Degradation of Microcystins Using Immobilized Microorganism Isolated in an Eutrophic Lake
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Chemosphere 65 (2006) 117–124 www.elsevier.com/locate/chemosphere Degradation of microcystins using immobilized microorganism isolated in an eutrophic lake Kiyomi Tsuji a,*, Miki Asakawa a, Yojiro Anzai b, Tatsuo Sumino c, Ken-ichi Harada d a Kanagawa Prefectural Institute of Public Health (Formerly, Kanagawa Prefectural Public Health Laboratory), 1-3-1 Shimomachiya, Chigasaki, Kanagawa 253-0087, Japan b Faculty of Pharmaceutical Sciences, Toho University, Miyama, Funabashi, Chiba 274-8510, Japan c Research Division, Hitachi Plant Engineering and Construction Corporation, Kamihongo 537, Matsudo, Chiba 271-0064, Japan d Graduate School of Environmental and Human Sciences and Faculty of Pharmacy, Meijo University, Tempaku, Nagoya 468-8503, Japan Received 28 September 2005; received in revised form 14 February 2006; accepted 14 February 2006 Available online 24 March 2006 Abstract The final purpose of our series of studies is to establish a biological removal method of cyanobacteria and their toxic products using immobilized microorganisms that can lyse cyanobacteria and decompose microcystins. To establish the biological removal method in non-point areas and water purification plants, as the first step, we explored bacteria active against the cyanobacterial hepatotoxin micro- cystin in the present study. Eleven active bacteria were isolated from samples taken from Lakes Tsukui and Sagami, Japan. Among 3 strains (B-9 to B-11) with degradative activity, strain B-9 exhibited the strongest activity. The 16S rDNA sequence of the strain B-9 showed the highest similarity to that of Sphingomonas sp. Y2 (AB084247, 99% similarity). Microcystins-RR and -LR were completely degraded by strain B-9 (SC16) within 1 d, which led to an immobilized microorganism with a polyester resin. The degradation of micr- ocystin-RR in a bioreactor using the immobilized strain B-9 was observed and microcystin-RR (>90%) was completely degraded after 24 h. Microcystin-RR was added to the lake water at regular intervals and the degradation after 24 h was observed in the bioreactor over a 72-d period. An over 80% removal efficiency continued for 2 months, showing that the life of the immobilized B-9 in terms of activity was at least 2 months under the optimized conditions. From these results, this immobilized B-9 is feasible for the practical treatment of microcystins in non-point areas and water purification plants. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Microcystin; Biodegradation; Immobilization; Cyanobacteria 1. Introduction tins, the cyclic heptapeptide toxins produced by cyanobac- teria, such as Microcystis, show a potent hepatotoxicity Cyanobacteria (blue-green algae) commonly occur in a and tumor-promoting activity by inhibition of the protein variety of water types throughout the world. A variable phosphatases 1 and 2A (Kuiper-Goodman et al., 1999; but high proportion of the cyanobacterial blooms and Sivonen and Jones, 1999). Although animal poisoning scums, which grow annually in lakes, reservoirs, canals and human health problems associated with the ingestion and slow-flowing rivers, contain potent toxins. Microcys- of or contact with cyanobacterial scums have long been recognized, a toxic incident involving the death of 50 peo- ple occurred in Brazil in 1996 due to microcystins in the * Corresponding author. Tel.: +81 467 83 4400; fax: +81 467 83 4457. water used for hemodialysis (Jochimsen et al., 1998; Pouria E-mail address: [email protected] (K. Tsuji). et al., 1998). Because toxic cyanobacteria containing 0045-6535/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2006.02.018 118 K. Tsuji et al. / Chemosphere 65 (2006) 117–124 microcystins threaten human health and life, we must The MA medium was prepared as follows: a mixture establish an effective method for the regulation of the of bicine, 500 (mg); Ca(NO3)2 Æ4H2O, 50; KNO3, 100; occurrence of cyanobacteria and their toxic metabolites. NaNO3, 50; Na2SO4, 40; MgClÆ6H2O, 50; b-Na2 glycero- Although many methods, such as biomanipulation and phosphate, 100, metal mixture composed of Na2EDTA algicides, have been tried for the elimination of cyanobac- (5 mg), FeCl3 Æ6H2O (0.5 mg), MnCl34H2O (5 mg), ZnCl2 teria in a lake, no suitable method has been developed, and (0.5 mg), CoCl2 Æ6H2O (5 mg), Na2MoO4 Æ2H2O (0.8 mg) it would be difficult to regulate the occurrence of cyanobac- and H3BO3 (20 mg) were dissolved in 1 l of purified water teria by conventional methods (Chorus and Mur, 1999). and the resulting solution was adjusted to pH 8.6. Furthermore, no effective method has been proposed for degrading microcystins in the natural environment. Our 2.3. HPLC laboratory is developing a biological control system using microorganisms co-existing in the same ecosystem to HPLC was carried out under the following conditions: decrease the outbreak of cyanobacteria (Sigee et al., pump, Shimadzu LC-9A (Kyoto, Japan); photodiode array 1999) and the decomposition of microcystins (Jones detector, Shimadzu SPD-M10A, CLASS-LC10 integrating et al., 1994; Bourne et al., 1996). Immobilized microorgan- system; Inertsil ODS-3 column (150 · 4.6 mm ID, GL Sci- isms have been assessed for water purification applications ence Inc, Tokyo, Japan), COSMOSIL 5C18 AR-II column (Sumino et al., 1985, 1991; Kokufuta et al., 1986; Hashi- (150 · 4.6 mm ID, Nacalai Tesque Inc, Kyoto, Japan); moto and Furukawa, 1987). In our system, suitable micro- mobile phase, methanol: 0.05 M NaH2PO4 (pH 3) = organisms are explored from eutrophic lakes, and they are 58:42; flow rate, 1.0 ml minÀ1; detection, UV 238 nm; col- improved for the purposes mentioned above. Additionally, umn temperature, 40 °C. these microorganisms are immobilized with appropriate resins and then applied to an eutrophic lake. After this 2.4. Isolation of microorganism with anticyanobacterial operation, the applied immobilized microorganism should activity and degradative activity be recovered from the lakes to avoid secondary pollution due to the microorganism used. Lake waters, sediments and soils were collected from In this study, lake waters, sediments and soils were col- Lakes Sagami and Tsukui in Kanagawa Prefecture, Japan, lected from Lakes Sagami and Tsukui in Kanagawa Prefec- from July 1997 to July 1998. The soil samples were sus- ture, Japan, from July 1997 to July 1998 in order to find pended in sterilized water (10 ml), and the supernatants suitable microorganisms. From these samples, the isolation and lake water were subjected to the soft-agar overlayer of microorganisms, which have a degradative activity method (Uchida et al., 1998). After incubation for several toward microcystins, was tried. To develop an immobiliza- d, the desired microorganisms were obtained from the tion method having a high degradative activity, the effect of resulting plaques and they were then transferred to Sakurai purified bacteria and the immobilization method on the (0.2% peptone, 0.1% yeast extract, 0.05% glucose, 1.5% degradative activity of the microcystin were investigated. agar) or ISP No. 2 medium. To isolate the degradative bac- For optimizing the immobilizing conditions, a continuous terium, the supernatants of the soil samples and lake water bioreactor using immobilized microcystin-degradative bac- were inoculated onto Sakurai medium. Single colonies teria was used for the practical treatment of lake water con- from these plates were transferred to a solution of microcy- taining the microcystins. stin-RR in distilled water (2 mg lÀ1), and the microcystin- RR degradation was monitored by HPLC. During this 2. Materials and methods operation, 23 bacteria, of which 12 bacteria were actinomy- cete, were collected. Among them, three bacteria showed 2.1. Toxins and other reagents microcystin-degradative activity. The OD value at 660 nm was used as index to the viable bacteria count of strain Microcystins-RR and -LR were purchased from Wako B-9. Pure Chemical Industries (Osaka, Japan). Microcystins- RR and -LR were also isolated and purified from the sur- 2.5. Identification of the microorganisms face blooms collected from Lake Suwa in Japan according to the method described by Harada et al. (1988). All One isolated single strain (B-9), which showed the high- reagents used were of analytical grade or HPLC grade. est degradative activity among the 3 strains, was selected for subsequent studies. Strain B-9 was routinely main- 2.2. Cyanobacteria tained on peptone–yeast extract agar. Strain B-9 was identified using morphological observations, chemotaxo- NIES-102 (toxic, Microcystis viridis) was obtained from nomical analyses, and 16S rRNA sequencing. The total the National Institute for Environmental Studies, DNA of strain B-9 was extracted from cells cultured on Tsukuba, Japan. This strain was cultivated in 500 ml Erlen- an agar plate by the benzyl chloride method according to meyer flasks containing 200 ml each of the modified MA Zhu et al. (1993). Amplification of the 16S rRNA coding medium at 25 °C for 8 d under continuous illumination. region of the DNA and sequencing of the rDNA using K. Tsuji et al. / Chemosphere 65 (2006) 117–124 119 the direct sequencing method was performed as described 2.8. Degradation of microcystins-LR and -RR in a by Anzai et al. (2000). The 16S rRNA gene sequence data cyanobacterium using immobilized adhesively B-9 of the related strains were obtained from the GenBank/ EMBL/DDBJ databases for comparison. The genetic dis- Thirty-five pieces of polyester (Fabios) were added to 6 À1 tances between the sequences were estimated using the Knuc the B-9 culture (8 · 10 cells ml ) and the suspension values (Kimura, 1980). A phylogenetic tree was then con- was shaken at 80 rpm and 27 °C for 30 min. After filtra- structed by the neighbor-joining method (Saitou and Nei, tion, the resulting resin was placed in a flask, in which a 1987), and the evaluation of the tree was carried out by cyanobacterium, NIES 102, was cultivated in lake water the bootstrap method using the Clustal W program and a (300 ml) for 6 d.