concentrated in a rotavapor. The concentrated filtrate was extracted using acetonitrile, butyl alcohol, ethyl acetate and chloroform separately three times. Crude extracts were obtained from the concentrate by extraction three times with an equal volume of organic solvent followed by concentration and drying under reduced pressure in an evaporator at 30 °C. All extracts were dissolved in DMSO (2 mg mL−1). The algicidal activity experiment was carried out as described above. Cultures with the addition of the same volume of DMSO served as a control, and a no-addition control was also involved in the experiments. All treatments and controls were performed in triplicate. Ethyl acetate extract was subjected for further isolation since it was tested to have the highest algicidal activity among the four extracts. The extract was applied to a Sephadex LH-20 (Amersham Biosciences) column with 100% methanol as eluent. Fractions (E1-5) which showed the highest activity in the algicidal assay, were further fractionated in an ODS column (ODS-A, YMC) with the following eluents (ultrapure water, 30% methanol, 50% methanol, 70% methanol, 100% methanol, acetone) successively at a flow rate of 25 mL min−1. Fraction E1-5-11 showed the highest activity and displayed sole spot in the Thin Layer Chromatography (TLC). However, the quantity is too limited to subject to further structure determination (e.g. 1 H and 13 C NMR analysis). E1-5-11 was preliminary analyzed using MALDI-TOF MS in order to determine its molecular weight since molecular weight is an important tag in the fractionation procedure. E1-5-11 was prepared as 7.5, 15, 30, 60, 90, 120 mg L−1 solutions with HPLC-grade methanol. Samples were combined with α-cyano-4-hydroxy cinnamic acid matrix and a carbon nanotubes matrix, and analyzed using the REFLEX III MALDI-TOF mass spectrometer. 2.9. Statistical analysis The data from each experiment were pooled and then evaluated by one-way ANOVA (SPSS 18.0 for Windows). 2.10. Nucleotide sequence accession number The 16S rRNA gene sequence has been deposited in GenBank under accession number KF631444. 3. Results 3.1. Isolation and identification of DH77-1 The bacterial isolate DH77-1 colony was somewhat circular, slightly raised, yellow and translucent. Its surface was smooth, moist and shiny. It was gram-negative, rod-shaped (0.3–0.4 μm × 1.3–1.5 μm), without a flagellum, under the electron microscope (Fig. 1). 16S rRNA gene sequence analysis revealed that DH77-1(KF631444) shared 99.5% homology with type strain Joostella marina En5T (EF660761) (Fig. 2). 3.2. Basic characterization of the active substances of DH77-1 3.2.1. Algicidal mode and specificity of DH77-1 The effect of algicidal bacteria on the growth of A. tamarense was tested. Bacterial cells and extracellular substances were separated and inoculated into A. tamarense cultures separately to investigate the algicidal mode of DH77-1. The results are shown in Fig. 3A. The addition of 2216 broth showed no effect on the growth of A. tamarense compared to the no-addition control (p N 0.05). The addition of washed bacterial cells showed no obvious inhibition on the growth of A. tamarense (p N 0.05), while the filtrate and bacterial cultures both showed an algicidal effect

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3.2.2. Effect of temperature, pH and storage condition on algicidal activity Heat stability of algicidal activity was investigated by comparing the different impacts between heat treated and non-treated bacterial filtrates on the growth of A. tamarense. All the supernatants retained algicidal ability after 1 h incubation in water baths with different temperatures, which indicated that the active substances of DH77-1 were heat tolerant even up to 100 °C (Fig. 3B). pH variation did not affect the algicidal activity of the supernatant (Fig. 3C) (p N 0.05). The results of dialysis suggested that the algicidal substance might be of low molecular weight (data not shown). As shown in Fig. 3D, the algicidal activity of lyophilized powder showed no obvious decrease compared to that of the fresh filtrate (p N 0.05). This indicated that it was possible to store the filtrate as the lyophilized powder which would be spatially efficient and suitable for exploring the environmental-safety algicidal substances. The high stability of the DH77-1 active substance should make the process of separation and purification relatively feasible.

3.3. Preliminary algicidal mechanism of the active substance

Fig. 1. Transmission electron micrography of strain DH77-1, Bar, 0.5 μm.

(p b 0.05). The cytolysis of algicidal bacteria on A. tamarense cells during the algicidal process were similar to that by Pseudoalteromonas sp. SP48 as previously described (Su et al., 2007a, 2007b). Algal cells were observed with reduced mobility, detached cell walls, disrupted intracellular structure and broken cell walls, and cellular substances were released and decomposed, resulting in the appearance of abundant broken thecae. These results indicated that DH77-1 exhibited algicidal activity through indirect attack, since no algicidal activity was observed after inoculation of bacterial cells. Excretion of unknown algicidal compounds in the filtrates by the bacteria was responsible for the algicidal activity. DH77-1 showed algicidal activity against 10 of the algae tested, namely Chlorella, Thalassiosira weissflogii, Thalassiosira pseudonana, Phaeodactylum tricornutum, Asterionella japonica, Alexandrium minutum TW01, Scrippsiella trochoidea XM01, Chattonella marina, Phaeocystis globosa, and Isochrysis galbana (Table 1).

3.3.1. Effect of DH77-1 supernatant on antioxidant enzymes activity To investigate whether exposure to different supernatant concentrations of DH77-1 affected the molecular function of algal cells, activities of antioxidant enzymes as well as membrane lipid peroxidation were measured. Cellular enzymatic activities including SOD and POD were determined to investigate the cellular defense response induced by DH77-1 supernatant. As illustrated in Fig. 4A, the total SOD (T-SOD) enzyme activity of the algal cells increased markedly compared to the control (p b 0.05). Furthermore, the activities of SOD increased significantly with incubation time and concentration, which indicated that the algal cells immediately responded and their antioxidant system began selfdefense when they were exposed to algicidally active substances. POD activity showed a similar pattern to SOD activity (Fig. 4B). POD enzyme activity of the algal cells was higher than that of the control, while no obvious variations were observed among the addition of different ratios (p b 0.05). The POD enzyme activity increased significantly after 24 h incubation which indicated that the algae might have undergone serious damage after exposure for a long time. The effect of the algicidal compounds on the MDA levels was illustrated in Fig. 4C. The levels in the treated groups were higher than that of the controls (p b 0.05). In addition, the MDA levels increased with the addition ratios and incubation time which meant that membrane lipid peroxidation occurred to varying degrees in the treated groups.

Fig. 2. Phylogenetic tree based on 16S rRNA gene sequences showing the relationship between strain DH77-1 and related genera in the family Flavobacteriaceae. The tree was constructed using the neighbor-joining method using MEGA5.0 program. The bootstrap values were evaluated from 1,000 replications. Segment scale (0.01) means the branch length where the sequence difference was 1%.

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Fig. 3. Characterization of algicidal substance of DH77-1. (A) algicidal mode, (B) temperature, (C) pH effect and (D) storage condition in the algicidal activity.

3.3.2. Effect of DH77-1 supernatant on protein production, pigment contents and membrane integrity Under the stress of the algicidally active ingredient of DH77-1, the total protein in treated groups under different concentration treatments were significantly less than that of the control group. The cellular protein contents decreased significantly (p b 0.05) with increased concentration and incubation time though there was no obvious change (p N 0.05) between the concentration of 2% and 2.5% (Fig. 5A). These indicated that the effect of algicidal metabolites on the cell protein content might be concentration-dependent and irreversible. As shown in Fig. 5B and C, both Chla and Car content decreased along with co-culture time when the addition ratio was 2%. While in the ratio was 1%, Chla and Car content decreased in the first 2 h and maintained a consistent value. In addition, the Chla/Car ratio of different treated groups showed small fluctuations with the incubation time and addition ratio (data not shown). This might indicate that the algicidal compounds of DH77-1 would change the amount of pigment content but without affecting its composition ratio. The OD260 ratio of all the different concentration treated groups were larger than 2 which mean that the value of OD260 in the treated groups were more than 2 times (p b 0.05) that of the controls (Fig. 5D). This indicated that the release of extracellular nucleic acids was seriously under the stress of the algicidal materials.

acetonitrile extracts demonstrated the weakest activity. Butyl alcohol and chloroform extracts showed almost the same level of algicidal activity, which was slightly lower than that of ethyl acetate extracts (data not shown). The results indicated that the active substance should be more soluble in ethyl acetate. Furthermore, ethyl acetate is less toxic compared to the other solvents. Therefore, the ethyl acetate extract was subjected to further isolation of the active substances. One major active fraction, approximately 881.1 mg, was obtained after elution with 100% methanol from a Sephadex LH-20 column (named as E1-5). This active fraction was vacuum-concentrated and further subjected to ODS column. Of the fractions eluted, fraction of E1-5-11(ca. 11.7 mg) was further purified due to its highest algicidal activity. The fraction was repeatedly tested in TLC by using different developing solvents, similar profile with sole spot were obtained. This might indicate that the fraction contain relatively pure compound. Due to the limited amount, the fraction was further analyzed to determine its molecular weight by mass spectrometer. The molecular mass spectra of all concentrations of E1-5-11 had a high absorption peak in 164.88 and 142.88 (Fig. 6). This demonstrated that the main substances of E1-5-11 ionized in the form of [M + Na] + 142.88 and [M + K] + 164.88. Thus,we initially determined the molecular weight to be about 125.88.

3.4. Isolation of the algicidal compound from DH77-1

4.1. Identification, algicidal mode and specificity of DH77-1

Of the four extracts from organic solvents mentioned above, ethyl acetate extracts showed the strongest algicidal activity, whereas

Bacteria which can directly or indirectly inhibit the growth of algae, lyse algae or dissolve algal cells are known collectively as algicidal

4. Discussion

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Table 1 Algicidal range of DH77-1. Algal species Chlorophyta Platymonas helgolandica Prasinophyceae Dunaliella salina Chlorella autotrophica Chlorella Diatom Chaetoceros comperssus Amphiprora alata Thalassiosira weissflogii Thalassiosira pseudonana Phaeodactylum tricornutum Asterionella japonica Dinoflagellates Alexandrium minutum TW01 Prorocentrum donghaiense Alexandrium catenella DH01 Scrippsiella trochoidea XM01 Yellow algae Chattonella marina Heterosigma akashiwo Cyanophyta Cyanobacteria Golden algae Phaeocystis globosa Isochrysis galbana Nannochloropsis Dicrateria inornata

Algicidal activity − − − − + − − + + + + + − − + + − − + + − −

+, algicidal activity over 75%; −algicidal activity lower than 5%.

bacteria (Mayali and Azam, 2004). Mayali and Azam (2004) summarized the affiliation of the reported algicidal bacteria, which fell into a few major groups of bacteria, including the phyla of Bacteroidetes, Proteobacteria and Actinobacteria. DH77-1 belongs to the Bacteroidetes, Flavobacteria, Flavobacteriales, Flavobacteriaceae, Joostella. Joostella was discovered as a new member of the Bacteroidetes, Flavobacteriaceae by Quan et al (2008) and as far as we know, DH77-1 is the first record of a Joostella sp. bacterium being algicidal to the harmful dinoflagellate A. tamarense. Bacteria that inhibit the growth of algae take effect through direct or indirect attack (Mayali and Azam, 2004). The former needs a direct cell to cell contact, while indirect attacks are thought to be chemically mediated. The growth of A. tamarense was little affected by cells of the DH77-1 but was affected by cell free filtrates, suggesting that the DH77-1 exhibited algicidal activity through indirect attack. In the group of reported algicidal bacteria and actinomycete, some exhibit algicidal activities in the same way, such as Alteromonas strain K and D (Imai, 1995), Pseudoalteromonas sp. T827/2B (Baker and Herson, 1978), Pseudoalteromonas sp. strain A28 (Lee et al., 2000), Pseudoalteromonas sp. SP48 (Su et al., 2007a, 2007b), Brevibacterium sp. BS01 (Bai et al., 2011) and Streptomyces sp. O4-6 (Zheng et al., 2013). In our study, DH77-1 has a relatively wide host range and showed algicidal activity against 10 species of algae (Table 1). While strain 5 N-3 which belongs to Flavobacterium sp. has a strong inhibitory effect on Gymnodinium nagasakiense and also a killing effect, it was ineffective on Chattonella antiqua, Heterosigma akashiwo and Skeletonema costatum. This indicates that strain 5 N-3 has a relatively specific algicidal effect on G. nagasakiense (Doucette et al., 1999). On the species-specific algicidal effect of bacteria, Doucette et al. (1999) found that the fact that the algicidal activity of Cytophaga 41-DBG2 is species-specific between different algae species may be due to different types and numbers of the extracellular secretions of the different algae. 4.2. Characterization of the algicidal substances There are no reports defining the algicidal substances of direct algicidal bacteria as yet. These may be extracellular enzymes from

Fig. 4. Variations of (A) SOD, (B) POD and(C) MDA contents of Alexandrium tamarense after exposure to DH77-1 supernatant with different concentrations.

bacteria (e.g. cellulase, protease, esterase) to destroy the algal cell wall or cell membranes. In indirect attacks, algicidal bacteria secrete soluble substances which can be ecto-proteases (Lee et al., 2000, 2002), peptides (Imamura et al., 2000), protein (Wang et al., 2012), biosurfactants (Ahn et al., 2003; Wang et al., 2005; Sun et al., 2004), antibiotic-like substances (Nakashima et al., 2006; Bai et al., 2011), or alkaloids (Kodani et al., 2002). So far, identification of algicidal substances from bacteria is limited, and makes it more difficult to determine the expression and regulation mechanism of the algicidal material (Doucette et al., 1999; Skerratt et al., 2002; Mayali and Azam, 2004). There are major difficulties in purifying algicidal substances as follows: 1) Usually algicidal substances are high-polar, can be quickly dissolved and distributed throughout the water environment, and so it is difficult to use traditional separation means; 2) Part of the algicidal substances may not be stable enough, and their biological activity may be lost during the extraction process; and 3) Following activity-guided bioassay, a large quantity of the components isolated are used for activity detection at each separation step and that may result in the residue of active substance not being enough for further separation, activity detection

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Fig. 5. Effects of DH77-1 supernatant on (A) protein, (B) chlorophyll a, (C) carotenoid content and (D) OD260 of A. tamarense with different addition ratio.

or identification. In present study, we can not obtain enough amount of active substance for further structure determination of the isolated compound. The molecular weight of the compound was determined

since molecular weight is an important tag in the fractionation procedure. In the future work, dialysis will be used to obtain the active fraction according to the known molecular weight of the algicidal

Fig. 6. MALDI-TOF mass spectrum of the isolated algicidal substance of DH77-1 on carbon nanotubes.

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substance. However, further identification is necessary since it is available to obtain enough substance via commercial pathway or chemical synthesis once the structure is determined, which will be much less time and labor consuming than fractionation procedure.

4.3. Isolation of the algicidal substance Physical characteristics of the active substances of DH77-1 against A. tamarense were determined for its further separation. The low molecular weight and high polarity of the active substance from DH77-1, make traditional “system-solvent extraction” difficult for its extraction and purification. The algicidal compounds of strain DH77-1 are mainly distributed in the water phase, in which inorganic salts would make it more difficult to separate the active substance. Ethyl acetate was finally chosen due to its highest algicidal activity, higher extraction rate and lower toxicity. Besides ethyl acetate extractions, other extracted phases also showed some algicidal activity which suggested that more than one algicidal compound might exist in the DH77-1 fermentation product. Of course, expanding the scale of fermentation and optimizing the extraction process are also necessary to obtain a determined structure of the compound, which will allow its synthesis and commercial production. Furthermore, the lyophilized powder of DH77-1 sterile filtrate, which showed similar algicidal activity compared to fresh material, could be used as a potential algicide other than the pure active substance for its low moisture content, high stability and an extended shelf life.

4.4. Physiological response of algal cells Our study of the physiological response of algal cells, exposed to the algicidal substances from DH77-1, was implemented in order to reveal the algicidal mechanism. Along with a series of responses by antioxidant system, cell protein contents, photosynthetic pigments and nucleic acid contents of the algal cells have been changed under stress from the algicidally active ingredients of DH77-1 in our study. Chlorophylls are the basic pigments involved in light absorption and photochemistry in algae, photosynthetic bacteria and plants (Eullaffroy and Vernet, 2003), and carotenoids as accessory pigments in dinoflagellates serve as light-harvesting pigments during photosynthesis (Douglas et al., 2003). In our study, the decreased photosynthetic pigments showed that pigment was deteriorated and photosynthesis was affected. SOD and POD are representatives of the most important antioxidant enzymes in cells and catalyzes the dismutation of the superoxide radical into H2O2 and O2 (Valentine et al., 1998). The enhancement of SOD and POD suggested that A. tamarense cells suffered from oxidant stress when exposed to the algicidal substances from DH77-1. A markedly increased MDA content and seriously leaked nucleic acids of the algal cell, further evidenced that algal cells might undergo severe lipid peroxidation, leading to a final cell lysis after exposure to the algicidal substances from DH77-1.

5. Conclusions In this study, bacterium DH77-1 exhibited high algicidal activity against the toxic dinoflagellate A. tamarense. Phylogenetic analysis of 16S rRNA gene sequence showed that it belonged to Joostella, which is the first record of a Joostella sp. bacterium being algicidal to A. tamarense. Furthermore, the algicidal compound was isolated based on the activity-guided bioassay and the molecular weight was determined to be 125.88. DH77-1 supernatant altered enzymatic antioxidant systems, pigment content, protein content as well as the membrane integrity in A. tamarense ultimately inducing cell death. However, the further structure determination of the algicidal substance and field application are required for its potential use in the mitigation of HABs.

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Conflict of interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled “Novel insights into the algicidal bacterium DH77-1 killing the toxic dinoflagellate Alexandrium tamarense”.

Acknowledgments This work was supported by the National Nature Science Foundation of China (40930847, 41376119), Public Science and Technology Research Funds Projects of Ocean (201305016, 201305022), Program for Changjiang Scholars and Innovative Research Team in University (41121091) and Xiamen University MEL Young Scientist Visiting Fellowship (MELRS1124). We would like to thank Prof. I. J. Hodgkiss from The University of Hong Kong for help with English.

References Ahn CY, Joung SH, Jeon JW, Kim HS, Yoon BD, Oh HM. Selective control of cyanobacteria by surfactin-containing culture broth of Bacillus subtilis C1. Biotechnol Lett 2003; 25(14):1137–42. Anderson DM. Turning back the harmful red tide. Nature 1997;388(6642):513–4. Bai SJ, Huang LP, Su JQ, Tian Y, Zheng TL. Algicidal effects of a novel marine actinomycete on the toxic dinoflagellate Alexandrium tamarense. Curr Microbiol 2011;62:1774–81. Baker KH, Herson DS. Interactions between the diatom Thallasiosira pseudonanna and an associated pseudomonad in a mariculture system. Appl Environ Microbiol 1978; 35(4):791–6. Bricelj VM, Malouf RE. Influence of algal and suspended sediment concentrations on the feeding physiology of the hard clam Mercenaria mercenaria. Mar Biol 1984;84: 155–65. Cai WW, Wang H, Tian Y, Chen F, Zheng TL. Bacteriophage influences algal bloom decline by modulating algicidal bacteria population dynamics. Appl Environ Microbiol 2011; 77:7837–40. Chen CZ, Cooper SL. Interactions between dendrimer biocides and bacterial membranes. Biomaterials 2002;23:3359–68. Choi HG, Kim PJ, Lee WC, Yun SJ, Kim HG, Lee HJ. Removal efficiency of Cochlodinium polykrikoides by yellow loess. Korean Fish Soc 1998;31:109–13. Doucette GJ, Kodama M, Franca S, Gallacher S. Bacterial interactions with harmful algal bloom species: bloom ecology, toxigenesis and cytology. Physiological Ecology of Harmful Algal Blooms. Berlin: Springer-Verlag; 1998. p. 619–47. Doucette GJ, McGovern ER, Babinchak JA. Algicidal bacteria active against Gymnodinium breve (Dinophyceae). I. Bacterial isolation and characterization of killing activity. J Phycol 1999;35(6):1447–54. Douglas SE, Larkum AWD, Raven JA. Photosynthesis in algae. The Netherlands: Kluwer Academic Publishers; 2003. Eullaffroy P, Vernet G. The F684/F735 chlorophyll fluorescence ratio: a potential tool for rapid detection and determination of herbicide phytotoxicity in algae. Water Res 2003;37(9):1983–90. Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 1985;39:783–91. Granéli E, Weberg M, Salomon PS. Harmful algal blooms of allelopathic microalgal species: The role of eutrophication. Harmful Algae 2008;8(1):94–102. Guillard RRL. Culture of phytoplankton for feeding marine invertebrates. In: Smith WL, Canley MH, editors. Culture of Marine Invertebrate Animals. New York: Plenum Press; 1975. p. 26–60. Imai I. Algicidal Marine-Bacteria Isolated from Northern Hiroshima-Bay, Japan. Fish Sci 1995;61(5):628–36. Imamura N, Motoike I, Noda M, Adachi K, Konno A, Fukami H. Argimicin A, a novel anticyanobacterial compound produced by an algae-lysing bacterium. J Antibiot 2000; 53(11):1317–9. Jeong JH, Jin HJ, Sohn CH, Suh KH, Hong YK. Algicidal activity of the seaweed Corallina pilulifera against red tide microalgae. J Appl Phycol 2000;12(1):37–43. Jeong HJ, Kim JS, Yoo YD, Kim ST, Song JY, Kim TH, et al. Control of the harmful alga Cochlodinium polykrikoides by the naked ciliate Strombidinopsis jeokjo in mesocosm enclosures. Harmful Algae 2008;7(3):368–77. Jin Q, Dong S. Comparative studies on the allelopathic effects of two different strains of Ulva pertusa on Heterosigma akashiwo and Alexandrium tamarense. J Exp Mar Biol Ecol 2003;293(1):41–55. Kodani S, Imoto A, Mitsutani A, Murakami M. Isolation and identification of the antialgal compound, harmane (1-methyl-beta-carboline), produced by the algicidal bacterium, Pseudomonas sp. K44-1. J Appl Phycol 2002;14(2):109–14. Lee SO, Kato J, Takiguchi N, Kuroda A, Ikeda T, Mitsutani A, et al. Involvement of an extracellular protease in algicidal activity of the marine bacterium Pseudoalteromonas sp. strain A28. Appl Environ Microbiol 2000;66(10):4334–9.

124

X. Yang et al. / Science of the Total Environment 482–483 (2014) 116–124

Lee SO, Kato J, Nakashima K, Kuroda A. Cloning and characterization of extracellular metal protease gene of the algicidal marine bacterium Pseudoalteromonas sp. strain A28. Biosci Biotechnol Biochem 2002;66(6):1366–9. Mayali X, Azam F. Algicidal bacteria in the sea and their impact on algal blooms. J Eukaryot Microbiol 2004;51(2):139–44. Nagasaki K, Tomaru Y, Katanozaka N, Shirai Y, Nishida K, Itakura S, et al. Isolation and characterization of a novel single-stranded RNA virus infecting the bloom-forming diatom Rhizosolenia setigera. Appl Environ Microbiol 2004;70(2):704–11. Nakai S, Inoue Y, Hosomi M, Murakami A. Growth inhibition of blue-green algae by allelopathic effects of macrophytes. Water Sci Technol 1999;39(8):47–53. Nakashima T, Miyazaki Y, Matsuyama Y, Muraoka W, Yamaguchi K, Oda T. Producing mechanism of an algicidal compound against red tide phytoplankton in a marine bacterium γ-proteobacterium. Appl Microbiol Biotechnol 2006;73(3):684–90. Quan ZX, Xiao YP, Roh SW, Nam YD, Chang HW, Shin KS, et al. Joostella marina gen. nov., sp. nov., a novel member of the family Flavobacteriaceae isolated from the East Sea. Int J Syst Evol Microbiol 2008;58:1388–92. Rhoads DC, Young DK. The influence of deposit-feeding organisms on sediment stability and community trophic structure. J Mar Res 1970;28:150–78. Skerratt JH, Bowman JP, Hallegraeff G, James S, Nichols PD. Algicidal bacteria associated with blooms of a toxic dinoflagellate in a temperate Australian estuary. Mar Ecol Prog Ser 2002;244:1–15. Su JQ, Yang XR, Zheng TL, Hong HS. An efficient method to obtain axenic cultures of Alexandrium tamarense—a PSP-producing dinoflagellate. J Microbiol Methods 2007a;69(3):425–30. Su JQ, Yang XR, Zheng TL, Tian Y, Jiao NZ, Cai LZ, et al. Isolation and characterization of a marine algicidal bacterium against the toxic dinoflagellate Alexandrium tamarense. Harmful Algae 2007b;6:799–810. Su JQ, Yang XR, Zhou YY, Zheng TL. Marine bacteria antagonistic to the Harmful Algal Bloom (HAB) species Alexandrium tamarense (Dinophyceae). Biol control 2011;56: 132–8.

Sun XX, Choi JK, Kim EK. A preliminary study on the mechanism of harmful algal bloom mitigation by use of sophorolipid treatment. J Exp Mar Biol Ecol 2004;304(1):35–49. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. The CLUSTAL-X Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 1997;25:4876–82. Valentine JS, Wertz DL, Lyons TJ, Liou LL, Goto JJ, Gralla EB. The dark side of dioxygen biochemistry. Curr Opin Chem Biol 1998;2(2):253–62. Wang XL, Gong LY, Liang S, Han X, Zhu C, Li Y. Algicidal activity of rhamnolipid biosurfactants produced by Pseudomonas aeruginosa. Harmful Algae 2005;4(2):433–43. Wang BX, Zhou YY, Bai SJ, Su JQ, Tian Y, Zheng TL, et al. A novel marine bacterium algicidal to the toxic dinoflagellate Alexandrium tamarense. Lett Appl Microbiol 2010a;51: 552–7. Wang X, Li ZJ, Su JQ, Tian Y, Yang XR, Hong HS, et al. Lysis of a red-tide causing alga, Alexandrium tamarense, caused by bacteria from its phycosphere. Biol Control 2010b;52:123–30. Wang BX, Yang XR, Zhou YY, Lv JL, Su JQ, Tian Y, et al. An algicidal protein produced by bacterium isolated from the Donghai Sea, China. Harmful Algae 2012;13:83–8. Widholm JM. The Use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells. Biotech Histochem 1972;47:189–94. Yang CY, Li Y, Zhou YY, Zheng W, Tian Y, Zheng TL. Bacterial community dynamics during a bloom caused by Akashiwo sanguinea in the Xiamen Sea area, China. Harmful Algae 2012;20:132–41. Zhang HJ, An XL, Zhou YY, Zhang BZ, Li D, Chen ZR, et al. Effect of oxidative stress induced by Brevibacterium sp. BS01 on a HAB causing species-Alexandrium tamarense. PLoS ONE 2013;5(8):e63018. Zheng XW, Zhang BZ, Zhang JL, Huang LP, Lin JP, Li XY, et al. A marine algicidal actinomycete and its active substance against the harmful algal bloom species Phaeocystis globosa. Appl Microbiol Biotechnol 2013;97:9207–15. Zhou LH, Chen XH, Zheng TL. Study on the ecological safety of algacides: a comprehensive strategy for their screening. J Appl Phycol 2010;22:803–11.

Novel insights into the algicidal bacterium DH77-1 killing the toxic dinoflagellate Alexandrium tamarense.

Algicidal bacteria may play a major role in controlling harmful algal blooms (HABs) dynamics. Bacterium DH77-1 was isolated with high algicidal activi...
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