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Lysing activity of an indigenous algicidal bacterium Aeromonas sp. against Microcystis spp. isolated from Lake Taihu a
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Fei Yang , Xiaoqin Li , Yunhui Li , Haiyan Wei , Guang Yu , Lihong Yin , Geyu Liang & Yuepu Pu
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Key Laboratory of Environmental Medicine Engineering, Ministry of Education, School of Public Health, Southeast University, Nanjing, 210009, China Accepted author version posted online: 26 Nov 2012.
To cite this article: Fei Yang , Xiaoqin Li , Yunhui Li , Haiyan Wei , Guang Yu , Lihong Yin , Geyu Liang & Yuepu Pu (2012): Lysing activity of an indigenous algicidal bacterium Aeromonas sp. against Microcystis spp. isolated from Lake Taihu, Environmental Technology, DOI:10.1080/09593330.2012.752872 To link to this article: http://dx.doi.org/10.1080/09593330.2012.752872
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Lysing activity of an indigenous algicidal bacterium Aeromonas sp. against Microcystis spp. isolated from Lake Taihu Fei Yang, Xiaoqin Li, Yunhui Li, Haiyan Wei, Guang Yu, Lihong Yin, Geyu Liang and Yuepu Pu * Key Laboratory of Environmental Medicine Engineering, Ministry of Education, School of
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Public Health, Southeast University, Nanjing 210009, China
Corresponding author: Mailing address: School of Public Health, Southeast University, 2 Sipailou Road, Nanjing 210009, China. Phone: +86-25-83794996. Fax: +86-25-83324322 E-mail: [email protected]
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Lysing activity of an indigenous algicidal bacterium Aeromonas sp. against Microcystis spp. isolated from Lake Taihu Abstract This study aimed to isolate and characterize an indigenous algicidal bacterium named LTH-1 and its algae-lysing compounds active against three Microcystis aeruginosa strains (toxic TH1, nontoxic TH2,
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and standard FACHB 905). The LTH-1 isolated from Lake Taihu near Wuxi City in China was identified as Aeromonas sp. based on its morphological characteristic features and phylogenetic analysis by sequencing of 16S rDNA. Extracellular compounds produced by LTH-1 showed strong algae-lysing activity, and they were water-soluble, heat-tolerant with molecular mass of lower than 2 kDa. Two algae-lysing compounds were isolated and purified from extracellular filtrate using silica gel column chromatography. One of them was identified as phenylalanine (C9H11NO2, m/z 166.0862) and the other one (C8H16N2O3, m/z 189.1232) was unidentified using hybrid ion trap/time-of-flight mass spectrometry coupled with a high-performance liquid chromatography (LC/MS-IT-TOF) system. The half maximal effective concentration (EC50) of phenylalanine produced by LTH-1 against FACHB 905 was 68.2 ± 8.2 μg mL-1 in 48h. These results suggested that the algicidal Aeromonas sp. LTH-1 could play a role in controlling Microcystis blooms, and its extracellular compounds are potentially useful for regulating blooms of harmful M. aeruginosa. Keywords: Algicidal bacteria, Microcystis aeruginosa, Aeromonas, algae-lysing, phenylalanine.
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1. Introduction Cyanobacterial harmful algal blooms (CyanoHABs) are proliferating worldwide primarily due to eutrophication and climate change. Environmental problems such as severe damage to aquatic ecosystems and human health caused by CyanoHABs in eutrophic lakes, rivers and drinking water reservoirs have been increasingly documented and received more and more
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attention [1]. Microcystis is the most commonly found bloom forming cyanobacterium in the world’s eutrophic and hypereutrophic waters [2]. Especially in China, Microcystis can be dominant year-round in areas of hypereutrophy. Many strains of Microcystis are known to produce cyanobacterial cyclic-peptide hepatotoxins called microcystins, which have been responsible for animal and human poisonings in almost every country that they have been found in [2]. Therefore, controlling Microcystis blooms is a very important goal in environmental health. In order to manage CyanoHABs, several techniques have been used including clay and chemical agents such as copper algicides [3]. These methods are effective in controlling CyanoHABs for a short period after application, but clays have secondary effects on bottom-dwelling organisms, and copper algicides often induce copper toxicity and trigger release of microcystins into water after cyanobacterial cell lysis [3, 4]. Biological control agents such as viruses, protozoa, and bacteria are considered to be a potential solution to regulate CyanoHABs [5]. Some bacteria have algae-lysing effects, which may play an important role in the decline of CyanoHABs. Most of the known algicidal bacteria belong to the Cytophaga-Flavobacterium-Bacteroides (CFB) phylum and the γ-subclass of the proteobacteria including members of genera Alteromonas, Cytophaga, Saprospira,
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Flavobacterium, and Pseudoalteromonas [6, 7]. The remaining strains represent the Gram-positive genera Micrococcus, Bacillus, and Planomicrobium [8]. In general, the bacteria lyse algae through production of extracellular algae-lysing products or by cell-to-cell contact [7]. Lake Taihu (119°54′-120°36′ N, 30°56′-31°33′ E) is the third largest freshwater lake in
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China. It has a total water surface area of about 2338 km2, an average water depth of 1.89 m and a water volume of approximately 4.43 × 1012 L. With the rapid development of industries and agriculture in this region, the lake is becoming more seriously polluted and the water quality decreased. Microcystis blooms have appeared more frequently and larger scales in recent years, especially in Meiliang Bay and Nanquan region of the lake coverage [9-11]. This paper aimed to isolate and characterize an indigenous algicidal bacterium named LTH-1 and its algae-lysing compounds active against three Microcystis aeruginosa strains (toxic TH1, nontoxic TH2, and standard FACHB 905). . 2. Materials and methods 2.1 M. aeruginosa culture Two strains of indigenous M. aeruginosa (TH1 and TH2) were isolated from Lake Taihu during blooms using single-colony isolation technique. The standard M. aeruginosa FACHB 905 was purchased from the Freshwater Algae Culture Collection of the Institute of Hydrobiology, Chinese Academy of Sciences (Figure S-1 of online supplementary material). All three strains of cyanobacteria used in the study contained phycocyanin intergenic spacer (PC-IGS) as determined by PCR method [12]. TH1 and FACHB 905 contained microcystin synthetase gene B (mcyB) and produced microcystin-LR. TH2 neither contained mcyB nor
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produced microcystin-LR. The M. aeruginosa cells were incubated in sterile BG-11 medium [13]. The M. aeruginosa strains were maintained as unialgal axenic culture at 28 oC, pH 7.2, with illumination at 150 μmol photons m-2 s-1 under a 12-h light-12-h dark regimen. 2.2 Isolation and screening of algicidal bacteria against M. aeruginosa The algicidal bacteria were isolated from an immobilized biosystem, which is a synthetic
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medium of Vinlon immobilizing indigenous microbes in Meiliang Bay of Taihu Lake, China [14]. Samples were serially diluted with sterile water and 0.1 mL aliquots of each dilution were inoculated onto a nutrient agar (NA, 2% agar) plates. Isolated bacteria were inoculated into 100 mL of liquid nutrient broth (NB) medium at 30 oC at 130 rpm/min for 48 h. For each bacterial culture, one mL of culture was added into 20 mL of M. aeruginosa FACHB 905 cultures. The mixed algal-bacterial cultures were incubated under algal culture conditions, as described above. Bacteria exhibiting algae-lysing activities against M. aeruginosa were selected for further study. 2.3 Identification of bacteria by 16S rDNA sequencing and phylogenetic analysis The results of conventional biochemical tests were identified according to Bergey's manual of systematic bacteriology [15]. The isolates were also identified by phylogenetic analysis. The bacterial 16S rDNA was amplified by PCR using sense primer 5'-CTA CTT TTG CCG GCG AGC GG -3' and antisense primer 5'-TGA TTC CGA AGG CAC TCC C -3' [16] in 50 μL reaction mixture, which contained 25 ng template DNA, 200 μM of dNTP Mixture, 1.5 mM MgCl2, 1 μM of each primer, and 1.0 unit of Taq DNA (Invitrogen, Shanghai, China) polymerase with the buffer (20 mM Tris-HCl, pH 8.4; 50 mM KCl). The PCR ran with 35 thermal cycles of denaturation for 60 seconds at 95 oC, annealing for 60 seconds at 55 oC,
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extension for 60 seconds at 72 oC and a final elongation for 10 min at 72 oC. The PCR products were purified with the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and sequenced at the Invitrogen Biotechnology Corporation in Shanghai. Comparisons of nucleotide sequences were performed using the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/BLAST) and the Ribosomal
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Database Project (RDP) database (http://rdp.cme.msu.edu/seqmatch). Sequences were aligned using the program CLUSTAL W, and a phylogenetic tree was constructed using the neighbor-joining method using the software MEGA 5 [17]. The phylogenetic tree was tested by bootstrap procedure using 1000 random samples. 2.4 Algae-lysing activity of LTH-1 against M. aeruginosa and the mode of algae-lysing action The bacterial culture of LTH-1 was prepared as above and diluted with sterile BG11 to initial densities of 3 × 107 CFU mL-1, then 1 mL was inoculated into 10 mL of exponentially growing cultures of the three strains of M. aeruginosa (3 × 106 cells mL-1 each). To investigate the mechanism of algae-lysing activity, bacterial culture was centrifuged at 5000 × g for 10 min. Then the supernatant was filtered through a 0.22 μm Millipore-Express PES membrane (Millipore, USA). The bacterial culture and bacteria-free filtrate were subsequently added to M. aeruginosa FACHB 905 cultures at the same final volume ratio as described above. In the case of control, equal volumes of sterile NB medium were added to the algal cultures. Cell numbers of M. aeruginosa were counted with a hemocytometer under a microscope (BX41TF, Olympus, Tokyo, Japan) daily for 48 h. The following formula was used to calculate the algae-lysing activity: algae-lysing activity (%) = (1-Tt/Ct) × 100, where T (treatment) and C
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(control) are the cell concentrations of treatments and the control, respectively, and t is the incubation time. All experiments were conducted in triplicate. 2.5 Characterization of algae-lysing substance in LTH-1 2.5.1 Stability of algae-lysing substance To investigate the effect of heat treatment on algae-lysing activity, cell-free filtrates from the
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bacterial cultures were incubated in a water bath at 30 oC, 60 oC, and 120 oC for 20 min. Additionally, to investigate the effect of pH, the filtrates were kept in buffer with pH of 5, 7, and 11 for 30 min as previously described [8]. The treated filtrates (10%, v/v) were subsequently inoculated into M. aeruginosa cultures (3 × 106 cells mL-1). As a control, sterile NB medium was added to the algal cultures instead of cell-free filtrates. 2.5.2 Molecular size of the algae-lysing substance Molecular size of the algae-lysing substance was estimated using a stirred ultrafiltration cell equipped with a 2,000-MW-cutoff and 10,000-MW-cutoff membranes (Sartorius, Germany) [18]. 2.6 Growth of the bacterium and purification of algae-lysing compound in the bacterium culture filtrate. The algae-lysing bacterium was cultured in a 1-liter Erlenmeyer flask containing 400 mL of NB medium. The cells were removed by centrifugation, and supernatant was concentrated by evaporation and stored at -20 oC. The concentrated cell-free supernatant was separated by silica gel column chromatography (2.5 × 30cm, kieselgel 60, Merck, Germany) with gradient elution consisting of chloroform and methanol (from 0 : 1 to 2 : 1, v/v). 2.7 Structural determination of the purified compound.
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For the characterization of the algae-lysing compounds, LC/MS-IT-TOF system was used (Shimadzu Corp., Kyoto, Japan). The separation was performed on a ODS-C18 column (150 mm×2.0 mm, particle size 5 μm) using an elution consisting of 0.1% formic acid and acetonitrile (9 : 1, v/v). The whole analysis took 10 min. The injection volume was 20 μLand the flow rate was 0.2 mL/min. The sample chamber in the autosampler was maintained at 4 oC,
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while the column was set at 40 oC. The total effluent from the detector was transferred directly to the hybrid IT/TOF mass spectrometer without splitting. The mass spectrometer was equipped with an electrospray ionization source and operated in the positive mode. Mass spectrometric analyses were carried out on full-scan MS with a mass range of m/z 100–2000 and data-dependent MS/MS acquisition on the suspected precursor ions. Nitrogen was used as the nebulizing gas set at 1.5 L min-1. The capillary and skimmer voltages were set at 4.5 KV and 1.6 KV, respectively. The curved desolvation line (CDL) and heat block temperatures were both maintained at 200 °C. The MS2 spectra were produced using collision-induced dissociation (CID) of the selected precursor ions using argon as collision gas with relative energy of 50%. Data acquisition and processing were carried out using the LC/MS solution version 3.41 software supplied with the instrument. Any mass numbers corresponding to particular elemental compositions were also calculated by the formula predictor [19]. 3. Results 3.1 Isolation, characterization and identification of algae-lysing bacteria A total of seventy-five bacterial strains were isolated, five isolates showed algicidal activity against M. aeruginosa FACHB 905. The average algae-lysing activities of strains LTH-1, 3, 4, 5, and 6 were 83.9%, 74.2%, 67.8%, 72.6%, and 75.0% in 48 h, respectively. Among these
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five isolates, LTH-1 showed the strongest algae-lysing activity against M. aeruginosa FACHB 905. To identify the strongest algicidal bacterium LTH-1, morphological, biochemical, and genetic analyses were performed. The algicidal bacterium LTH-1 was Gram-negative, rod-shaped, and non-pigmented in a NA plate. LTH-1 cells were elongated and thin according
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to a scanning electron microscope (SEM) (Figure S-2 of online supplementary material). The results of biochemical reactions were as follows: Indole (+), Methyl red (MR) (+), Voges-Proskauer (VP) (-), Catalase (+), Oxidase (+). A phylogenetic tree showed the phylogenetic relationship between LTH-1 strain and the closest relatives (Figure 1). The LTH-1 strain was identified as Aeromonas sp. via culture characters, scanning electron micrograph, and phylogenetic analysis based on 16S rDNA. 3.2 Algae-lysing activity of LTH-1 against M. aeruginosa and the mode of algae-lysing action Rapid lysis of M. aeruginosa TH1, TH2, and FACHB 905 occurred in the presence of LTH-1 cells (Figure 2). After 48 h treatment, the lysis of M. aeruginosa TH1, TH2, and FACHB 905 were 78.0% ± 3.6, 82.7% ± 3.2, and 81.3% ± 5.6, respectively. In addition, the algae-lysing effect of cell-free filtrates against FACHB 905 was 66.5% ± 4.7. These results showed that the bacterium LHT-2 exhibited Microcystis-lysing effects mainly through production of extracellular algae-lysing products. 3.3 Characterization of algae-lysing substances in LTH-1 3.3.1 Stability of algae-lysing substances All cell-free filtrates exhibited algae-lysing activity after incubation at 30 oC, 60 oC, and 120
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o
C for 20 min. The lysis of M. aeruginosa FACHB 905 cells were 22.72% ± 3.13,
33.63%±3.79, and 40.91%±2.34 in 24 h, and 40.45%±2.74, 46.82%±2.66, and 62.27%±4.33 in 48 h, respectively, which indicated that algae-lysing substances were heat-tolerant. When pH of filtrates was adjusted to 5, 7, and 11, the lysis of M. aeruginosa FACHB 905 cells were 23.04%±3.47, 21.51%±3.24, and 42.97%±4.22 in 24 h, and 55.68%±3.93, 51.82%
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±4.77, and 80.18%±5.15 in 48 h, respectively. 3.3.2 Molecular size The results indicated that the molecular size of the algae-lysing compounds from LTH-1 was smaller than 2 kDa (Figure 3). 3.3.3 Hydrophilicity of the algae-lysing substances The cell-free filtrate of LTH-1 was extracted with an equal volume of chloroform or ethyl acetate. As aqueous fraction from the filtrate of LTH-1 had much higher activity against M. aeruginosa FACHB 905 than the organic fraction (Figure 4), this indicated that the active compounds were water-soluble. 3.4 Structural determination of the purified compounds. Two fractions (compound 1 and compound 2) exhibiting algae-lysing activities were collected using silica gel column chromatography. The algae-lysing activity of compound 1 was 28.9% ± 5.1 in 48 h, and the algae-lysing activity of compound 2 was 62.5% ± 3.5 in 48 h. The accurate MS2 spectra of the purified compounds were acquired using LC/MS-IT-TOF (Figure 5). Compound 1 had a retention time of 2.5 min and had a measured elemental composition of C8H16N2O3 ([M+H]+ ion at m/z 190.1232). The fragment ions at m/z 112.0775, 130.0865 and 172.0966 were C6H9NO (predicted 112.0757 Da), C6H11NO2 (predicted 130.0863 Da), and
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C8H13NO3 (predicted 172.0968 Da), respectively. According to the formula predictor software, indicating that NH3 was lost from m/z 189 to form m/z 172, HCOOH was lost from m/z 172 to form m/z 130, and H2O was lost from m/z 130 to form m/z 112. However, the exact structure couldn’t be proposed due to the lack of matching structure in the database of LC/MS-IT-TOF. Compound 2 had a retention time of 3.5 min and had a measured elemental
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composition of C9H11NO2 ([M+H]+ ion at m/z 167.0862), which corresponds to phenylalanine. Moreover, the fragment ions at m/z 147.0463 and 120.0808 were C9H6O2 (predicted147.0441 Da) and C8H9N (predicted 120.0808 Da), respectively. Therefore, compound 2 was identified as phenylalanine. 3.5 Algae-lysing effects of the compound 2 from LTH-1 and standard phenylalanine To confirm the structure and detect the algae-lysing effect of compound 2, compound 2 and standard phenylalanine purchased from Sigma Company were added to 10 mL of exponentially growing cell suspensions of FACHB 905, respectively. The final concentrations of compound 2 and standard phenylalanine in M. aeruginosa cultures were from 12.5 μg mL-1 to
200.0
μg
mL-1.
M.
aeruginosa
FACHB
905
responded
to
them
in
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concentration-dependent manner. The EC50 values of compound 2 and standard phenylalanine were 68.2 ± 8.2 μg mL-1 and 59.2 ± 5.5 μg mL-1 in 48h calculated using SPSS 13.0. 3.6 Nucleotide sequence accession number The nucleotide sequence of LTH-1, 3, 4, 5, and 6 have been deposited in the NCBI nucleotide sequence database and the corresponding accession number are EU814519, EU814515, EU814516, EU814517, and EU814518, respectively. 4. Discussion
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Members of the genus Aeromonas are widely distributed in various aquatic environments, most of which have been associated with human and other animal diseases such as gastroenteritis and wound infections, therefore, Aeromonas species are often studied as human and animal pathogens [20]. However, an indigenous bacterium Aeromonas sp. LTH-1 from Lake Taihu, coexisting with M. aeruginosa, showed strong Microcystis-lysing activities
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against local and standard M. aeruginosa. Aeromonas sp. was reported as a Microcystis-lysing bacterium for the first time in this study. The finding increases our knowledge about the multiple roles of Aeromonas sp. in the aquatic environments. Most of the known algicidal bacteria belong to the CFB phylum and the γ-subclass of the proteobacteria. Moreover, the available data suggested that γ-Proteobacteria employ primarily an indirect mode of attack [7, 21-24]. The cell-free supernatant of LTH-1 showed strong algae-lysing activity and was only a little weaker than the bacterial culture, therefore, the results revealed that LTH-1 lysed M. aeruginosa mainly through extracellular substances. Additionally, the identification showed LTH-1 belonged to the γ-proteobacteria group. Thus, these findings in the study are consistent with the conclusion that γ--proteobacteria primarily employ an indirect mode of attack. LTH-1 is the first finding that an Aeromonas sp. is algae-lysing to M. aeruginosa, and this finding broadens our knowledge on which strains from γ—proteobacteria have algae-lysing activity. The isolation, purification and characterization of algae-lysing compounds are often difficult because of their various characteristic features. An extracellular protease with molecular mass of about 50 kDa had potent algae-lysing activity [18].. An extracellular algae-lysing substance produced by KWR2 was water-soluble with molecular mass < 8 kDa [1]. In this study, two
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Microcystis-lysing compounds produced by Aeromonas sp. LTH-1 were isolated, both of which were water-soluble with molecular mass < 2 kDa. These characters were similar to the anti-Microcystis substance secreted by Streptomyces strain NT0401 (water-soluble with molecular mass < 1 kDa) [25]. The algae-lysing compounds were alkalophilic because the activity against M. aeruginosa FACHB 905 was much higher when the filtrate of LTH-1 was
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in pH =11 instead of pH =5 and 7. Moreover, the filter treated at 30 oC, 60 oC, and 120 oC for 20 min exhibited similar algae-lysing activities, so the algae-lysing compounds excreted by the bacterium LTH-1 were heat-tolerant and unlikely to be enzymes [8]. According to LC/MS-IT-TOF spectra, one Microcystis-lysing compound (compound 1) had a measured elemental composition of C8H16N2O3, the molecular mass of which was 189.1232. The other Microcystis-lysing compound (compound 2) was identified as phenylalanine, which had a measured elemental composition of C9H11NO2. Furthermore, algae-lysing effects are not significantly different between compound 2 and standard phenylalanine. Thus, compound 2 was confirmed as phenylalanine. Our finding that phenylalanine could lyse algae was in accord with the conclusion that some amino acids could selectively kill Microcystis and inhibit Synechocystis effectively [25-28]. In this study, the purified phenylalanine showed a significant algae-lysing activity against Microcystis. The results showed that the phenylalanine from LTH-1 may become useful control agents for removing Microcystis blooms. This work furthers our knowledge regarding the algae-lysing activities of phenylalanine, and other bacterial metabolites, against CyanoHABs. In the future, our work is needed to determine the field application of phenylalanine in lakes and reservoirs. A floating biodegradable plastic carrier immobilizing
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cyanobacteriolytic bacteria, B.cereus N-14, for effective treatment of floating cyanobacteria blooms was demonstrated by Nakamura [29], so it may realize an effective in-situ control of natural cyanobacterial blooms using some floating biodegradable media for immobilization of phenylalanine. 5. Conclusion
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An indigenous bacterium Aeromonas sp. LTH-1 was isolated from Lake Taihu, which showed strong algae-lysing activities against M. aeruginosa, strains local toxic TH1, nontoxic TH2, and standard FACHB 905. Two algae-lysing compounds were isolated and purified from extracellular filtrate of LTH-1. One of them was determined as phenylalanine (C9H11NO2, m/z 166.0862), and the other one (C8H16N2O3, m/z 189.1232) was unidentified. The algicidal Aeromonas sp. LTH-1 could play a role in controlling Microcystis blooms, and its extracellular compounds are potentially useful for regulating blooms of harmful M. aeruginosa. Acknowledgements This work was supported by National Science and Technology Major Project (2009ZX07101-011), National Natural Science Foundation of China (30972440), and Jiangsu Province Postgraduate Innovation Project (CX10B-087Z). We thank Dr. John Pisciotta in Pennsylvania State University for polishing the manuscript.
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degradation products of danofloxacin under stressed conditions, Anal. Bioanal. Chem. 399 (2011), pp. 2475-2486. [20] A. Aberoum and H. Jooyandeh, A Review on Occurrence and Characterization of the Aeromonas Species from Marine, Fishes World Journal of Fish and Mari. 2 (2010), pp. 519-523. [21] J.D. Kim, B. Kim, and C.G. Lee, Alga-lytic activity of Pseudomonas fluorescens against the red tide causing marine alga Heterosigma akashiwo (Raphidophyceae), Biol. Control. 47 (2007), pp. 296-303. [22] P.B. Roth, M.J. Twiner, C.M. Mikulski, A.B. Barnhorst, and G.J. Doucette, Comparative analysis of two algicidal bacteria active against the red tide dinoflagellate Karenia brevis, Harmful Algae. 7 (2008), pp. 682–691. [23] J.Q. Su, X.R. Yang, Y.Y. Zhou, and T.L. Zheng, Marine bacteria antagonistic to the harmful algal bloom strains Alexandrium tamarense (Dinophyceae), Biol. Control. 56 (2011), pp. 132–138. [24] B.X. Wang, Y.Y. Zhou, S.J. Bai, J.Q. Su, Y. Tian, T.L. Zheng, and X.R. Yang, A novel marine bacterium algicidal to the toxic dinoflagellate Alexandrium tamarense, Lett. Appl. Microbiol. 51 (2010), pp. 552-557.
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cyanobacterium, Microcystis, by a Streptomyces sp, Biotechnol. Lett. 31 (2009), pp. 1531-1535. [26] G.C. Hall and R.A. Jensen, Enzymological basis for growth inhibition by L-phenylalanine in the cyanobacterium Synechocystis sp. 29108, J. Bacteriol. 144 (1980), pp. 1034-1042.
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[27] A. Hehmann, K. Kaya, and M.M. Watanabe, Selective control of Microcystis using an amino acid – a laboratory assay, J. Appl. Phycol. 14 (2002), pp. 85–89. [28] K. Kaya, Y.D. Liu, Y.W. Shen, B.D. Xiao, and T. Sano, Selective control of toxic Microcystis water blooms using lysine and malonic acid: an enclosure experiment, Environ. Toxicol. 20 (2005), pp. 170-178. [29] N. Nakamura, K. Nakano, N. Sugiura, and M. Matsumura, A novel control process of cyanobacterial bloom using cyanobacteriolytic bacteria immobilized in floating biodegradable plastic carriers, Environ. Technol. 24 (2003), pp.1569-1576.
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Figures Legends Figure 1. Phylogenetic tree based on bacterial 16S rDNA sequence of the isolated LTH-1 strain and closely related members. Numbers at nodes are levels of bootstrap support (%),
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based on neighbor-joining analyses of 1,000 resampled datasets. Scale bar represents 0.001 nucleotide substitution per position.
Figure 2. The algae-lysing activities of Aeromonas strain LTH-1 on different M. aeruginosa strains in 24 h and 48 h. A: TH1- LTH-1 cultures; B: TH2- LTH-1 cultures; C: FACHB 905LTH-1 cultures; D: FACHB 905- LTH-1 cell-free filtrates. Data are expressed as Mean ± SD.
Figure 3. Algae-lysing activities of different molecular fractions of filtrates from Aeromonas strain LTH-1 against M. aeruginosa FACHB 905 in 24 h and 48 h. Initial filtrate, the bacteria-free supernatant; < 10,000 Da, ulfiltrate containing substances passed through 10,000-MW-cutoff ultrafiltration membranes; < 2,000 Da, ulfiltrate containing substances passed through 2,000-MW-cutoff ultrafiltration membranes.
Figure 4. Algae-lysing activities of different organic solvent extractions from filtrates of Aeromonas sp. LTH-1 on M. aeruginosa FACHB 905 in 24 h and 48 h. H/C, hydrophobic phase extracted by chloroform; A/C, aqueous phase extracted by chloroform; H/E, hydrophobic phase extracted by ethyl acetate; A/E, aqueous phase extracted by ethyl acetate.
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Figure 5. Accurate MS2 spectrum of compound. (a) Accurate MS2 spectrum of compound 1.
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(b) Accurate MS2 spectrum of compound 2.
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Environmental Technology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tent20
Lysing activity of an indigenous algicidal bacterium Aeromonas sp. against Microcystis spp. isolated from Lake Taihu a
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Fei Yang , Xiaoqin Li , Yunhui Li , Haiyan Wei , Guang Yu , Lihong Yin , Geyu Liang & Yuepu Pu
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Key Laboratory of Environmental Medicine Engineering, Ministry of Education, School of Public Health, Southeast University, Nanjing, 210009, China Accepted author version posted online: 26 Nov 2012.
To cite this article: Fei Yang , Xiaoqin Li , Yunhui Li , Haiyan Wei , Guang Yu , Lihong Yin , Geyu Liang & Yuepu Pu (2012): Lysing activity of an indigenous algicidal bacterium Aeromonas sp. against Microcystis spp. isolated from Lake Taihu, Environmental Technology, DOI:10.1080/09593330.2012.752872 To link to this article: http://dx.doi.org/10.1080/09593330.2012.752872
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Lysing activity of an indigenous algicidal bacterium Aeromonas sp. against Microcystis spp. isolated from Lake Taihu Fei Yang, Xiaoqin Li, Yunhui Li, Haiyan Wei, Guang Yu, Lihong Yin, Geyu Liang and Yuepu Pu * Key Laboratory of Environmental Medicine Engineering, Ministry of Education, School of
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Public Health, Southeast University, Nanjing 210009, China
Corresponding author: Mailing address: School of Public Health, Southeast University, 2 Sipailou Road, Nanjing 210009, China. Phone: +86-25-83794996. Fax: +86-25-83324322 E-mail: [email protected]
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Lysing activity of an indigenous algicidal bacterium Aeromonas sp. against Microcystis spp. isolated from Lake Taihu Abstract This study aimed to isolate and characterize an indigenous algicidal bacterium named LTH-1 and its algae-lysing compounds active against three Microcystis aeruginosa strains (toxic TH1, nontoxic TH2,
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and standard FACHB 905). The LTH-1 isolated from Lake Taihu near Wuxi City in China was identified as Aeromonas sp. based on its morphological characteristic features and phylogenetic analysis by sequencing of 16S rDNA. Extracellular compounds produced by LTH-1 showed strong algae-lysing activity, and they were water-soluble, heat-tolerant with molecular mass of lower than 2 kDa. Two algae-lysing compounds were isolated and purified from extracellular filtrate using silica gel column chromatography. One of them was identified as phenylalanine (C9H11NO2, m/z 166.0862) and the other one (C8H16N2O3, m/z 189.1232) was unidentified using hybrid ion trap/time-of-flight mass spectrometry coupled with a high-performance liquid chromatography (LC/MS-IT-TOF) system. The half maximal effective concentration (EC50) of phenylalanine produced by LTH-1 against FACHB 905 was 68.2 ± 8.2 μg mL-1 in 48h. These results suggested that the algicidal Aeromonas sp. LTH-1 could play a role in controlling Microcystis blooms, and its extracellular compounds are potentially useful for regulating blooms of harmful M. aeruginosa. Keywords: Algicidal bacteria, Microcystis aeruginosa, Aeromonas, algae-lysing, phenylalanine.
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1. Introduction Cyanobacterial harmful algal blooms (CyanoHABs) are proliferating worldwide primarily due to eutrophication and climate change. Environmental problems such as severe damage to aquatic ecosystems and human health caused by CyanoHABs in eutrophic lakes, rivers and drinking water reservoirs have been increasingly documented and received more and more
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attention [1]. Microcystis is the most commonly found bloom forming cyanobacterium in the world’s eutrophic and hypereutrophic waters [2]. Especially in China, Microcystis can be dominant year-round in areas of hypereutrophy. Many strains of Microcystis are known to produce cyanobacterial cyclic-peptide hepatotoxins called microcystins, which have been responsible for animal and human poisonings in almost every country that they have been found in [2]. Therefore, controlling Microcystis blooms is a very important goal in environmental health. In order to manage CyanoHABs, several techniques have been used including clay and chemical agents such as copper algicides [3]. These methods are effective in controlling CyanoHABs for a short period after application, but clays have secondary effects on bottom-dwelling organisms, and copper algicides often induce copper toxicity and trigger release of microcystins into water after cyanobacterial cell lysis [3, 4]. Biological control agents such as viruses, protozoa, and bacteria are considered to be a potential solution to regulate CyanoHABs [5]. Some bacteria have algae-lysing effects, which may play an important role in the decline of CyanoHABs. Most of the known algicidal bacteria belong to the Cytophaga-Flavobacterium-Bacteroides (CFB) phylum and the γ-subclass of the proteobacteria including members of genera Alteromonas, Cytophaga, Saprospira,
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Flavobacterium, and Pseudoalteromonas [6, 7]. The remaining strains represent the Gram-positive genera Micrococcus, Bacillus, and Planomicrobium [8]. In general, the bacteria lyse algae through production of extracellular algae-lysing products or by cell-to-cell contact [7]. Lake Taihu (119°54′-120°36′ N, 30°56′-31°33′ E) is the third largest freshwater lake in
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China. It has a total water surface area of about 2338 km2, an average water depth of 1.89 m and a water volume of approximately 4.43 × 1012 L. With the rapid development of industries and agriculture in this region, the lake is becoming more seriously polluted and the water quality decreased. Microcystis blooms have appeared more frequently and larger scales in recent years, especially in Meiliang Bay and Nanquan region of the lake coverage [9-11]. This paper aimed to isolate and characterize an indigenous algicidal bacterium named LTH-1 and its algae-lysing compounds active against three Microcystis aeruginosa strains (toxic TH1, nontoxic TH2, and standard FACHB 905). . 2. Materials and methods 2.1 M. aeruginosa culture Two strains of indigenous M. aeruginosa (TH1 and TH2) were isolated from Lake Taihu during blooms using single-colony isolation technique. The standard M. aeruginosa FACHB 905 was purchased from the Freshwater Algae Culture Collection of the Institute of Hydrobiology, Chinese Academy of Sciences (Figure S-1 of online supplementary material). All three strains of cyanobacteria used in the study contained phycocyanin intergenic spacer (PC-IGS) as determined by PCR method [12]. TH1 and FACHB 905 contained microcystin synthetase gene B (mcyB) and produced microcystin-LR. TH2 neither contained mcyB nor
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produced microcystin-LR. The M. aeruginosa cells were incubated in sterile BG-11 medium [13]. The M. aeruginosa strains were maintained as unialgal axenic culture at 28 oC, pH 7.2, with illumination at 150 μmol photons m-2 s-1 under a 12-h light-12-h dark regimen. 2.2 Isolation and screening of algicidal bacteria against M. aeruginosa The algicidal bacteria were isolated from an immobilized biosystem, which is a synthetic
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medium of Vinlon immobilizing indigenous microbes in Meiliang Bay of Taihu Lake, China [14]. Samples were serially diluted with sterile water and 0.1 mL aliquots of each dilution were inoculated onto a nutrient agar (NA, 2% agar) plates. Isolated bacteria were inoculated into 100 mL of liquid nutrient broth (NB) medium at 30 oC at 130 rpm/min for 48 h. For each bacterial culture, one mL of culture was added into 20 mL of M. aeruginosa FACHB 905 cultures. The mixed algal-bacterial cultures were incubated under algal culture conditions, as described above. Bacteria exhibiting algae-lysing activities against M. aeruginosa were selected for further study. 2.3 Identification of bacteria by 16S rDNA sequencing and phylogenetic analysis The results of conventional biochemical tests were identified according to Bergey's manual of systematic bacteriology [15]. The isolates were also identified by phylogenetic analysis. The bacterial 16S rDNA was amplified by PCR using sense primer 5'-CTA CTT TTG CCG GCG AGC GG -3' and antisense primer 5'-TGA TTC CGA AGG CAC TCC C -3' [16] in 50 μL reaction mixture, which contained 25 ng template DNA, 200 μM of dNTP Mixture, 1.5 mM MgCl2, 1 μM of each primer, and 1.0 unit of Taq DNA (Invitrogen, Shanghai, China) polymerase with the buffer (20 mM Tris-HCl, pH 8.4; 50 mM KCl). The PCR ran with 35 thermal cycles of denaturation for 60 seconds at 95 oC, annealing for 60 seconds at 55 oC,
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extension for 60 seconds at 72 oC and a final elongation for 10 min at 72 oC. The PCR products were purified with the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and sequenced at the Invitrogen Biotechnology Corporation in Shanghai. Comparisons of nucleotide sequences were performed using the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/BLAST) and the Ribosomal
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Database Project (RDP) database (http://rdp.cme.msu.edu/seqmatch). Sequences were aligned using the program CLUSTAL W, and a phylogenetic tree was constructed using the neighbor-joining method using the software MEGA 5 [17]. The phylogenetic tree was tested by bootstrap procedure using 1000 random samples. 2.4 Algae-lysing activity of LTH-1 against M. aeruginosa and the mode of algae-lysing action The bacterial culture of LTH-1 was prepared as above and diluted with sterile BG11 to initial densities of 3 × 107 CFU mL-1, then 1 mL was inoculated into 10 mL of exponentially growing cultures of the three strains of M. aeruginosa (3 × 106 cells mL-1 each). To investigate the mechanism of algae-lysing activity, bacterial culture was centrifuged at 5000 × g for 10 min. Then the supernatant was filtered through a 0.22 μm Millipore-Express PES membrane (Millipore, USA). The bacterial culture and bacteria-free filtrate were subsequently added to M. aeruginosa FACHB 905 cultures at the same final volume ratio as described above. In the case of control, equal volumes of sterile NB medium were added to the algal cultures. Cell numbers of M. aeruginosa were counted with a hemocytometer under a microscope (BX41TF, Olympus, Tokyo, Japan) daily for 48 h. The following formula was used to calculate the algae-lysing activity: algae-lysing activity (%) = (1-Tt/Ct) × 100, where T (treatment) and C
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(control) are the cell concentrations of treatments and the control, respectively, and t is the incubation time. All experiments were conducted in triplicate. 2.5 Characterization of algae-lysing substance in LTH-1 2.5.1 Stability of algae-lysing substance To investigate the effect of heat treatment on algae-lysing activity, cell-free filtrates from the
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bacterial cultures were incubated in a water bath at 30 oC, 60 oC, and 120 oC for 20 min. Additionally, to investigate the effect of pH, the filtrates were kept in buffer with pH of 5, 7, and 11 for 30 min as previously described [8]. The treated filtrates (10%, v/v) were subsequently inoculated into M. aeruginosa cultures (3 × 106 cells mL-1). As a control, sterile NB medium was added to the algal cultures instead of cell-free filtrates. 2.5.2 Molecular size of the algae-lysing substance Molecular size of the algae-lysing substance was estimated using a stirred ultrafiltration cell equipped with a 2,000-MW-cutoff and 10,000-MW-cutoff membranes (Sartorius, Germany) [18]. 2.6 Growth of the bacterium and purification of algae-lysing compound in the bacterium culture filtrate. The algae-lysing bacterium was cultured in a 1-liter Erlenmeyer flask containing 400 mL of NB medium. The cells were removed by centrifugation, and supernatant was concentrated by evaporation and stored at -20 oC. The concentrated cell-free supernatant was separated by silica gel column chromatography (2.5 × 30cm, kieselgel 60, Merck, Germany) with gradient elution consisting of chloroform and methanol (from 0 : 1 to 2 : 1, v/v). 2.7 Structural determination of the purified compound.
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For the characterization of the algae-lysing compounds, LC/MS-IT-TOF system was used (Shimadzu Corp., Kyoto, Japan). The separation was performed on a ODS-C18 column (150 mm×2.0 mm, particle size 5 μm) using an elution consisting of 0.1% formic acid and acetonitrile (9 : 1, v/v). The whole analysis took 10 min. The injection volume was 20 μLand the flow rate was 0.2 mL/min. The sample chamber in the autosampler was maintained at 4 oC,
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while the column was set at 40 oC. The total effluent from the detector was transferred directly to the hybrid IT/TOF mass spectrometer without splitting. The mass spectrometer was equipped with an electrospray ionization source and operated in the positive mode. Mass spectrometric analyses were carried out on full-scan MS with a mass range of m/z 100–2000 and data-dependent MS/MS acquisition on the suspected precursor ions. Nitrogen was used as the nebulizing gas set at 1.5 L min-1. The capillary and skimmer voltages were set at 4.5 KV and 1.6 KV, respectively. The curved desolvation line (CDL) and heat block temperatures were both maintained at 200 °C. The MS2 spectra were produced using collision-induced dissociation (CID) of the selected precursor ions using argon as collision gas with relative energy of 50%. Data acquisition and processing were carried out using the LC/MS solution version 3.41 software supplied with the instrument. Any mass numbers corresponding to particular elemental compositions were also calculated by the formula predictor [19]. 3. Results 3.1 Isolation, characterization and identification of algae-lysing bacteria A total of seventy-five bacterial strains were isolated, five isolates showed algicidal activity against M. aeruginosa FACHB 905. The average algae-lysing activities of strains LTH-1, 3, 4, 5, and 6 were 83.9%, 74.2%, 67.8%, 72.6%, and 75.0% in 48 h, respectively. Among these
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five isolates, LTH-1 showed the strongest algae-lysing activity against M. aeruginosa FACHB 905. To identify the strongest algicidal bacterium LTH-1, morphological, biochemical, and genetic analyses were performed. The algicidal bacterium LTH-1 was Gram-negative, rod-shaped, and non-pigmented in a NA plate. LTH-1 cells were elongated and thin according
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to a scanning electron microscope (SEM) (Figure S-2 of online supplementary material). The results of biochemical reactions were as follows: Indole (+), Methyl red (MR) (+), Voges-Proskauer (VP) (-), Catalase (+), Oxidase (+). A phylogenetic tree showed the phylogenetic relationship between LTH-1 strain and the closest relatives (Figure 1). The LTH-1 strain was identified as Aeromonas sp. via culture characters, scanning electron micrograph, and phylogenetic analysis based on 16S rDNA. 3.2 Algae-lysing activity of LTH-1 against M. aeruginosa and the mode of algae-lysing action Rapid lysis of M. aeruginosa TH1, TH2, and FACHB 905 occurred in the presence of LTH-1 cells (Figure 2). After 48 h treatment, the lysis of M. aeruginosa TH1, TH2, and FACHB 905 were 78.0% ± 3.6, 82.7% ± 3.2, and 81.3% ± 5.6, respectively. In addition, the algae-lysing effect of cell-free filtrates against FACHB 905 was 66.5% ± 4.7. These results showed that the bacterium LHT-2 exhibited Microcystis-lysing effects mainly through production of extracellular algae-lysing products. 3.3 Characterization of algae-lysing substances in LTH-1 3.3.1 Stability of algae-lysing substances All cell-free filtrates exhibited algae-lysing activity after incubation at 30 oC, 60 oC, and 120
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o
C for 20 min. The lysis of M. aeruginosa FACHB 905 cells were 22.72% ± 3.13,
33.63%±3.79, and 40.91%±2.34 in 24 h, and 40.45%±2.74, 46.82%±2.66, and 62.27%±4.33 in 48 h, respectively, which indicated that algae-lysing substances were heat-tolerant. When pH of filtrates was adjusted to 5, 7, and 11, the lysis of M. aeruginosa FACHB 905 cells were 23.04%±3.47, 21.51%±3.24, and 42.97%±4.22 in 24 h, and 55.68%±3.93, 51.82%
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±4.77, and 80.18%±5.15 in 48 h, respectively. 3.3.2 Molecular size The results indicated that the molecular size of the algae-lysing compounds from LTH-1 was smaller than 2 kDa (Figure 3). 3.3.3 Hydrophilicity of the algae-lysing substances The cell-free filtrate of LTH-1 was extracted with an equal volume of chloroform or ethyl acetate. As aqueous fraction from the filtrate of LTH-1 had much higher activity against M. aeruginosa FACHB 905 than the organic fraction (Figure 4), this indicated that the active compounds were water-soluble. 3.4 Structural determination of the purified compounds. Two fractions (compound 1 and compound 2) exhibiting algae-lysing activities were collected using silica gel column chromatography. The algae-lysing activity of compound 1 was 28.9% ± 5.1 in 48 h, and the algae-lysing activity of compound 2 was 62.5% ± 3.5 in 48 h. The accurate MS2 spectra of the purified compounds were acquired using LC/MS-IT-TOF (Figure 5). Compound 1 had a retention time of 2.5 min and had a measured elemental composition of C8H16N2O3 ([M+H]+ ion at m/z 190.1232). The fragment ions at m/z 112.0775, 130.0865 and 172.0966 were C6H9NO (predicted 112.0757 Da), C6H11NO2 (predicted 130.0863 Da), and
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C8H13NO3 (predicted 172.0968 Da), respectively. According to the formula predictor software, indicating that NH3 was lost from m/z 189 to form m/z 172, HCOOH was lost from m/z 172 to form m/z 130, and H2O was lost from m/z 130 to form m/z 112. However, the exact structure couldn’t be proposed due to the lack of matching structure in the database of LC/MS-IT-TOF. Compound 2 had a retention time of 3.5 min and had a measured elemental
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composition of C9H11NO2 ([M+H]+ ion at m/z 167.0862), which corresponds to phenylalanine. Moreover, the fragment ions at m/z 147.0463 and 120.0808 were C9H6O2 (predicted147.0441 Da) and C8H9N (predicted 120.0808 Da), respectively. Therefore, compound 2 was identified as phenylalanine. 3.5 Algae-lysing effects of the compound 2 from LTH-1 and standard phenylalanine To confirm the structure and detect the algae-lysing effect of compound 2, compound 2 and standard phenylalanine purchased from Sigma Company were added to 10 mL of exponentially growing cell suspensions of FACHB 905, respectively. The final concentrations of compound 2 and standard phenylalanine in M. aeruginosa cultures were from 12.5 μg mL-1 to
200.0
μg
mL-1.
M.
aeruginosa
FACHB
905
responded
to
them
in
a
concentration-dependent manner. The EC50 values of compound 2 and standard phenylalanine were 68.2 ± 8.2 μg mL-1 and 59.2 ± 5.5 μg mL-1 in 48h calculated using SPSS 13.0. 3.6 Nucleotide sequence accession number The nucleotide sequence of LTH-1, 3, 4, 5, and 6 have been deposited in the NCBI nucleotide sequence database and the corresponding accession number are EU814519, EU814515, EU814516, EU814517, and EU814518, respectively. 4. Discussion
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Members of the genus Aeromonas are widely distributed in various aquatic environments, most of which have been associated with human and other animal diseases such as gastroenteritis and wound infections, therefore, Aeromonas species are often studied as human and animal pathogens [20]. However, an indigenous bacterium Aeromonas sp. LTH-1 from Lake Taihu, coexisting with M. aeruginosa, showed strong Microcystis-lysing activities
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against local and standard M. aeruginosa. Aeromonas sp. was reported as a Microcystis-lysing bacterium for the first time in this study. The finding increases our knowledge about the multiple roles of Aeromonas sp. in the aquatic environments. Most of the known algicidal bacteria belong to the CFB phylum and the γ-subclass of the proteobacteria. Moreover, the available data suggested that γ-Proteobacteria employ primarily an indirect mode of attack [7, 21-24]. The cell-free supernatant of LTH-1 showed strong algae-lysing activity and was only a little weaker than the bacterial culture, therefore, the results revealed that LTH-1 lysed M. aeruginosa mainly through extracellular substances. Additionally, the identification showed LTH-1 belonged to the γ-proteobacteria group. Thus, these findings in the study are consistent with the conclusion that γ--proteobacteria primarily employ an indirect mode of attack. LTH-1 is the first finding that an Aeromonas sp. is algae-lysing to M. aeruginosa, and this finding broadens our knowledge on which strains from γ—proteobacteria have algae-lysing activity. The isolation, purification and characterization of algae-lysing compounds are often difficult because of their various characteristic features. An extracellular protease with molecular mass of about 50 kDa had potent algae-lysing activity [18].. An extracellular algae-lysing substance produced by KWR2 was water-soluble with molecular mass < 8 kDa [1]. In this study, two
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Microcystis-lysing compounds produced by Aeromonas sp. LTH-1 were isolated, both of which were water-soluble with molecular mass < 2 kDa. These characters were similar to the anti-Microcystis substance secreted by Streptomyces strain NT0401 (water-soluble with molecular mass < 1 kDa) [25]. The algae-lysing compounds were alkalophilic because the activity against M. aeruginosa FACHB 905 was much higher when the filtrate of LTH-1 was
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in pH =11 instead of pH =5 and 7. Moreover, the filter treated at 30 oC, 60 oC, and 120 oC for 20 min exhibited similar algae-lysing activities, so the algae-lysing compounds excreted by the bacterium LTH-1 were heat-tolerant and unlikely to be enzymes [8]. According to LC/MS-IT-TOF spectra, one Microcystis-lysing compound (compound 1) had a measured elemental composition of C8H16N2O3, the molecular mass of which was 189.1232. The other Microcystis-lysing compound (compound 2) was identified as phenylalanine, which had a measured elemental composition of C9H11NO2. Furthermore, algae-lysing effects are not significantly different between compound 2 and standard phenylalanine. Thus, compound 2 was confirmed as phenylalanine. Our finding that phenylalanine could lyse algae was in accord with the conclusion that some amino acids could selectively kill Microcystis and inhibit Synechocystis effectively [25-28]. In this study, the purified phenylalanine showed a significant algae-lysing activity against Microcystis. The results showed that the phenylalanine from LTH-1 may become useful control agents for removing Microcystis blooms. This work furthers our knowledge regarding the algae-lysing activities of phenylalanine, and other bacterial metabolites, against CyanoHABs. In the future, our work is needed to determine the field application of phenylalanine in lakes and reservoirs. A floating biodegradable plastic carrier immobilizing
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cyanobacteriolytic bacteria, B.cereus N-14, for effective treatment of floating cyanobacteria blooms was demonstrated by Nakamura [29], so it may realize an effective in-situ control of natural cyanobacterial blooms using some floating biodegradable media for immobilization of phenylalanine. 5. Conclusion
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An indigenous bacterium Aeromonas sp. LTH-1 was isolated from Lake Taihu, which showed strong algae-lysing activities against M. aeruginosa, strains local toxic TH1, nontoxic TH2, and standard FACHB 905. Two algae-lysing compounds were isolated and purified from extracellular filtrate of LTH-1. One of them was determined as phenylalanine (C9H11NO2, m/z 166.0862), and the other one (C8H16N2O3, m/z 189.1232) was unidentified. The algicidal Aeromonas sp. LTH-1 could play a role in controlling Microcystis blooms, and its extracellular compounds are potentially useful for regulating blooms of harmful M. aeruginosa. Acknowledgements This work was supported by National Science and Technology Major Project (2009ZX07101-011), National Natural Science Foundation of China (30972440), and Jiangsu Province Postgraduate Innovation Project (CX10B-087Z). We thank Dr. John Pisciotta in Pennsylvania State University for polishing the manuscript.
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Figures Legends Figure 1. Phylogenetic tree based on bacterial 16S rDNA sequence of the isolated LTH-1 strain and closely related members. Numbers at nodes are levels of bootstrap support (%),
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based on neighbor-joining analyses of 1,000 resampled datasets. Scale bar represents 0.001 nucleotide substitution per position.
Figure 2. The algae-lysing activities of Aeromonas strain LTH-1 on different M. aeruginosa strains in 24 h and 48 h. A: TH1- LTH-1 cultures; B: TH2- LTH-1 cultures; C: FACHB 905LTH-1 cultures; D: FACHB 905- LTH-1 cell-free filtrates. Data are expressed as Mean ± SD.
Figure 3. Algae-lysing activities of different molecular fractions of filtrates from Aeromonas strain LTH-1 against M. aeruginosa FACHB 905 in 24 h and 48 h. Initial filtrate, the bacteria-free supernatant; < 10,000 Da, ulfiltrate containing substances passed through 10,000-MW-cutoff ultrafiltration membranes; < 2,000 Da, ulfiltrate containing substances passed through 2,000-MW-cutoff ultrafiltration membranes.
Figure 4. Algae-lysing activities of different organic solvent extractions from filtrates of Aeromonas sp. LTH-1 on M. aeruginosa FACHB 905 in 24 h and 48 h. H/C, hydrophobic phase extracted by chloroform; A/C, aqueous phase extracted by chloroform; H/E, hydrophobic phase extracted by ethyl acetate; A/E, aqueous phase extracted by ethyl acetate.
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Figure 5. Accurate MS2 spectrum of compound. (a) Accurate MS2 spectrum of compound 1.
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(b) Accurate MS2 spectrum of compound 2.
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