Department of Oceanography, Kunsan National University, Kunsan Korea. Korea Environmental Research Center for Hydrosphere, Ansan Korea

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1 The Sea Journal of the Korean Society of Oceanography Vol. 11, No. 4, pp , November 2006 y x û³ (1) š û³ Anabaena spiroides v. crassa w 1 Á½¼ 2 Áx 3 Á½ 4 Áy 1 * 1 w ww, 2 w wy, 3 w er y œw, 4 w y w Bloom-forming Cyanobacteria in Yongdam Lake (1) Nutrient limitation in a Laboratory Strain of a Nitrogen-fixing Cyanobacterium, Anabaena spiroides v. crassa JONG WOO PARK 1, YOUNG GEEL KIM 2, WOOMYUNG HEO 3, BOMCHUL KIM 4 AND WONHO YIH 1 * 1 Department of Oceanography, Kunsan National University, Kunsan Korea 2 Korea Environmental Research Center for Hydrosphere, Ansan Korea 3 Department of Environmental Disaster Prevention Engineering, Kangwon National University, Samchock Korea 4 Department of Environmental Science, Kangwon National University, Choonchon Korea l w y 8.2m ü 5 ³ y, y w ew œy. ƒ œ w, y vjm y y x w w. y» y w û³ x ùkû, x k Anabaena, Microcystis, Aphanizomenon. ƒ û³ Anabaena s sù {s p» s ƒ sw. y x ùkù x w Anabaena spiroides v. crassa KNU-YD0310 yw, ü x mw w. w s s w, w ƒw. š KNU-YD0310 w ù,» œ w ùkü. w {s x ƒw, w x ƒ s w ww y» j ƒ q. Anabaena spiroides v. crassa k p wz y x w kw» y. Yongdam Lake is the fifth largest artificial lake in Korea newly formed by the first impounding the Yongdam Multi-purpose Dam on December, Yongdam Lake, with her total water storage of 820 million M/T, is located at the roof-top region of the streams flowing into the just-constructed new Saemankeum Lake. Seasonal succession of phytoplakton in Yongdam Lake might affect cyanobacterial blooms in Saemankeum Lake by inoculating seasonal dominants. During when the first impounding after the constrcution of Yongdam Multipurpose Dam was still undergoing, summer cyanobacterial blooms by Anabaena, Microcystis, and Aphanizomenon were observed. Among these three, filamentous Anabaena is well known to have its species with N 2-fixing ability and special cells such as heterocysts and akinetes as well as the vegetative cells. We established a clonal culture of Anabaena spiroides v. crassa (KNU-YD0310) from the live water samples collected at the bloom site of Yongdam Lake. The N- and P-nutrient requirement of the KNU-YD0310 was explored by the experimental cultivation of the laboratory strain. Ratio of heterocysts to vegetative cells increased as N-deficiency extended with its maximum at N 2-fixing condition. The strain KNU-YD0310 exhibited considerable growth under N-limiting conditions while its growth was proportional to the initial phosphate-p concentration under P-deficient conditions. Under P-limiting conditions akinete density increased, which could be interpreted as an adaptation strategy to survive severe environment by transforming into resting stage. The above eco-physiological characteristics of *Corresponding author: ywonho@kunsan.ac.kr 158

2 y x û³ (1) š û³ Anabaena spiroides v. crassa w 159 Anabaena spiroides v. crassa might be useful as an ecological criterion in controlling cyanobacterial blooms at Shaemankeum Lake in near future. Keywords: Cyanobacterial Bloom, Yongdam Lake, N 2-fixation, Inorganic nutrients, Laboratory strain y 8.2m ü 5 ³, y w ew œy. y 32 mù, ƒ 50%(16m) «œ w (w œ, 2000)., ƒ œ yƒ w, y v jm y y ü x w w. y w ù, 2002l x û³ xw. p, w û³ w x ùkù 2-3, x k Anabaena(2002, 2003), Microcystis (2002, 2003), Aphanizomenon(2003) (½, 2005). ƒ s s {s û ³ Anabaena š 280,000 cells ml 1 ùkü(½, 2005). y 20 w y,» 1973 ³ 7 6 š(, 1974), 1968 y ³ƒ v jmš š (, 1968) j. ù y 1985 zw Anabaenaƒ v jm y (Kim, 1987;,1998), yü ƒƒ y w (, 1990; x, 1991; x ½, 1992;, 1994)., ü Anabaena w ƒ w, - p s (Choi et al. 1998) z(, 1999; Lee and Yoo, 2001) s w šƒ. wì y» y w, w nm»ƒ š ù z x y ì (x,1997).» nm d œ w w y p ùküš (, 1997), Anabaena š x (, 1982) w ƒ. y w x vw» x w, x k k p qw z ym w vƒ (, 1997). mw, y z x d x yw Anabaena spiroides v. crassa w ww w šwš w. w, w s, s s w y, {s s w y w. mw ³ Anabaena spiroides v. crassa p wz y x w y. y y 1, 2, 3(Fig. 1) x (live sample) w, x w. x z, 24 ü s ww. x Anabaena spiroides v. crassa s wù w» w, Ÿwx w vr w ww(guillard, 1973). w s CB media multi-well culture chamber wù, Fig. 1. Location of sampling stations (indicated by arrows) and dike (shown as a dark bar) at Yongdam Lake.

3 160 Á½¼ÁxÁ½Áy s» g. 2-3 z, s ƒ y 2-3 w 20 w yw( : Anabaena spiroides v. crassa KNU-YD0310). x y 20 o C 60 µe m 2 s 1 Ÿ. x CB media(½, 1999)» w, w x w CB media w w(table 1)., (β-sodium glycerophosphate) (Ca(NO 3) 2-4H 2O, KNO 3) ƒƒ CB media» w 0, 0.2, 1 w, w x w(table 1). 3 ƒƒ w w 125 ml PC 20 o C, 100 µe m 2 s 1 Ÿ w š w. x sww 3 w ƒ 3ml subsamplew z Lugol s solution šw w. q Sedgwick-Rafter slide w Ÿwx 100 s (counting) w. s {s s(heterocyst) {s(akinete) j»,, n ƒ p w, Ÿwx 100 s w(whitton and Potts, 2000) w. š Lugol s solution š w, s j» š nw kx, s j» š nw x s ó polar plug(sherman et al. 2000) Table 1. Chemical composition of CB Medium. Compound Concentration Ca(NO 3) 24H 2O 150 mg KNO mg MgSO 47H 2O 40 mg β-na 2glycerophosphate 50 mg Vitamin B 1 10 µg Vitamin B µg Biotin 0.1 µg HCl-thiamine 10 µg PIV Metals 3 ml Bicine 500 mg Distilled water to 997 ml PIV Metals FeCl 3 6H 2O 196 mg MnCl 2 4H 2O 36 mg ZnSO 4 7H 2O 22 mg CoCl 2 6H 2O 4 mg Na 2MoO 4 2H 2O 2.5 mg Na 2EDTA 2H 2O 1000 mg Distilled water to 1000 ml (NO 3-N: 21.6 mg/l, PO 4-P: max. 8.0 mg/l) Fig. 2. Light microscopic image of a clonal culture of Anabaena spiroides v. crassa (KNU-YD0310 strain). Vegetative cells are relatively small and dark-brown colored when compared with the half-transparent cells of small and bright heterocysts (A) and large and pale akinetes (B). Scale bars = 10 µm. ƒ(fig. 2A), {s s 3-5 j» n w kx ùkù(fig. 2B) w. š x w 3 3 yw w s s³e Fig. 3 w. x 1600 cell ml 1» ƒ cells ml 1 (Table 2, 3). ƒw x x ùkù (Fig. 3 d, Table 3)., x ƒw x ƒ» 4.5 wš(fig. 3 d ), k 1/2 w(table 2). x š w, š y œ y w j û (Fig. 3 d)., y w, ww w

4 y x û³ (1) š û³ Anabaena spiroides v. crassa w 161 Fig. 3. Time course of N-limited (left) and P-limited (right) vegitative growth of Anabaena spiroides v. crassa (KNU-YD0310 strain; mean cell density). N-0 and P-0 means experimental CB-N and CB-P medium without any added nitrate-n and phosphate-p, respectively. One fifth of nitrate-n and phosphate-p for the enriched CB medium (CB-1.0 with 21.6 mg NO 3 -N l 1 and 8.0 mg PO 3 4 -P l 1 ) was added to prepare medium N-0.2 and P-0.2, repectively. Table 2. Maximum biomass yield and maximal growth rate of Anabaena spiroides v. crassa (KNU-YD0310 strain) under different NO 3 -N concentrations. NO 3 -N (mg l 1 ) Y max (cells/ml) GR max (divisions/d) Table 3. Maximum biomass yield and maximal growth rate of Anabaena spiroides v. crassa (KNU-YD0310 strain) under different PO 3 4 -P concentrations. PO 3 4 -P (mg l 1 ) Y max(cells/ml) GR max(divisions/d) »» w Anabaena ãw w x ùkú. w w œ y k y nd yy w(, 1997), y k» Anabaena û³ w» x ƒv w. w» sw y ƒ œ, w» y x ƒ j. w s s e s³ Fig. 4 w. t w (Fig. 4 d CB-1.0) ƒ CB media 1/5 (Fig. 4 d N-0.2) s ù,» 21» 15 e»w CB (Fig. 4 d). w, x ƒw s ù,» ƒ (12). p, s s w w š,» (Fig. 5 d)., (Fig. 4 d) ƒ s š». ù, s s w ƒ w (Fig. 5 d), w. w l, x w, w w» w š s w (Zhang et al. 2006). ù ww w ùk ùš(fig. 4 d) s s w w ƒ (Fig. 5 d), w w w w š w» q. y xl ƒ w» w y ³ w w. w {s Fig. 6 {s e s³, ƒ CB media 1/5 (Fig. 6 d P-0.2) ƒ {s ùkü (Van Dok and Hart, 1996). s (Fig. 4, 5) w w (survival strategy) w(olli et al., 2005). w xƒ w pw ù, s g(={s x) œ»¾ s»

5 162 Á½¼ÁxÁ½Áy Fig. 4. Time course of mean heterocyst density under N-limited (left) and P-limited (right) condition by Anabaena spiroides v. crassa (KNU- YD0310 strain). N-0 and P-0 means experimental CB-N and CB-P medium without any added nitrate-n and phosphate-p, respectively. One fifth of nitrate-n and phosphate-p for the enriched CB medium (CB-1.0 with 21.6 mg NO 3 -N l 1 and 8.0 mg PO 4 3 -P l 1 ) was added to prepare medium N-0.2 and P-0.2, respectively. Fig. 5. Fluctuation of (Heterocysts)/(Vegetative cells) ratio in N-limited (left) and P-limited (right) condition by Anabaena spiroides v. crassa (KNU-YD0310 strain). N-0 and P-0 means experimental CB-N and CB-P medium without any added nitrate-n and phosphate-p, respectively. One fifth of nitrate-n and phosphate-p for the enriched CB medium (CB-1.0 with 21.6 mg NO 3 -N l 1 and 8.0 mg PO 4 3 -P l 1 ) was added to prepare medium N-0.2 and P-0.2, respectively. w k., x Anabaena š û³ w x w» w, y ƒ ³ w» w. y -k» x ƒ x w y l w wù, 2002 l x û³ xw. y 20 w y j» x. y zl(, 1968;, 1974) 1986 ¾ 12 û³, ³, r w»» e z (Kim, 1987;, 1998), û³ w ³ x ù kù» w(, 1990; x, 1991; x ½, 1992;, 1994). w» zw œ œy., y e ü w w, y ü sw ¾ w w q., 10 z ƒ w w w(x, 1992) y, y zl

6 y x û³ (1) š û³ Anabaena spiroides v. crassa w 163 Fig. 6. Time course of mean akinete density under N-limited (left) and P-limited (right) condition by Anabaena spiroides v. crassa (KNU- YD0310 strain). N-0 and P-0 means experimental CB-N and CB-P medium without any added nitrate-n and phosphate-p, respectively. One fifth of nitrate-n and phosphate-p for the enriched CB medium (CB-1.0 with 21.6 mg NO 3 -N l 1 and 8.0 mg PO 3 4 -P l 1 ) was added to prepare medium N-0.2 and P-0.2, respectively. mw w w j (x, 2005;, 2005). y w» w y ü ƒ d (x, 2005). y w y w œ z l k-y, ³ s³ û y yw (x, 1997). œy wƒ w. w» y k» ¾ w d. w nd y(, 1997). w w, y» x ƒ. p, w š û³ (, 2002; Kim et al., 2004)» k w. y w x vw» x w, mw ³ Anabaena spiroides v. crassa - w k p x wz w w x x w w» w y w xl( y l) w. w» ½x, wì ¾. w š w mw w Ì. šx ½, ½, y, ³(œ), y û.». ½¼, w y y vjm kw. w w. w » Anabaena flosaquae Ÿw p w wz 25(4): ,, w, ³, ½, xz, yy y-y y w,» yw yw. w wwz 2(2): ,, y,,, k z y k. /w y wz, 21(4): , ½, ³ y () v jm (1984~1997) w wz 31(2): , ½, ³ y y y t x. w wz, 23(4): 291., ³ y vjm y. w wz, 27(1): 9 22.,, ½ û 5- Aminolevulinic Acid Dehydratase. Algae. 14(4): ³ w 3 œy w w. 1 : qy, y y y Plankton w. w wz 1(1): ³ Dam y w. 1.» plankton. w wz 7(1-2): , y, ù w û Anabaena. Algae, 17(2): »,, z, xz yy yy w vjm w. w ww

7 164 Á½¼ÁxÁ½Áy z 2(2): xz,, œy yy w k :. w wwz 2(2): x, «, ½¼, y, ½, y w. w wz, 38(3): x, ½ y y (P) w l (N) w y. w wz, 25(4): 301. x, ½, ³ y y N/P y û Bloom. w wz, 24(4): x, ½, k,», y ƒ l w (Phosphorus Budget). w wz, 25(4): w œ, š. p. 17. Choi, C.W., S.A. Yoo, I.H. Oh and S.H. Park Characterization of extracellular flocculation substances produced by a planktonic cyanobacterium, Anabaena flos-aquae. Biotechnology Letters. 20(7): Guillard, R.R.L., Methods for microflagellates and nanoplankton. In (J.R. Stein ed.) Handbook of phycological methods, Cambridge University Press, New York, pp.g Kim, B.C., An ecological study of phytoplankton in Lake Soyang. Ph.D. Thesis, Department of Oceanography, Seoul National University, 125pp. Kim, Y.G., G.O. Myung, W. Yih and Y.K. Shin, Optimal growth conditions for the two euryhaline cyanobacterial clones, Anabaena sp. CB-MAL21 and CB-MAL22 isolated from Mankyeong Estuary, Korea. Algae, 19(2): Lee, I.C. and S.A. Yoo, Characterization of 5-Aminolevulinic Acis Dehydratase purified from Anabaena cylindrica. Algae, 16(1): Olli, K., K. Kangro and M. Kabel, Akinete production of Anabaena lemmermannii and A. cylindrica(cyanophyceae) in natural populations of n- and p-limited coastal mesocosms. J. Phycol. 41: Sherman, D.M., D. Tucker and L.A. Sherman, Heterocyst development and localization of cyanophycin in N 2-fixing cultures of Anabaena sp. pcc 7120 (Cyanobacteria) J. Phycol. 36: Van Dok, W., and Hart, B.T Akinete differentiation in Anabaena circinalis (Cyanophyta). Journal of Phycology, 32: Whitton, B.A. and M. Potts (ed.), The ecology of cyanobaceria. The diversity in time and space. Kluwer Academic Publishers, London. 669pp. Zhang, C., S. Laurent, S. Sakr, L. Peng and S. Bedu, Heterocyst differentiation and pattern formation in cyanobacteria: a chorus of signals. Molecular Microbiology, 59(2): š k r: y