Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
LIMNOLOGY and OCEANOGRAPHY: METHODS Limnol. Oceanogr.: Methods 6, 2008, 513–522 © 2008, by the American Society of Limnology and Oceanography, Inc. In situ bacterial mitigation of the toxic cyanobacterium Microcystis aeruginosa: implications for biological bloom control Baik-Ho Kim1*, Miao Sang2,3, Soon-Jin Hwang1, and Myung-Soo Han2* 1 Department of Environmental Science, Konkuk University, Seoul 143-701, Republic of Korea Department of Life Science, Hanyang University, Seoul 133-791, Republic of Korea 3 Department of Environmental Microbiology, Harbin Institute of Technology, Harbin 150-001, China 2 Abstract The algicidal bacterium Xanthobacter autotrophicus HYS0201-SM02 (SM02) was isolated from the surface water of a eutrophic lake (Lake Daechung, Korea). In vivo and in situ experiments showed that SM02 had algicidal activity against both a cultured strain and natural colonial morphs of the toxic cyanobacterium, Microcystis aeruginosa. Both the SM02 bacteria and its culture filtrate showed anti-algal activity against M. aeruginosa, indicating that an algicidal substance was released from SM02. The threshold concentration of SM02 for maximal algicidal activity against a natural bloom of M. aeruginosa was 107 CFU/mL. In situ co-culture of SM02 and M. aeruginosa showed that SM02 did not benefit from the massive decay of M. aeruginosa. In fact, repeated inoculations with a low concentration of SM02 were required for optimal algicidal activity, suggesting that water quality worsened during co-culture (i.e., nutrients and microcystin-LR concentration increased). These results suggest a role for the algicidal bacterium X. autotrophicus SM02 in biorestoration but probably not in treating outdoor Microcystis blooms. When developing a biological agent to control M. aeruginosa blooms in the field, it will be important to screen for specific agents with low threshold concentrations and high algicidal activity. Introduction Direct application of chemicals to control algal blooms can harm aquatic ecosystems by killing beneficial organisms such as plankton and fish (McGuire et al. 1984; Reynolds 1984; Reyssac and Pletikosic 1990). Therefore, many researchers have taken pan-ecological and environmental approaches to lake conservation by investigating the potential for biological control of cyanobacterial blooms, mainly using Microcystis aeruginosa as a model system (Redhead and Wright 1978; Brabrand et al. 1983; Manage et al. 2000; Choi et al. 2005; Kim et al. 2007). So far, most reports have been from laboratory studies, and the biological control of algal blooms in situ has not been adequately assessed. There is ongoing debate, particularly among Japanese researchers, regarding the effect of heterotrophic bacteria on the growth dynamics of harmful freshwater and marine algae (Yamamoto et al. 1998; Manage et al. 2000; Kodani et al. 2002). Although numerous laboratory studies have demonstrated anti-algal effects of some bacteria on marine and freshwater algae species (Daft et al. 1975; Sigee et al. 1999; Lee et al. 2000; Mayali and Azam 2004; Kim et al. 2007), the use of M. aeruginosa as an agent of biological control in situ, that is, under natural conditions or in bloom water, is complicated. Choi et al. (2005) demonstrated that the Microcystis-killing bacterium Streptomyces did not continue to grow as the number of algae declined and that bacterial growth required the As a result of eutrophication, cyanobacterial blooms have become common in lakes and reservoirs worldwide (EkmanEkebom et al. 1992). Such blooms result in foul odors, decreased aesthetic value, deteriorated water quality, and water deoxygenation (Falconer 1999; Oberholster et al. 2004). Cyanobacterial blooms can also lead to the production of microcystins (MCs) that can be toxic to aquatic organisms including fish, birds, wild animals, livestock, waterfowl, as well as to humans (Carmichael et al. 1975; Codd et al. 1989; Jacquet et al. 2004; Ernst et al. 2006; Palikova et al. 2007). MCs are also associated with allergies, irritant reactions, gastroenteritis, liver disease, and tumors in humans (An and Carmichael 1994; Bell and Codd 1994; Dawson 1998; Almeida et al. 2006). *Corresponding authors: E-mail: [email protected] (B.H.K), [email protected] (M.S.H) Acknowledgments We are grateful to Dr. N. Takamura and J. P. Gaur for valuable comments on earlier versions of the manuscript. S.W. Jung and J.K. Seo provided field and technical assistance. Han-River Environmental Research Laboratory (HERL) provided a small pond for the field study. This work was supported by a Korea Research Foundation Grant (KRF-2004-C00018). 513 Kim et al. Bacterial mitigation of M. aeruginosa developed on the lawn. Bacteria associated with these zones were isolated by serial streaking onto nutrient agar (NA) plates. The isolated anti-algal bacteria were cultured in nutrient broth (NB) in the dark at the optimal temperature of 40°C, with gentle orbital shaking (120 rpm). Bacterial biomass was determined by dry weight (APHA 1998). The isolates were maintained axenically in the dark on NA plates and cryopreserved at –76°C in NB containing 20% glycerol. Identification of algicidal bacteria—To identify the algicidal bacterial isolates, bacterial chromosomal DNA was isolated according to the method described by Choi et al. (2005), and molecular identification was performed as in Wellinghausen et al. (2005). Briefly, 16S rDNA was amplified using primers 27F, 5′-GAGTTTGATCATGGCTCAG-3′ and 1492R, 5′-GGTTACCTTGTTACGACTT-3′, in a 50 μL reaction volume containing 20 ng of template DNA, 1× PCR buffer (Sigma), 5 mM MgCl2, 10 mM dNTPs, 10 pM of each primer, and 2.5 units of Taq DNA polymerase. The PCR consisted of 35 cycles of denaturation for 1 min at 94°C, annealing for 2.5 min at 55°C, extension for 2.5 min at 72°C, and a final elongation step of 7 min at 72°C in a DNA thermal cycler (Genetic analyzer 377; Perkin-Elmer). The PCR products were purified with the QIA quick PCR Purification Kit (Qiagen) and sequenced by automated DNA sequencing (Bionex). The resulting 16S rDNA sequences were aligned using CLUSTAL W software (Thompson et al. 1994), and the distance matrices were calculated using the DNADIST program within the PHYLIP package (http://evolution.genetics.washington.edu/phylip.html). A phylogenetic tree was constructed by the neighbor-joining method based on the calculated distance matrix. Identification of the HYS0201-SM02 isolate (SM02) was determined by full-length 16S rDNA sequence comparison with sequences in the DDMJ/EMBL/Genbank database. The sequence of the SM02 isolate was similar to the sequences of various strains of Xanthobacter and was identical to those of X. autotrophicus T101 (GenBank accession no. U62887), T102 (U62888), and 7c (X94201) (Fig. 1). The sequences of the SM02 isolate were deposited in GenBank under the accession number EU982303. These aerobic gram-negative bacteria are known to decompose 1,2-dichloroethane, a toxic, halogenated compound commonly used in the production of vinyl chloride (Baptista et al. 2006) and have been reported to produce the acetone-using enzyme, acetone carboxylase (Sluis and Ensign 1997; Sluis et al. 2002). Optimal growth of algicidal bacteria—To determine the optimal growth conditions for SM02, the isolate was grown with gentle shaking in NB (pH 7) at 25°C, 30°C, 35°C, 40°C, 45°C, and 50°C. It was also grown in NB at 40°C at various pHs (5, 6, 7, 8, 9, and 10). Bacterial growth was quantified by 4’,6diadimino-2-phenylindole (DAPI) staining (Porter and Feig 1980). Briefly, bacteria fixed with 2% glutaraldehyde (final concentration) were filtered onto black polycarbonate filters (0.6-μm pore diameter) and counted at 1000× magnification using an epifluorescence microscope (Karl Zeiss). The optimal growth conditions determined by these experiments were addition of nutrients (e.g., casitone), because the target algae were not a sufficient source of food. The need for added nutrients when applying bacteria for algal bloom control in eutrophic waters is an important constraint. Sigee et al. (1999) and Mayali and Azam (2004) also reported that the conditions optimal for growth of algicidal bacteria can differ from those used in experiments and those used for algal growth control. Typically, conditions optimal for the growth of the target alga are used in co-culture experiments involving bacteria, potentially resulting in low algicidal activity. Evidence that algicidal bacteria can control blooms of M. aeruginosa is based on reports of increased bacterial abundance correlating with the termination of cyanobacterial blooms (Shilo 1967; Daft et al. 1975; Rashidan and Bird 2001). Investigation into the algicidal mechanisms of bacteria has shown that some bacteria actually increase the concentration of dissolved MCs via lysis of Microcystis cells (Choi et al. 2005; Kim et al. 2007). The mechanism of MC-mediated destruction of Microcystis and the relationship between MC and algicidal bacteria remain unclear. Moreover, there are no reports about using algicidal bacteria to control colonial morphs of M. aeruginosa in situ. In this report, we describe bacteria-mediated control of M. aeruginosa. Specifically, we isolated and characterized a Microcystis-lytic bacterium (Xanthobacter autotrophicus) with host specificity. The algicidal activity of X. autotrophicus was assessed at different bacterial densities as well as at different bacterial and algal growth phases. Finally, we performed an in situ test to determine whether the algicidal activity (AA) persisted in suboptimal bacterial growth conditions within a natural phytoplankton community. Materials and procedures Study of algae and algicidal bacterial isolation—The toxic cyanobacteria M. aeruginosa NIER-100001 were obtained from The National Institute for Environmental Research (NIER, Korea) and cultured in modified Cary-Blair (CB) medium (Watanabe and Hiroki 1997) on an orbital shaker (120 rpm) at 25°C with a 12 h:12 h light:dark cycle (light intensity 50 μE m–2 s–1). The M. aeruginosa cells used in this study were spherical or ovoid, 3.7 to 5.7 μm in diameter, and 27 to 97 ìm3 in volume. Bacteria with algicidal activity against M. aeruginosa NIER100001 were isolated from an algal lawn or through the use of a modified soft agar overlay method (Sakata et al. 1991). Surface water samples were collected from Lake Daechung, Korea, during an algal bloom on June 12, 2004, and were then filtered through 0.8-μm nucleopore membrane filters. Cultures of M. aeruginosa N-100001 were grown in CB medium for 7 d and harvested by centrifugation at 3000×g for 20 min. The cell pellet was mixed with molten CB soft agar medium and poured into a plastic Petri dish (87-mm diameter). A cyanobacterial lawn developed after 2 to 3 d, and 200-μL water samples were spread onto the lawn. After incubation for 5 to 10 d, the plates were examined for the presence of clear zones (indicating inhibition of cyanobacterial growth) around bacterial colonies that 514 Kim et al. Bacterial mitigation of M. aeruginosa 100 mL flasks containing 50 mL of mid-exponential phase M. aeruginosa (13.57 × 105 cells mL–1, cultured for 13 d). The M. aeruginosa cells were counted daily, as described below. Anti-algal activity of SM02 against other algae—We tested the AA of SM02 in liquid cultures of cyanobacteria Anabaena cylindrica (NIES-19; NIES; National Institute for Environmental Studies), A. flos-aquae (KCTC-AG10026; KCTC; Korean Collection for Type Cultures, KRIBB, Daejeon, Korea), and M. aeruginosa (strains NIES-44, NIES-101, and NIES-298), and the diatoms Aulacoseira granulata (CCAP 1002/1; CCAP; Culture Collection of Algae and Protozoa), Cyclotella meneghiniana (previously isolated by Han, M.S.), and Stephanodiscus hantzschii (UTCC267; UTCC; University of Toronto Culture Collection of Algae and Cyanobacteria). Briefly, SM02 was cultured in NB in the dark (24 h, 40°C, orbital shaking at 150 rpm). The bacteria were harvested by centrifugation at 14,000×g for 20 min, and inoculated to the cultures of each algal species, while the final bacterial density was adjusted to 1×108 cells mL–1 for all algal species. Algae samples were collected from exponential-phase cultures. The cyanobacteria were cultured in 300-mL flasks containing 100 mL sterilized CB medium, as described previously (25°C, light intensity 50 μE m–2 s–1, 12 h:12 h light:dark cycle, orbital shaking at 120 rpm). The three types of diatoms were cultured at 20°C in 300-mL flasks containing 100 mL sterilized diatom medium (DM), pH 6.9 (Beakes et al. 1988). The light intensity, light:dark cycle, and shaking conditions were as for cyanobacteria. The AA of SM02 was calculated using the same equation as for the cyanobacteria. In situ anti-algal activity of SM02 on natural Microcystis bloom water—In 1981, we constructed a small artificial concrete pond (ca. 300 m3) directly connected to the lower part of the Han River (Paltang reservoir, Korea) for the purpose of field experiments. Since its construction, the study pond has developed Microcystis blooms every year between April and November. The cyanobacterial blooms are composed of over 90% M. aeruginosa and ~10% other phytoplankton species. To assess the AA of SM02 in natural Microcystis bloom water, we conducted an in situ experiment over 11 d (June 14 to June 24, 2004) in 12 cone-shaped PVC tanks. The PVC tanks (0.8 m high; total volume 120 L) contained 100 L water supporting a natural Microcystis bloom. The tanks were treated with SM02 as follows: 1) no added bacteria (Control); high concentration of bacteria (HD, 1 × 108 CFU/mL); midconcentration of bacteria (MD, 5 × 107 CFU/mL); low concentration of bacteria (LD, 1 × 107 CFU/mL), and a bi-daily low-concentration (1 × 107 CFU/mL) inoculation performed five times during the study (BLD, final added concentration, 5 × 107 CFU/mL, the same as for MD). All experiments were performed in triplicate. The SM02 bacteria, prepared as described below, were sprayed onto the water surface of each tank on the fifth day of the 11-day experiment. Axenic cultures of SM02 were maintained in the dark on NA plates (1.5% agar; pH 7) at 20°C, or Fig. 1. Phylogenetic tree based on 16S rDNA sequences, showing the relative positions of the Microcystis-killing bacterium HYS0201-SM02, the type strains of some Xanthobacter species, and the type strain of Xanthobacter autotrophicus. Scale bar represents 0.01 substitutions per nucleotide position. used for all subsequent bacterial cultures. Anti-algal activity of SM02 on M. aeruginosa—Two methods, a paper disc test and a liquid culture test, were used to assess the anti-algal activity of the bacterial isolates and to select the isolate most effective against M. aeruginosa. The paper disc method involved growing the isolates in NB medium at 40°C with shaking for 2 d. Discs of Whatman GF/F filter paper (pore size = 0.7 μm; 5-mm diameter) were soaked with 100 μL of each cultured bacterial isolate, placed on lawns of M. aeruginosa, and incubated for 5 d. Anti-algal activity was recorded as the diameter of the clear zone that formed in the algal lawn. For the liquid culture test, a bacterial culture was washed with algal medium and then inoculated into a 50-mL test tube containing 25 mL of M. aeruginosa N-100001 culture. The co-culture was incubated for 5 d. Each day, bacterial growth was determined by the DAPI method, and algal growth was measured by direct counting using an inverted microscope (Utermöhl 1958). To assess the effect of SM02 on M. aeruginosa during different phases of algal and bacterial growth, a culture of SM02 was prepared as described above, adjusted to an optical density of 1.8 at 660 nm, and 2.5 mL inoculated into 100 mL flasks containing 50 mL of lag, exponential, or stationary phase M. aeruginosa cells (2.3, 20.7, and 22.7 × 105 cells mL–1, respectively). We also measured the AA of SM02 at different growth phases (lag, exponential, and stationary phase) on M. aeruginosa in exponential phase. Aliquots of SM02 were incubated for 3 h (lag phase), 18 h (exponential phase), and 36 h (stationary phase) in 100 mL flasks containing 50 mL of NB. The cells were harvested by centrifugation at 18,000×g for 20 min, the cell concentration was adjusted to 1.8 at 660 nm, and 2.5 mL (approximately 1 × 108 cells mL–1) of culture was inoculated into 515 Kim et al. Bacterial mitigation of M. aeruginosa each sample, filtered with glass filters (25 mm diameter, Whatman GF/C), and extracted with 10 mL of 90% methanol for 24 h at room temperature. Each methanol-extracted sample (10 to 50 μL) was neutralized or diluted with distilled water (90 to 50 μL). The samples or standards were mixed with an appropriate dilution of M8H5 MAb and dispensed in a 96-well microtiter plate (Coaster) pre-coated with an MC-bovine serum albumin conjugate. The plates were washed, and the bound MAb was detected with horseradish peroxidase-labeled goat anti-mouse IgG (TAGO 4550) and its substrate (0.1 mg/mL of 3,3′,5,5′-tetramethylbenzidine, 0.05% H2O2 in 0.1 M acetate buffer, pH 5.0). Finally, absorbance was measured at 450 nm. The MC concentration was determined from a standard competitive curve with a detection limit of 50 pg/mL. For the d-MC, small quantity (50-100 μL) of above 2-L filtered water was used directly to measurement without methanol extraction and water dilution. The next step was the same that of c-MC measurement. Data analysis—The AA of SM02 on cultured or natural Microcystis blooms and/or other algae was calculated using the equation AA (%) = (1–T/C) × 100, where T and C are the cell densities of Microcystis in the presence or absence of SM02, respectively. Differences in cell densities between treated and control cultures were analyzed by analysis of variance (ANOVA), and data were compared using linear contrasts. Values of P < 0.05 were considered statistically significant. Statistical analyses were performed using the SPSS package (SPSS Inc. 1989-2003). cryopreserved at –76°C in NB medium (Difco, South Royal) containing 20% glycerol. Prior to the experiment, frozen bacteria were inoculated into 300 mL fresh NB medium and incubated at 25°C with shaking (120 rpm). After 48 h, 2.7 L NB medium was added, and incubation was continued for 60 h. The bacterial cells were then harvested by centrifugation at 18,000×g for 20 min and washed twice with distilled water. The SM02 bacteria were introduced to the experimental tanks within 24 h of harvesting. Phytoplankton community and water chemistry—Before taking water samples from the tanks, the physiochemical parameters (water temperature, dissolved oxygen, conductivity, turbidity, and pH) in each tank were measured at a depth of 50 cm using a YSI portable meter (YSI-61). For measuring nutrient concentrations and phytoplankton densities and/or biomass, water samples were collected from a depth of 50 cm in each tank, transferred to a 10-L plastic bucket, and mixed. To enumerate the phytoplankton, 250 mL subsamples were fixed with Lugol’s solution (1% final concentration) in polypropylene bottles and stored in the dark at 4°C until analysis. A 1-mL sample of fixed phytoplankton was placed in a SedgwickRafter Counting Chamber (PhycoTech Inc.) and observed by light microscopy (Olympus microscope, Tokyo, Japan) at 4001000× magnification. Phytoplankton were enumerated based on counts of 200-300 cells per sample and identified at the genus or species level. The total biomass (carbon content) of phytoplankton was calculated using a conversion factor and biovolume values (Mullin et al. 1966; Strathmann 1967). For inorganic nutrient analysis, water samples were filtered (Whatman GF/F; Whatman International) and stored frozen (–10°C) until analysis. Ammonium (NH4), nitrate nitrogen (NO3), and soluble reactive phosphorus (PO4) were measured with an automatic analyzer (TRAACS800, Bran-Luebbe) after pretreatment with ascorbic acid, indophenol blue, and cadmium reduction (APHA 1998). The silicone concentration was measured by inductively coupled plasma-atomic emission spectrometry (ICAP 61E Trace; Thermo Jarreell-Ash). To measure the concentration of chlorophyll a (Chl a), 100-mL subsamples were filtered (Whatman GF/F) and extracted overnight with 90% acetone at 4°C. The fluorescence of each extract was measured using a spectrophotometer calibrated to Chl a standards (Sigma-Aldrich Co.). Microcystin-LR measurements—The concentration of cellular (c-MC) and dissolved microcystin-LR (d-MC) was quantified from each sample by enzyme-linked immunosorbent assay (ELISA; Nagata et al. 1997). The total concentration (t-MC) was calculated as the sum of the c-MC and d-MC values. Because the anti-microcystin monoclonal antibody (MAb) M8H5 used in the ELISA assay reacts equally with all major microcystin derivatives (i.e., microcystin-LR, microcystin-RR, and microcystin-YR), we used 250 μg of the most widely studied microcystin, microcystin-LR (C49H74N10O12, MW = 995.17, Wako) as the ELISA standard. Briefly, to quantify the concentrations of c-MC, 2-L samples of the water were taken from Assessment Algicidal activity of SM02 bacteria on toxic Microcystis aeruginosa—Optimal growth of the SM02 isolate occurred at 40°C and pH 7, and the isolate was characterized by rod-shaped cells, yellowish colonies, gram-negative staining, oxidase-positivity, and growth on D-maltose (data not shown). We tested the AA of SM02 against the toxic cyanobacterium M. aeruginosa NIER100001 in both the paper-disc and liquidculture tests. SM02 was co-incubated with M. aeruginosa for 5 d. When the starting concentration of SM02 was 108 cells mL–1, the algal cell density after 5 d was decreased by approximately 95.6% relative to the uninoculated control; that is, the algal cell density was only 4.6% of the cell density in the control culture. When the starting concentration of SM02 was 107 cells mL–1, the algal cell density after 5 d was only 33.9% of the uninoculated control (Fig. 2A). In contrast, inoculation of SM02 at concentrations below 106 cells mL–1 did not effectively inhibit M. aeruginosa (ANOVA, P > 0.5) as compared with the control. There were significant differences between the AA of bacterial cell culture at 108 cells mL–1 and the AA of the corresponding culture filtrate (ANOVA, P < 0.0001). Interestingly, the filtrate from SM02 cultures at 107 cells mL–1 effectively suppressed M. aeruginosa cell growth (to 51.5% of the control after 5 d; Fig. 2B), exceeding the suppression by SM02 cell culture at 107 cells mL–1 and indicating that the bacteria secreted 516 Kim et al. Bacterial mitigation of M. aeruginosa Fig. 3. Anti-algal effects of Xanthobacter autotrophicus HYS0201-SM02 on Microcystis aeruginosa N-10001 (A) at different bacterial growth phases (lag, exponential, and stationary) against exponential phase M. aeruginosa N-1000, and (B) at different algal growth phases (lag, exponential, and stationary). The arrow indicates the bacterial inoculation time. Each value is the mean ±SD of three replicates. Fig. 2. Anti-algal effects of Xanthobacter autotrophicus HYS0201-SM02 for exponential phase Microcystis aeruginosa N-10001 (A) at different bacterial cell concentrations, and (B) by bacterial cells and the corresponding culture filtrate. The arrow indicates the bacterial inoculation time. Each value is the mean ±SD of three replicates. meneghiniana (2.7%). Therefore, SM02 effectively suppressed the growth of toxic M. aeruginosa strains but had little effect on the growth of diatoms or some other cyanobacteria, including a different strain of M. aeruginosa, implying interand intra-specific host susceptibility. In situ algicidal activity of SM02 on natural Microcystis water— In situ, the biomass of M. aeruginosa was significantly reduced (to approximately 73.9% of control) by the addition of a high concentration (HD) of SM02 and by bi-daily additions of low concentration (BLD) of SM02 (to approximately 54.7% of control; ANOVA, P < 0.001 for both treatments). Addition of a low concentration (LD) of SM02 enhanced M. aeruginosa cell growth significantly, to approximately 121.4% of control (ANOVA, P < 0.001), while treatment with the middle concentration (MD) of SM02 did not have a significant effect (ANOVA, P > 0.5; Fig. 4). The effects of co-culturing SM02 at various concentrations with other phytoplankton species varied (Fig. 4). The carbon biomass of some phytoplankton increased after bacterial an algicidal substance into the media. Notably, the bacterial biomass did not change substantially after inoculation, but fluctuated over the 7 d of incubation. Inoculates of SM02 in the exponential and/or stationary growth phase had greater AA as compared to cells in lag phase (Fig. 3A). In contrast, exponential phase SM02 effectively suppressed M. aeruginosa in all growth phases (Fig. 3B). Therefore, active SM02 at a density greater than 107 cells mL–1 controlled Microcystis growth in all culture stages tested. Algicidal activity of SM02 on other phytoplankton species— During 5 d of co-cultivation, SM02 strongly inhibited two M. aeruginosa strains, NIER100001 (97.5% growth inhibition) and NIES101 (90.2%), and moderately inhibited NIES298 (46.6%; Table 1). In contrast, SM02 had little effect on Anabaena cylindrica (31.7% growth inhibition), M. aeruginosa NIES44 (26.0%), Aulacoseira granulata (23.5%), Stephanodiscus hantzschii (16.4%), Anabaena flos-aquae (8.8%), or Cyclotella 517 Kim et al. Bacterial mitigation of M. aeruginosa Table 1. Antialgal activity (AA) of the isolate HYS0201-SM02 against various phytoplankton species measured by direct counting (or Chl a concentration for Anabaena species) after 5-d co-culture. Host strain AA* Anabaena cylindrica Lemmermann NIES19† Anabaena flos-aquae (Lyngb.) Brebisson KCTCAG10026‡ Aulacoseira granulata (Ehr.) Simonsen CCAP1002/1§ Cyclotella meneghiniana Kützing HYK0210-CM01# Microcystis aeruginosa (Kütz.) Lemmermann NIES44 Microcystis aeruginosa (Kütz.) Lemmermann NIES101 Microcystis aeruginosa (Kütz.) Lemmermann NIES298 Microcystis aeruginosa (Kütz.) Lemmermann NIER100001** Stephanodiscus hantzschii Grunow UTCC267†† 31.7 (25.2–42.4) 8.8 (6.6–11.4) 23.5 (18.2–27.0) 2.7 (–0.8–4.4) 26.0 (24.5–28.3) 90.2 (88.1–92.6) 46.6 (41.4–53.7) 97.5 (95.3–99.6) 16.4 (15.7–18.2) All algal strains were cultured with gentle shaking at a light intensity of 50 μE m–2 s–1on a 12 h:12 h light:dark cycle. AA is presented as the mean (range) of triplicate experiments. * AA (%) = (1 – T/C) × 100, where T and C are the algal cell densities in the presence and absence of HYS0201-SM02, respectively. † NIES; National Institute for Environmental Studies, Tsukuba, Japan. † KCTC; Korean Collection for Type Cultures, KRIBB, Daejeon, Korea. § CCAP; Culture Collection of Algae and Protozoa, Scotland, UK. # Strains were isolated by capillary method and deposited at Hanyang University, Seoul, Korea ** NIER; National Institute of Environmental Research, Incheon, Korea. †† UTCC; University of Toronto Culture Collection of Algae and Cyanobacteria, Toronto, ON, Canada. inoculation: Chroococcus turgidus (approximately 8.5–10.4-fold greater growth than control, P < 0.001 for all concentrations tested), Scenedesmus quadricauda (approximately 21% greater than control, P < 0.01 for only the LD treatment), and Ankistrodesmus falcatus (approximately 17% greater than control, P < 0.001 for only the BLD treatment). Interestingly, the carbon biomass of the green alga Coelastrum sphaericum decreased in the presence of all concentrations of SM02. In particular, the BLD treatment suppressed algal biomass by approximately 92%. Thus, suppression of Coelastrum sphaericum by SM02 was greater than the suppression of natural M. aeruginosa, suggesting algicidal potential for SM02 beyond Microcystis. All physicochemical water quality parameters tested changed in the presence of SM02, including decreased dissolved oxygen, increased nutrients (N, P, and Si), and increased turbidity (data not shown). As expected, there was a significant increase in d-MC (ANOVA, P < 0.001 for HD, MD, and BLD) and a significant decrease in c-MC concentration (ANOVA, P < 0.0001 for BLD, P < 0.001 for HD and MD) after 5 d of co-culture with SM02 (Fig. 5), possibly due to algal cell lysis or degradation. Interestingly, the concentration of Chl a significantly increased in parallel with decreasing concentrations of M. aeruginosa after bacterial inoculation (ANOVA, P < 0.0001 for HD and MD, P < 0.001 for LD and BLD), suggesting a phytoplankton succession (e.g., Microcystis to Chroococcus in cyanobacteria; Coelastrum to Ankistrodesmus in green algae). inhibition (> 90% growth inhibition) evident only in two M. aeruginosa strains (NIES-101 and NIER-100001), and moderate or low inhibition evident only in M. aeruginosa strains NIES298 and NIES-44. SM02 also showed little inhibitory activity against the toxic cyanobacterium Anabaena flos-aquae and the small centric diatoms Stephanodiscus hantzschii and Cyclotella meneghiniana. It is generally accepted that these intra- and interspecific differences susceptibility of alga to algicidal bacteria are due to physiological differences among algal strains (Manage et al. 2000; Walker and Higginbotham 2000; Yasuno et al. 2000), bacterial density, and bacterial growth rates (Mayali and Azam 2004; Choi et al. 2005). Our results suggest that SM02 not be effective against all species of M. aeruginosa. Therefore, the goal of isolating bacteria that can mitigate the harmful effects of M. aeruginosa remains elusive and vitally important. In the in situ experiments with the Microcystis bloom water, two concentrations of SM02 (HD and BLD) showed AA against M. aeruginosa. Although SM02 (LD) significantly increased the cell density of M. aeruginosa (P < .01), repeated LD treatments (i.e., BLD treatment) was more effective in reducing M. aeruginosa than MD treatment of SM02. Interestingly, the total inoculated bacterial density and the corresponding AA from five inoculations of SM02 LD were less than those of a single HD treatment. Taken together, these results indicate that for optimal AA against a natural bloom of M. aeruginosa in situ, the concentration of SM02 must be at least 107 CFU/mL. However, it remains to be seen whether repeated treatment with SM02, resulting in a higher total bacterial density than a single HD treatment, will be effective against M. aeruginosa. In particular, all bacterial concentrations tested strongly increased the biomass of cyanobacterium Chroococcus turgidus (approximately 8.5~11.5 fold-higher-growth) as compared with the control. Discussion We found that the bacterium X. autotrophicus SM02 strongly suppressed the growth of M. aeruginosa NIER-10001 in the laboratory. However, there was distinct variability in algal susceptibility to the AA of SM02 (Table 1), with strong 518 Kim et al. Bacterial mitigation of M. aeruginosa Fig. 4. Phytoplankton biomass before and after addition of Xanthobacter autotrophicus HYS0201-SM02 to an isolated natural bloom of Microcystis aeruginosa. Control, no bacteria added into the tank; HD, high concentration of bacteria (1 × 108 CFU/mL); MD, mid-level concentration (5 × 107 CFU/mL); LD, low concentration (1 × 107 CFU/mL); and BLD, bidaily low-concentration (1 × 107 CFU/mL) inoculation performed five times during the study (a total of 5 × 107 CFU/mL, equivalent to MD). Fig. 5. Concentrations of Chl a, cellular (c-MC), and dissolved microcystin-LR (d-MC) before and after addition of Xanthobacter autotrophicus HYS0201-SM02 into enclosed waters containing a natural bloom of Microcystis aeruginosa. Values are means of triplicate experiments for the five day periods 14–18 June (PRE) and 19–24 June 2005 (POST). Moreover, BLD treatment increased the biomasses of green algae such as Ankistrodesmus and Scenedesmus, while one-time inoculations with other concentrations did not. We propose that repeated treatment with algicidal bacteria, regardless of the amount added, decreases the water quality due to the increase in organic materials such as bacteria and medium debris. During co-cultivation of SM02 and M. aeruginosa, the bacterial biomass fluctuated but did not increase substantially. Thus, the bacterial population did not benefit from the massive decay of the M. aeruginosa population. The bacterial diversity, as determined by DGGE and FISH assay, did not change when the addition of the filtered Microcystis bloom water to the natural water (Kim, unpublished data). This phenomenon could be related to the effect of the growth conditions on bacterial behavior or to the effect of toxic metabolites produced by M. aeruginosa. As in a previous study using Streptomyces (Choi et al. 2005), SM02 grew optimally at pH 7 and 40°C, conditions different from those optimal for the cyanobacterium and different from those used during co-cultivation. In addition, the concentration of dissolved d-MC gradually increased relative to c-MC following SM02 inoculation (Fig. 5). Kim et al. (2007) demonstrated that the release of d-MC from Microcystis following cell lysis by algicidal bacteria could suppress growth of the anti-algal bacteria. Although a detoxification role for microcystin-degrading bacteria residing in the outer mucilage of Microcystis has been proposed (Rashidan and Bird 2001; Maruyama et al. 2003), the bacteriolytic activity of M. aeruginosa via MC derivatives has not been tested in situ. Therefore, we hypothesize that the low growth or biomass of X. autotrophicus SM02 was due to unfavorable growth conditions (e.g., water temperature that was too low or too high). Further studies are necessary to better understand the mechanisms of bacteria-mediated inhibition of M. aeruginosa. In particular, toxin production and bacteriolytic or alga-lytic processes (e.g., Kitaguchi et al. 2001) following bacterial 519 Kim et al. Bacterial mitigation of M. aeruginosa 72:4411–4418. Beakes, G., H. M. Canter, and G. H. M. Jaworski. 1988. Zoospores ultrastructure of Zygorhizidium affluens Canter and Z. planktonicum Canter, two chytrids parasitizing the diatom Asterionella formosa Hassall. Can. J. Bot. 66:1054–1067. Bell, S.G., and G. A. Codd.1994. Cyanobacterial toxins and human health. Rev. Med. Microbiol. 5:256–264. Brabrand A., B. A. Faafeng, T. Kallquist, and J. P. Nilssen. 1983. Biological control of undesirable cyanobacteria in culturally eutrophic lakes. Oecologia 60:1–5. Carmichael, W. W., D. F. Biggs, and P. R. Gorham. 1975. Toxicology and paharmacological action of Anabaena flos-aquae toxin. Science 187:542–544. Choi, H. J., B. H. Kim, J. D. Kim, and M. S. Han. 2005. Streptomyces neyagawaensis as a control for the harzardous biomass of Microcystis aeruginosa (Cyanobacteria) in eutrophic freshwaters. Biol. Control 33:335–343. Chung, I. M., and others. 2007. Steroidal constituents of rice (Oryza sativa) hulls with algicidal and herbicidal activity against blue–green algae and duckweed. Phytochem. Anal. 18:133–145. Codd, G. A., S. G. Bell, and W. P. Brooks. 1989. Cyanobacterial toxins in water. Water Sci. Technol. 21:1–13. Daft, M. J., S. B. McCord, and W. D. P. Stewart. 1975. Ecological studies on algal-lysing bacteria in fresh waters. Freshwater Biol. 5:577–596. Dawson, R. M. 1998. The toxicology of microcystins. Toxicon 36:953–962. Ekman-Ekebom, M., M. Kauppi, K. Sivonen, M. Niemi, and L. Lepisto. 1992. Toxic cyanobacteria in some Finnish lakes. Environ. Toxicol. Water Qual. 7:201–203. EPA (Environmental Protection Agency). 2002. Implementation guidance for ambient water quality criteria for bacteria. United States Environmental Protection Agency. Ernst, B., S. J. Hoeger, E. O’Brien, and D. R. Dietrich. 2006. Oral toxicity of the microcystin-containing cyanobacterium Planktothrix rubescens in European whitefish (Coregonus lavaretus). Aquat. Toxicol. 79:31–40. Falconer, I. R. 1999. An overview of problems caused by toxic blue-green algae (cyanobacteria) in drinking and recreational water. Environ. Toxicol. 14:5–12. Jacquet, C., V. Thermes, A. Luze, S. Puiseux-Dao, C. Bernard, J. S. Joly, F. Bourrat, and M. Edery. 2004. Effects of microcystin-LR on development of Medaka (Oryzias latipes). Toxicon 43:141–147. Kim, B. H., H. J. Choi, and M. S. Han. 2003. Potential in the application for biological control of harmful algal bloom cause by Microcystis aeruginosa. Kor. J. Limnol. 37:64–69. ———, S. J. Hwang, Y. O. Kim, S. O. Hwang, N. Takamura, and M. S. Han. 2007. Effects of biological control agents on nuisance cyanobacterial and diatom blooms in freshwater systems. Microbes. Environ. 22:52–58 Kim, J. S., J. C. Kim, S. O. Lee, B. H. Lee, and K. Y. Cho. 2006. inoculation remain to be elucidated. Since the initial report by Shilo in 1967, many different Microcystis-killing bacteria have been isolated, including Streptomycetes phaeofaciens (Yamamoto et al. 1998), Alcaligenes denitrificans (Manage et al. 2000), Pseudomonas sp. (Kodani et al. 2002), and Streptomycetes neyagawaensis (Choi et al. 2005). However, there have been no previous reports of the application of anti-algal bacteria to natural cyanobacterial blooms (M. aeruginosa) with the goal of biorestoration, with the exception of a recent study by Takamura et al. (2004) involving the direct spraying of the algicidal amino acid, L-lysine. The difficulties and limitations of in situ experiments experienced by most researchers are most likely consequences of the unpredictability of bacterial activity in freshwater ecosystems after treatment with allelochemicals and xenobiotics (EPA 2002; Kim et al. 2007), including alterations in AA and host specificities in Microcystis blooms. Mayali and Azam (2004) recommended that before the application of anti-algal bacteria to freshwater systems, the following information should be determined: 1) the AA of xenobiotics and other chemicals against the target alga; 2) the effects of these chemicals on other organisms in the freshwater ecosystem; and 3) algal dynamics after removal of the target alga. Most relevant studies conducted in Korea (Kim et al. 2003; Kim et al. 2006; Park et al. 2006; Chung et al. 2007), including the present study, have been carried out in confined enclosures within reservoirs rather than in open water. Technical limitations associated with in situ studies include difficulties associated with enclosure construction and the availability of sufficient nutrients in the water and sediment to sustain the cyanobacterial bloom. These limitations present major challenges to the control of cyanobacterial blooms and to the determination of criteria for the use of xenobiotics and other chemicals in research in situ. The use of xenobiotics is often necessary, particularly in preservation areas where there is a need to expedite water treatment for biological remediation of algal bloom–affected ecosystems. References Almeida, V. P. S., K. Cogo, S. M.Tsai, and D. H. Moon. 2006. Colorimetric test for the monitoring of microcystins in cyanobacterial culture and environmental samples from southeast-Brazil. Brazil. J. Microbiol. 37:192–198. An J., and W.W. Carmichael. 1994. Use of a colorimetric protein phosphatase inhibition linked immunisorbent assay for the study of microcystins and nodularins. Toxicon 32:1495–1507. APHA (American Public Health Association). 1998. Standard methods for the examination of water and wastewater, 20th ed. American Public Health Association. Baptista, I. I. R., L. G. Peeva, N. Y. Zhou, D. J. Leak, A. Mantalaris, and A. G. Livingston. 2006. Stability and performance of Xanthobacter autotrophicus GJ10 during 1,2dichloroethance biodegradation. Appl. Environ. Microbiol. 520 Kim et al. Bacterial mitigation of M. aeruginosa in the termination of a cyanobacterial bloom. Microb. Ecol. 41:97–105. Redhead, K., and S. J. Wright. 1978. Isolation and properties of fungi that lyse blue-green algae. Appl. Environ. Microbiol. 35:962–969. Reynolds, C. S. 1984. The ecology of freshwater phytoplankton. Cambridge Univ. Press. Reyssac, S. J., and M. Pletikosic. 1990. Cyanobacteria in fishponds. Aquaculture 88:1–20. Sakata, T., Y. Fujita, and H. Yamamoto. 1991. Plaque formation by algicidal Saprospira sp. on a lawn of Chaetoceros ceratosporum. Nippon Suisan Gakkaishi 57:1147–1152. Shilo, M. 1967. Formation and mode of action of algal toxins. Bacteriol. Rev. 31:180–193. Sigee, D. C., R. Glenn, M. J. Andrews, E. G. Bellinger, R. D. Butler, H. A. S. Epton, and R. D. Hendry. 1999. Biological control of cyanobacteria: principles and possibilities. Hydrobiologia 395/396:161–172. Sluis, M. K., and S. A. Ensign. 1997. Purification and characterization of acetone carboxylase from Xanthobacter strain Py2. Proc. Natl. Acad. Sci. USA 98:8456–8461. ———, R. A. Larsen, J. G. Krum, R. Anderson, W. W. Metcalf, and S. A. Ensign. 2002. biochemical, molecular, and genetic analyses of the acetone carboxylases from Xanthobacter autotrophicus Strain Py2 and Rhodobacter capsulatus Strain B10. J. Bacteriol. 184:2969–2977. Strathmann, R. R. 1967. Estimating the organic carbon content of phytoplankton from cell volume or plasma volume. Limnol. Oceanogr. 12:411–418. Takamura, Y., T. Yamada, A. Kimoto, N. Kanehama, T. Tanaka, S. Nakadaira, and O. Yagi. 2004. Growth inhibition of Microcystis cyanobacteria by L-lysine and disappearance of natural Microcystis blooms with spraying. Microbes. Environ. 19:31–39. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positionspecific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673–4680. Utermöhl, H. 1958. Zur Vervollkommnung der quantitativen Phytoplankton-Methodik. Internationale Vereinigung fur Theoretische und Angewandte Limnologie Mitteilungen 9:1–38. Watanabe, M. M., and M. Hiroki. 1977. NIES-Collection. List of Strains, Algae and Protozoa. 5th ed. National Institute for Environmental Studies, Environment Agency, Japan. Walker, H. L., and L. R. Higginbotham. 2000. An aquatic bacterium that lyses cyanobacteria associated with off-flavor of channel catfish (Ictalurus punctatus). Biol. Control 18:71–78. Wellinghausen, N., J. Köthe, B. Wirths, A. Sigge, and S. Poppert. 2005. Superiority of molecular techniques for identification of gram-negative, oxidase-positive rods, including morphologically nontypical Pseudomonas aeruginosa, from Biological activity of L-2-azetidinecarboxylic acid, isolated from Polygonatum odoratum var. pluriflorum, against several algae. Aquat. Bot. 85:1–6. Kitaguchi, H., Hiragushi, N., Mitsutani, A., Yamaguchi, M., Ishida, Y.2001. Isolation of an algicidal bacterium with activity against the harmful dinoflagellate Heterocapsa circularisquama (Dinophyceae). Phycologia 40:275–279. Kodani, S., A. Imoto, A. Mitsutani, M. Murakami. 2002. Isolation and identification of the antialgal compound, harmane (1-methyl-â-carboline), produced by the algicidal bacterium, Pseudomonas sp. K44-1. J. Appl. Phycol. 14:109–114. Lee, S. O., J. Kato, N. Takiguchi, A. Kuroda, T. Ikeda, M. Atsushi, and H. Ohtake. 2000. Involvement of an extracellular protease in algicidal activity of the marine bacterium Pseudoalteromonas sp. strain A28. Appl. Environ. Microbiol. 66:4334–4339. Mayali, X., and F. Azam. 2004. Algicidal bacteria in the sea and their impact on algal blooms. J. Eukaryot. Microbiol. 51:139–144. Manage, M. P., Z. Kawabata, and S. Nakano. 2000. Algicidal effect of the bacterium Alcaligenes denitrificans on Microcystis spp. Aquat. Microb. Ecol. 22:111–117. Maruyama, T., K. Kato, A. Yokoyama, T. Tanaka, A. Hiraishi, and H. D. Park. 2003. Dynamics of microcystin-degrading bacteria in mucilage of Microcystis. Microb. Ecol. 46:279–288. McGuire, R. M., J. M. Jones, E. G. Means, G. Lzaquive, and A. E. Reston. 1984. Controlling attached blue-green algae with copper sulfate. Res. Technol. 27:60–65. Mullin, M. M., P. R. Sloan, and R. W. Eppley. 1966. Relationship between carbon content, cell volume, and area in phytoplankton. Limnol. Oceanogr. 11:307–311. Nagata, S., T. Tsutusmi, A. Hasegawa, F. Yoshida, Y. Ueno, and M. F. Watanabe. 1997. Enzyme immunoassay for direct determination of microcystins in environmental water. J. Assoc. Off. Anal. Chem. Int. 80:408–417. Oberholster, O. J., A. M. Botha, and J. U. Grobbelaar. 2004. Microcystis aeruginosa: source of toxic microcystins in drinking water. Afr. J. Biotechnol. 3:159–168. Palikova, M., R. Krejci, K. Hilscherova, P. Babica, S. Navratil, R. Kopp, and L. Blaha. 2007 Effects of different cyanobacterial biomasses and their fractions with variable microcystin content on embryonal development of carp (Cyprinus carpio L.). Aquat. Toxicol. 81:312–318. Park, M. H., S. J. Hwang, C. Y. Ahn, B. H. Kim, and H. M. Oh. 2006. Screening of seventeen oak extracts for the growth inhibition of the cyanobacterium Microcystis aeruginosa Kütz. em. Elenkin. Bull. Environ. Contam. Toxicol. 77:9–14. Porter, K. G., and Y.S. Feig. 1980. The use of DAPI for identifying and counting aquatic microflora. Limnol. Oceanogr. 25:943–948. Rashidan, K. K., and D. F. Bird. 2001. Role of predatory bacteria 521 Kim et al. Bacterial mitigation of M. aeruginosa patients with cystic fibrosis. J. Clin. Microbiol. 43:4070–4075. Yamamoto, Y., T. Kouchiwa, Y. Hodoki, K. Hotta, H. Uchida, and K. Harada. 1998. Distribution and identification of actinomycetes lysing cyanobacteria in a eutrophic lake. J. Appl. Phycol. 10:391–397. Yasuno, M., Y. Sugaya, K. Kaya, and M. M. Watanabe. 2000. Variations in the toxicity of Microcystis species to Moina macrocopa, pp. 43–51. In M. M.Watanabe and K. Kaya [eds.] Advances in microalgal and protozoal studies in Asia. Global Environmental Forum, Tsukuba, National Institute for Environmental Studies, Japan. Submitted 3 March 2008 Revised 15 August 2008 Accepted 2 September 2008 522