Growth and fatty acid composition of three heterotrophic Chlorella species

Transcription

1 Rsr Artil Alg 2013, 28(1): ttp://x.oi.org/1490/lg Opn Ass Growt n ftty i omposition of tr trotropi Clorll spis D Gun Kim 1 n Sung Bum Hur 1, * 1 Dprtmnt of Mrin Bio-mtrils n Aquultur, Puyong Ntionl Univrsity, Busn , Kor Som Clorll spis grow trotropilly wit orgni sustrt in r onition. Howvr, trotropi Clorll spis r limit n tir optimum ultur onitions r not fully nown. In tis stuy, tr trotropi Clorll spis, two strins (C4-3 n C4-4) of C. vulgris n on Clorll sp. (C4-8) wr xmin on optimum ultur onitions su s ron sour, tmprtur, n onntrtions of nitrogn n posporus in Jworsi s mium (JM). An t growt n ftty i omposition of Clorll wr nlyz. For tr trotropi Clorll spis, gluos (1-2%) s ron sour only inrs t growt n t rng of optimum ultur tmprtur ws C. Doul onntrtions of t nitrogn or posporus in JM mium lso improv t growt of Clorll. Clorll ultur trotropilly sow signifintly igr growt rt n iggr ll siz tn tos utotropilly i. C. vulgris (C4-3) ultur trotropilly sow t igst iomss in ry wigt ( g L -1 ) mong tr spis. Wit rspt to ftty i omposition, t ontnts of C16:0 n n-3 igly unsturt ftty i (HUFA) wr signifintly igr in utotropi Clorll tn in trotropi on n tos of totl lipi wr not iffrnt twn iffrnt onntrtions of nitrogn n posporus in JM mium. Among tr Clorll spis in tis stuy, C. vulgris (C4-3) ppr to t most il trotropi Clorll spis for inustril pplition sin it ig iomss n lipi ontnt. Ky Wors: iomss; ftty i; growt; trotropi Clorll; JM mium INTRODUCTION Htrotropi mirolgl spis r limit in numr n tir ultur mtos r not s fully nown s utotropi mirolgl spis (Olizol 2003). Altoug trotropi ultur of mirolg s vntg for pur ig-nsity ultur u to no potoiniition fft n sy rvst, t ost of initil invstmnt is vry ig. Trfor, ty n to ig vlu spis tt n grow rpily in frmntr witout ligt (Brly t l. 1994, Wn n Cn 2003). Clorll, typil gnus tt n trotropilly ultur, s ig growt rt n is loo forwr to s futur foo s it ontins onsirl mount of protin, itry fir, vitmin, n minrl, t. (Kim 2004). A lorll growt ftor (CGF), in prtiulr, is nown to fftiv in growt n rovry ftr illnss (Cn 1996, L 1997). Clorll s n lso rport to v ntirinogni, ntioxitiv, n sin-ligtning fft (Hsgw t l. 1994, Justo t l. 2001, Kim t l. 2008). In ition, it is in t spotligt s n ingrint of ioisl sin it ontins vrity of lipi omposition (Mio n Wu 2004, Mio t l. 2004, Xiong t l. 2008). Altoug mny rsults v n rport in trms of Tis is n Opn Ass rtil istriut unr t trms of t Crtiv Commons Attriution Non-Commril Lins (ttp://rtivommons.org/linss/y-n/3.0/) wi prmits unrstrit non-ommril us, istriution, n rproution in ny mium, provi t originl wor is proprly it. Riv Jnury 6, 2013, Apt Frury 8, 2013 *Corrsponing Autor E-mil: urs@pnu..r Tl: , Fx: Copyrigt T Korn Soity of Pyology 101 ttp://-lg.r pissn: ISSN:

2 Alg 2013, 28(1): trotropi ultur of Clorll (Si t l. 1999, 2002, Xiong t l. 2008), nw Clorll spis wit outstning growing ility unr trotropi ulturing-onition soul vlop mor for inustril purpos. In tis stuy, t optimum ultur onitions su s ron sour, tmprtur, n nutrint of tr trotropi Clorll spis tt v n ollt from Korn wtrs wr xmin on growt rt. An iomss n ftty i omposition of trotropilly ultur Clorll n utotropilly ultur on wr ompr otr. MATERIALS AND METHODS Cultur of spis n optimum ron sour Two strins (C4-3 n C4-4) of C. vulgris n on Clorll sp. (C4-8) wr riv from Kor Mrin Mirolg Cultur Cntr (KMMCC) in Puyong Ntionl Univrsity. Ty wr ultur in xni onition. As for ultur, 100 ml siz-stning ultur mto ws us wit Jworsi s mium (JM) (Tompson t l. 1988) t 25 C wit ontinuous ligt of 60 mol potons m -2 s -1 in 250 ml fls. T lls in xponntil growt ps wr us s inoul. Tr orgni mttrs, gluos, glyrol, n tt wr tst s ron sours. Tn mililitrs of Clorll, wi ws utotropilly ultur in xni stt ws inoult to 100 ml of JM mium in 250 ml fls. T initil ll nsity ws 10 6 lls ml -1 n ron sour wit 1 n 2% onntrtion ws. Tn, ty wr ultur unr ontinuously r onition t 28 C for 6 ys wit tr rptitions. T numr of Clorll lls ws ount tr tims y t t sm tim using mytomtr n t most il ron sour for trotropi ultur ws trmin y ompring spifi growt rt [SGR = log(n 2 /N 1 )/ (t 2 - t 1 ); N 1, numr of lls t t 1 ultur strting t; N 2, numr of lls t t 2 t igst ll nsity t] (Guillr 1973). Efft of tmprtur n gluos, nitrogn, n posporus onntrtion on growt T SGR of Clorll wit iffrnt tmprturs (24, 26, 28, 30, n 32 C) n gluos onntrtions (1, 2, 3, 4, n 5%) wr xmin. T xprimnt t 32 C ws onut only on t spis tt grw t 30 C. T otr xprimntl mtos wr t sm s for. To fin t optiml onntrtion of nitrogn n posporus for trotropi Clorll, t onntrtions of C(NO 3 ) 2 (2 g L -1 ) n NNO 3 (8 g L -1 ) s nitrogn sour n KH 2 PO 4 (124 g L -1 ) n N 2 HPO 4 (36 g L -1 ) s posporus sour in JM mium, wr opt s ontrol plot, rsptivly. Tr wr svn iffrnt xprimntl plots tt wr prpr y stting nitrogn n posporus onntrtion s oul n tripl tt of t ontrol plot, n ing t ontrol, oul, n tripl mount of posporus wn nitrogn is oul n tripl, rsptivly. Clorll ws trotropilly ultur wit iffrnt onntrtions of nitrogn n posporus in mium 1% gluos. T otr xprimntl mtos wr t sm s for. Efft of trotropi n utotropi ultur on growt n ftty i omposition T SGR n ftty i omposition of Clorll tt wr ultur in trotropi or utotropi onition wr xmin. For trotropi ultur, 1% gluos ws n r ultur mto ws us t 26 C. For utotropi ultur, ligt wit 60 µmol potons m -2 s -1 ws ontinuously suppli, rpling gluos. T rst of t prours for xprimntl mtos rmin t sm. T siz of Clorll from 100 lls slt rnomly ws msur wit Motim Pro 205A (Moti Instrumnts In., Rimon, BC, Cn). For ry wigt msurmnt, 1.2 µm glss mirofir filtr (GF/C; Wtmn, Knt, UK) ws ri t 60 C for 2 n its wigt ws msur. T lls from 25 ml of ultur mium wr rvst troug filtrtion n ri vi t sm mto. T iomss in ry wigt of lls ws msur rptly for tr tims. For nlysis of ftty i, Clorll ws ultur n rvst in t sm mnnr s ov n pt t -80 C until t tim of nlysis. Ftty i mtyl strs (FAME) wr prpr y trns-strifition wit 10% BF3-MOH t 85 C for 1 (Morrison n Smit 1964). A gs romtogrpy (HP 6890N; Agilnt, Snt Clr, CA, USA) quipp wit n Auto Smplr (Agilnt) ws us for ftty i nlysis. A w-wx olumn (30 m long, 5 mm i, 5 μm film tinss; Suplo, Bllfont, PA, USA) ws us for sprtions. Nitrogn ws us s t rri gs n t flow rt ws st t 30 ml min -1. T olumn tmprtur profil ws sm s t follows: stning t 200 C for 3 min, inrs to 1 C min -1 from 200 to 230 C, n tn ol t 230 C for 25 min. Tmprtur of injtor ws 250 C n flm ioniztion wit t ttor (FID) ws l t 250 C. Ftty i ps wr ttp://x.oi.org/1490/lg

3 Kim & Hur Growt n Ftty Ai of Htrotropi Clorll intgrt using t GC (HP 6890N). A gs romtogrpy softwr n intifition ws m wit rfrn to nown stnrs (PUFA 37 omponnt FAME Mix; Suplo). Sttistil nlysis T rsults of tis stuy wr nlyz y on-wy ANOVA n Dunn s multipl rng tst (Dunn 1955) ws ppli for t signifin lvl (p < 5). T SPSS vrsion 17 (SPSS In., Cigo, IL, USA) progrm ws us for ll sttistil nlyss. RESULTS Efft of ron sour on growt of Clorll As rsult of ulturing Clorll troug trotropi mto using tr iffrnt ron sours, it ws trmin tt gluos ws propr ron sour sin no growt ws onfirm in t xprimntl plots using glyrol n tt. In gluos, ll tr Clorll spis sow growt rt ov 0.5 in 1% n 2% onntrtion. C. vulgris (C4-3) sow igr growt rt ompr to t otr spis n its growt rt ws signifintly igr in t 2% tn 1% gluos. Howvr, no signifint iffrn ws osrv in gluos onntrtion of t otr spis (Fig. 1). Efft of tmprtur n gluos onntrtion on growt T growt rt influn y tmprtur n gluos onntrtion is sown in Fig. 2. As t tmprtur lvt from 24 to 28 C, t growt rt of C. vulgris (C4-3) ws signifintly nn wit ll iffrnt gluos onntrtions, ut it rpily rs to 0.1 t 30 C. A igr growt rt ws osrv in t xprimntl plot in wi 1% or 2% gluos ws tn wn 3-5% gluos ws. T igst growt rt (1) ws osrv t 28 C wit 1% or 2% gluos onntrtion. C. vulgris (C4-4) sow t igst growt rt mong tr spis, n prtiulrly, t growt rt t 32 C ws ovr. T growt rt of tis spis t 32 C ws out t sm s tt of 24 C or 26 C, wi suggsts tt tis spis is mor trmopili n urytrml tn t otr two spis. T growt rt in 1-2% gluos tn to igr tn tt in 3-5% gluos. T growt rt of Clorll sp. (C4-8) ws lwys Spifi growt rt Gluos Att Glyrol 1% 2% C4-3 C4-4 C4-8 1% 2% 1% 2% Cron sours n onntrtions Fig. 1. Spifi growt rt of tr trotropi Clorll spis (C4-3, C. vulgris; C4-4, C. vulgris; C4-8, Clorll sp.) wit iffrnt ron sours n onntrtions in Jworsi s mium (JM) t 28 C for six ys. Diffrnt lttrs on t r in gluos mn signifintly iffrns (p < 5). Spifi growt rt Spifi growt rt Spifi growt rt g f g 1.2 l C. vulgris (C4-3) f fg i g ij j j m 1% 2% 3% 4% 5% f g g n C. vulgris (C4-4) Clorll sp. (C4-8) 24 C 26 C 28 C 30 C f f f f g g g g j i i j 1% 2% 3% 4% 5% j i 24 C 26 C 28 C 30 C 32 C 1% 2% 3% 4% 5% Gluos onntrtions 24 C 26 C 28 C 30 C Fig. 2. Spifi growt rt of tr trotropi Clorll spis wit iffrnt tmprturs n gluos onntrtions in Jworsi s mium (JM) for six ys. Diffrnt lttrs on t r in spis mn signifintly iffrn (p < 5). f g 103 ttp://-lg.r

4 Alg 2013, 28(1): Spifi growt rt C. vulgris (C4-3) Spifi growt rt Htrotropi Spifi growt rt JM 2N1P 2N2P 2N3P 3N1P 3N2P 3N3P C. vulgris (C4-4) f JM 2N1P 2N2P 2N3P 3N1P 3N2P 3N3P JM 2N JM JM 2N JM JM 2NP JM C4-3 C4-4 C4-8 Spis of Clorll Fig. 4. Spifi growt rt of Clorll vulgris (C4-3 n C4-4) n Clorll sp. (C4-8) ultur unr utotropi n trotropi onition t 26 C wit iffrnt onntrtions of nitrogn n posporus in Jworsi s mium (JM) for six ys. 2N JM, oul onntrtions of nitrogn of JM mium; 2NP JM, oul onntrtions of nitrogn n posporus of JM mium. Diffrnt lttrs on t r mn signifintly iffrn (p < 5). Spifi growt rt Clorll sp. (C4-8) In ll xprimntl plots, t growt rt of trotropilly ultur ons ws signifintly igr tn tt of utotropilly ultur ons (p < 5). In trotropi ultur, t growt rt of ll tr spis ws igr in oul onntrtions of nitrogn or posporus tn in t ontrol JM. But for utotropi ultur, t growt rt ws rtr igr in t ontrol JM. Tis suggsts tt t rquir onntrtions of nitrogn n posporus r iffrnt in trotropi n utotropi ultur (Fig. 4). On t otr n, t siz of ll ws mu iggr in trotropi ultur tn in utotropi ultur in ll xprimntl plots (p < 5). In ition, t onigr t 26 C, rgrlss of t gluos onntrtion. T igst growt rt (2) ws in 1% n 2% gluos t 26 C (p < 5) n t rt rs signifintly t 30 C s C. vulgris (C4-3). Efft of nitrogn n posporus on growt T fft of nitrogn n posporus onntrtion on t growt rt of Clorll is illustrt in Fig. 3. T growt rt of two strins (C4-3 n C4-4) of C. vulgris ws 0.57 n 0.56, rsptivly, in oul onntrtion of nitrogn sours [C(NO 3 ) 2 n NNO 3 ] of JM mium. T growt rt ws signifintly inrs y oul JM 2N1P 2N2P 2N3P 3N1P 3N2P 3N3P Conntrtions of nitrogn n posporus Fig. 3. Spifi growt rt of tr trotropi Clorll spis wit iffrnt onntrtions of nitrogn n posporous in Jworsi s mium (JM) t 26 C for six ys. T onntrtion of ontrol JM mium (1N1P) is 2 g C(NO 3) 2 L -1 n 8 g NNO 3 L -1 in nitrogn n 124 g KH 2PO 4 L -1 n 36 g N 2HPO 4 L -1 in posporus. 2N n 3N, oul n tripl onntrtions of nitrogn of JM mium; 1P, originl onntrtion of posporus of JM mium; 2P n 3P, oul n tripl onntrtions of posporus of JM mium. Diffrnt lttrs on t r mn signifintly iffrn (p < 5). f tt of t originl mium (p < 5). Howvr, in s of Clorll sp. (C4-8), t igst growt rt (8) ws in mium ontining oul onntrtion of nitrogn n posporus sours (p < 5). Wn t onntrtion of nitrogn ws inrs twofol, Clorll sp. (C4-8), sow no signifint iffrn twn t xprimntl plot ontining posporus of t originl onntrtion n t plot ontining posporus wos onntrtion ws tripl. Trfor, it ws trmin tt tis spis ws mor ptl to posporus onntrtion ompr to t otr two spis. T growt rt of ll tr spis ws t lowst wn t onntrtion of ot nitrogn n posporus ws tripl tt of JM mium (p < 5). Efft of trotropi n utotropi ultur on growt n ftty i omposition ttp://x.oi.org/1490/lg

5 Kim & Hur Growt n Ftty Ai of Htrotropi Clorll Cll siz (µm) JM C. vulgris (C4-3) Htrotropi 2N JM Dry wigt (g L -1 ) f f JM 2N JM JM 2N JM JM 2NP JM C4-3 C4-4 C4-8 f f Htrotropi f Cll siz (µm) C. vulgris (C4-4) Htrotropi Spis of Clorll Fig. 6. Biomss in ry wigt of Clorll vulgris (C4-3 n C4-4) n Clorll sp. (C4-8) ultur in utotropi n trotropi onition t 26 C wit iffrnt onntrtions of nitrogn n posporus in Jworsi s mium (JM) for six ys. 2N JM, oul onntrtions of nitrogn of JM mium; 2NP JM, oul onntrtions of nitrogn n posporus of JM mium. Diffrnt lttrs on t r mn signifintly iffrn (p < 5). Cll siz (µm) JM JM Clorll sp. (C4-8) 2N JM ntrtions of nitrogn n posporus i not v mu impt on t siz of ll (Fig. 5). T iomss in ry wigt of tr spis is sown in Fig. 6. In ll xprimntl plots, t iomss in trotropi ultur ws signifintly igr tn tt of utotropi on (p < 5). T ry wigt of two strins (C4-3 n C4-4) of C. vulgris ultur wit oul onntrtion of nitrogn ws signifintly igr tn tt of t ontrol JM. But no signifint iffrn ws osrv in Clorll sp. (C4-8). Among tr spis, t iomss in ry wigt of C. vulgris (C4-3) ws t igst wit g L -1. T ftty i omposition of Clorll spis ultur utotropilly n trotropilly is sown in Tls 1-3. T ftty i ontnt of C16:0, C18:2n6, n 2NP JM Htrotropi Fig. 5. Cll siz of Clorll spis ultur in utotropi n trotropi onition t 26 C wit iffrnt onntrtions of nitrogn n posporus in Jworsi s mium (JM) for six ys. 2N JM, oul onntrtions of nitrogn of JM mium; 2NP JM, oul onntrtions of nitrogn n posporus of JM mium. Diffrnt lttrs on t r mn signifintly iffrn (p < 5). C18:3n3 ws t igst in ll tr spis. In s of C16:0 n C18:3n3, t omposition of utotropilly ultur on ws mu igr tn tt of trotropilly ultur on (p < 5). As for C18:2n6, tr ws not ny iffrn in C. vulgris (C4-3), ut t rsults of C. vulgris (C4-4) n Clorll sp. (C4-8) wr onsirly igr wit trotropi ultur. T onntrtion of nitrogn i not sm to v mu impt on ftty i omposition. In tis stuy, oosxnoi i (C22:6n3) ws not tt in ny of t xprimntl plots, wrs iospntnoi i (C20:5n3) of trotropilly ultur C. vulgris (C4-3) ontining oul onntrtion of nitrogn ws msur to 1.4 µg mg -1 wi ws t igst vlu msur out of ll. Arioni i (C20:4n6) of utotropilly ultur C. vulgris (C4-3) ws µg mg -1, ut it ws not tt t ll in Clorll sp. (C4-8). T mount of n-3 igly unsturt ftty i (HUFA) ws mu igr wit utotropi ultur in ll tr spis (p < 5). Wn ultur utotropilly, n-3 HUFA ws foun to t igst in C. vulgris (C4-3) wit 22 µg mg -1, ut wn ultur trotropilly, Clorll sp. (C4-8) sow t igst ontnt of n-3 HUFA wit 8-10 µg mg -1. T onntrtion of t otr HUFA su s n-6 n n-9 HUFA of t tr spis tn to igr wit trotropi ultur, ut no notil iffrn ws osrv wit iffrnt onntrtions of nitrogn. Totl lipi of C. vulgris (C4-3) ws signifintly lowr in trotropi ultur, ut t nitrogn onntrtion of mium i not us t signifint iffrn (p < 105 ttp://-lg.r

6 Alg 2013, 28(1): Tl 1. Ftty i ompositions (μg mg -1 ry mttr) of Clorll vulgris (C4-3) ultur in utotropi n trotropi onition t 26 C wit JM n 2N JM mium Ftty i Htrotropi JM 2N JM JM 2N JM C11:0 2.5 ± ± C12:0 2.0 ± ± C13:0 2.2 ± ± ± ± 4 C14:0 2.8 ± ± ± ± 6 C14:1 2.0 ± ± ± ± 0.78 C15:0 ± ± ± ± 2 C15:1 3.3 ± ± ± C16: ± ± ± ± 3.48 C16:1 1.9 ± ± ± ± 0.31 C17:0 1.3 ± ± ± ± 3 C17: ± ± 0.37 C18:0 1.7 ± ± ± ± 0.59 C18:1n9 4.9 ± ± ± ± 5 C18:2n ± ± ± ± 2.75 C18:3n C18:3n ± ± ± ± 1.48 C20:0 3.0 ± ± ± ± 0.14 C20:1n9-0.5 ± ± ± 1 C20: ± C20:4n6 1.2 ± ± C20:5n3-0.5 ± ± 6 C22:6n n-3 HUFA 22.1 ± ± ± ± 1.48 n-6 HUFA 15.2 ± ± ± ± 2.75 n-9 HUFA 4.9 ± ± ± ± 4 Totl lipi (mg g -1 ) ± ± ± ± Vlus in row wit iffrnt lttrs r signifintly iffrnt (p < 5). 2N JM mium, oul onntrtions of nitrogn of Jworsi s mium (JM); HUFA, igly unsturt ftty i. Tl 2. Ftty i ompositions (μg mg -1 ry mttr) of Clorll vulgris (C4-4) ultur in utotropi n trotropi onition t 26 C wit JM n 2N JM mium Ftty i Htrotropi JM 2N JM JM 2N JM C11:0 2.1 ± ± C12: C13:0 1.9 ± ± ± ± 0.74 C14:0 2.4 ± ± ± ± 1.45 C14:1 2.0 ± ± ± ± 5 C15:0 ± 5-3 ± 5 ± C15:1 2.1 ± ± ± 5 5 ± C16: ± ± ± ± 2.34 C16:1 1.6 ± ± ± ± 0.10 C17:0 ± ± ± ± 4 C17: ± 0.54 C18:0 1.4 ± ± ± ± 0.35 C18:1n9 4.0 ± ± ± ± 5.42 C18:2n ± ± ± ± 3.55 C18:3n C18:3n ± ± ± ± 1.38 C20:0 2.6 ± ± ± ± 0.14 C20:1n C20:2 - - ± 1 - C20:4n6 0.3 ± C20:5n ± 0.76 C22:6n n-3 HUFA 15.6 ± ± ± ± 1.97 n-6 HUFA 13.6 ± ± ± ± 3.55 n-9 HUFA 4.0 ± ± ± ± 5.42 Totl lipi (mg g -1 ) ± ± ± ± Vlus in row wit iffrnt lttrs r signifintly iffrnt (p < 5). 2N JM mium, oul onntrtions of nitrogn of Jworsi s mium (JM); HUFA, igly unsturt ftty i. ttp://x.oi.org/1490/lg

7 Kim & Hur Growt n Ftty Ai of Htrotropi Clorll 5). T ultur mto n mium i not v ny notil impt on t totl lipi of C. vulgris (C4-4). T totl lipi of Clorll sp. (C4-8) ws trmin to lowr in trotropi ultur wit oul onntrtion of nitrogn. Among utotropilly ultur ons, t totl lipi ws t igst wit mg g -1 in C. vulgris (C4-3) in oul onntrtion of nitrogn. An mong trotropilly ultur ons, it ws t igst wit 200 mg g -1 in t ontrol JM mium of Clorll sp. (C4-8). DISCUSSION Monosri su s gluos n mtoli sustns su s glyrol n tt r wily us s ron sour for trotropi ultur of mirolg (L 2001, D Swf t l. 2003, 2003, Ip n Cn 2005). A ron sour s n impt on growt n mtoli tivity for trotropi mirolgl spis (Yooi t l. 1998, D Swf t l. 2003). In tis stuy, tr spis of Clorll wr l to utiliz gluos s ron sour. Howvr, ty wr inpl of growing in t xprimnt plot in wi glyrol n tt wr us for ultur (Si t l. 1999, Ip n Cn 2005). Wit rgr to growt rt, tr Clorll spis sow similr tnny in t xprimnt on gluos onntrtion. T growt of lls sm mor lrt in t rlir stgs in t xprimntl plots in wi 1-2% gluos ws, ut t growt ws mu wr in 4-5% gluos. Tis tnny sms to us y t growt of lls in t rly stg tt ws inr wn gluos onntrtion ws 4% or ov (Xiong t l. 2008). Altoug t growt of t lls wit 1-2% gluos slow own roun y 3-4, t gluos onntrtion wit 4-5%, in turn, l to rltivly longr growt prio ompr to 1-2% gluos onntrtion n it too longr tims to r t xponntil ps (Si t l. 1999). T optimum onntrtion of gluos vri wit ll nsity n spis of Clorll. But ontinuous growt n xpt itionl quntity of gluos is in vry 3-4 ys ftr initil ultur wit 1-2% onntrtion. As for t ultur tmprtur, t optimum tmprtur for C. vulgris (C4-3) sms to 28 C sin it grw most rpily t 28 C, ut slowly t 30 C. But tt for ot C. vulgris (C4-4) n Clorll sp. (C4-8) ws trmin to 26 C. Altoug C. vulgris (C4-3) n Clorll sp. Tl 3. Ftty i ompositions (μg mg -1 ry mttr) of Clorll sp. (C4-8) ultur in utotropi n trotropi onition t 26 C wit JM n 2NP JM mium Ftty i Htrotropi JM 2NP JM JM 2NP JM C11:0-2.8 ± C12:0 3.5 ± ± C13:0 1.9 ± ± ± ± 0.31 C14:0 4.8 ± ± ± ± 0 C14:1 1.6 ± ± ± ± 4 C15:0 2.0 ± ± ± 1 ± 4 C15:1 1.3 ± ± C16:0 2 ± ± ± ± 6.12 C16:1 2.8 ± ± ± ± 0.34 C17:0 2.1 ± 0.32 ± ± ± 8 C17:1 - - ± 0.19 ± 0.19 C18:0 2.9 ± ± ± ± 0.55 C18:1n9 7.9 ± ± ± ± 1.28 C18:2n ± ± ± ± 7.94 C18:3n C18:3n ± ± ± ± 3.63 C20:0 5.0 ± ± ± ± 0.59 C20:1n9 - - ± C20: ± ± 0.58 C20:4n C20:5n C22:6n n-3 HUFA 16.0 ± ± ± ± 3.63 n-6 HUFA 12.2 ± ± ± ± 7.94 n-9 HUFA 7.9 ± ± ± ± 1.28 Totl lipi (mg g -1 ) ± ± ± ± Vlus in row wit iffrnt lttrs r signifintly iffrnt (p < 5). 2NP JM mium, oul onntrtions of nitrogn n posporus of Jworsi s mium (JM); HUFA, igly unsturt ftty i. 107 ttp://-lg.r

8 Alg 2013, 28(1): (C4-8) wr unl to grow t 30 C or ov tmprtur, C. vulgris (C4-4) grw wll t 30 C n its growt rt rmin firly ig t 32 C ompr to tt of 24 C. Tis l to onlusion tt C. vulgris (C4-4) ws most rsistnt to ig tmprtur. T optimum tmprtur C for tr trotropi Clorll spis in tis stuy ws onsistnt wit t prvious rports (Si t l. 2002, Wu n Si 2007, Xiong t l. 2008). It s n rport tt t rtio of nitrogn to posporus, long wit onntrtion of nitrogn n posporus ontin in mium, s n impt on growt of mirolg n syntsis of lipi n ftty i (Liu n L 2000, Coi t l. 2002, Tripti t l. 2002, Wn n Cn 2003). In tis stuy, only t onntrtion of nitrogn n posporus ws oul n tripl tt of JM mium, rsptivly, ut no signifint iffrn ws osrv. Tis n xplin y t ft tt t initil mount of gluos wit 1% s ron sour us in tis xprimnt ws insuffiint for rpi growt (Ip n Cn 2005). Wn t growt of Clorll spis ws ompr twn trotropi n utotropi ultur, it ws onlu tt t growt rt n ll siz wr signifintly igr wit trotropi ultur tn utotropi on (Griffits 1965). Wit t trotropi ultur in tis stuy, t ll nsity of C. vulgris (C4-4) n Clorll sp. (C4-8) r pproximtly 2, lls ml -1, wrs t ll nsity of C. vulgris (C4-3) ws pproximtly 1, lls ml -1. Howvr, t iomss in ry wigt of C. vulgris (C4-3) ws t igst mong tr spis wit g L -1. It n xplin y t ft tt t ll siz of tis spis ws 0.5 µm lrgr tn tt of t otr two spis. As for t ftty i ompositions, t ontnt of 16:0 n n-3 HUFA ws rmtilly lowr wn Clorll ws ultur trotropilly. Altoug t totl lipi of C. vulgris (C4-3) n Clorll sp. (C4-8) ws lowr wn trotropilly ultur, tr ws not ny iffrn in C. vulgris (C4-4). Tis l to onlusion tt t totl lipi vri wit spis. Howvr, in ll tr spis, t onntrtion of nitrogn i not sm to v mu influn on t ftty i ompositions. Evn in t sm mirolgl spis, t lipi ontnt of mirolg pns on tmprtur, nutrint, ulturing mto, t. (Rtlg n Wynn 2002, Wn n Cn 2003, Cisti 2007). Tus, furtr stuy on mil omposition of mirolg is n in til. As rsult of tis stuy, it ws onlu tt wn Clorll ws ultur trotropilly, t growt rt, ll siz, n ftty i ompositions wr fft y tmprtur, gluos onntrtion, nutrint in mium n strin. In ition, w suggst C. vulgris (C4-3) mong tr spis to t most il trotropi Clorll spis for inustril pplition sin it ig iomss n lipi ontnt. ACKNOWLEDGEMENTS Tis rsr ws support y grnt from Mrin Biotnology Progrm Fun y Ministry of Ln, Trnsport n Mritim Affirs of Korn Govrnmnt. REFERENCES Brly, W. R., Mgr, K. M. & Aril, J. R Htrotropi proution of long in omg-3 ftty is utilizing lg n lg-li miroorgnisms. J. Appl. Pyol. 6: Cn, F Hig ll nsity ultur of mirolg in trotropi growt. Trns Biotnol. 14: Cisti, Y Bioisl from mirolg. Biotnol. Av. 25: Coi, Y. E., Yun, Y. -S. & Pr, J. M Evlution of ftors promoting stxntin proution y unillulr grn lg, Hmtoous pluvilis, wit frtionl ftoril sign. Biotnol. Prog. 18: D Swf, M. E., Pron, J. T. & Sijtsm, L F-t ultivtion of t oosxnoi-i-prouing mrin lg Cryptoinium onii on tnol. Appl. Miroiol. Biotnol. 61: D Swf, M. E., Sijtsm, L. & Pron, J. T Hig-llnsity f-t ultivtion of t oosxnoi i prouing mrin lg Cryptoinium onii. Biotnol. Biong. 81: Dunn, D. B Multipl rng n multipl F tst. Biomtris 11:1-42. Grffits, D. J T umultion of royrt in Clorll vulgris unr trotropi onitions. Ann. Bot. 29: Guillr, R. L Growt msurmnt. In Stin, J. R. (E.) Hnoo of Pyologil Mtos. Cmrig Univrsity Prss, Lonon, pp Hsgw, T., Ou, M., Nomoto, K. & Yosii, Y Augmnttion of t rsistn ginst Listri monoytogns y orl ministrtion of ot wtr xtrt of Clorll vulgris in mi. Immunoprmol. Immunotoxiol. 16: Ip, P. -F. & Cn, F Proution of stxntin y t ttp://x.oi.org/1490/lg

9 Kim & Hur Growt n Ftty Ai of Htrotropi Clorll grn mirolg Clorll zofinginsis in t r. Pross Biom. 40: Justo, G. Z., Silv, M. R. & Quiroz, M. L Effts of t grn lg Clorll vulgris on t rspons of t ost mtopoiti systm to intrpritonl rli sits tumor trnsplnttion in mi. Immunoprmol. Immunotoxiol. 23: Kim, H. J., Kim, I. H. & L, J. H Biologil tivitis of tnol xtrt from t swtr lg, Clorll lliposi C020. Korn J. Biotnol. Biong. 23: Kim, J. -S Prprtion of lorll rins n its qulity rtristis. Korn J. Foo Nutr. 17: L, Y. -K Commril proution of mirolg in t Asi-Pifi rim. J. Appl. Pyol. 9: L, Y. -K Mirolgl mss ultur systm n mtos: tir limittion n potntil. J. Appl. Pyol. 13: Liu, B. -H. & L, Y. -K Sonry rotnois formtion y t grn lg Clorooum sp. J. Appl. Pyol. 12: Mio, X. & Wu, Q Hig yil of io-oil proution from fst pyrolysis y mtoli ontrolling of Clorll prototois. J. Biotnol. 110: Mio, X., Wu, Q. & Yng, C Fst pyrolysis of mirolg to prou rnwl fuls. J. Anl. Appl. Pyrolysis. 71: Morrison, W. R. & Smit, L. M Prprtion of ftty i mtyl strs n imtyltls from lipis wit oron fluori-mtnol. J. Lipi Rs. 5: Olizol, M Commril vlopmnt of mirolgl iotnology: from t tst tu to t mrtpl. Biomol. Eng. 20: Rtlg, C. & Wynn, J. P T iomistry n mo- lulr iology of lipi umultion in olginous miroorgnisms. Av. Appl. Miroiol. 51:1-51. Si, X. M., Jing, Y. & Cn, F Hig-yil proution of lutin y t grn mirolg Clorll prototois in trotropi f-t ultur. Biotnol. Pro. 18: Si, X. -M., Liu, H. -J., Zng, X. -W. & Cn, F Proution of iomss n lutin y Clorll prototois t vrious gluos onntrtions in trotropi ulturs. Pross Biom. 34: Tompson, A. S., Ros, J. C. & Pttmn, I Nturl Environmntl Rsr Counil Cultur Colltion of lg n protozo: tlogu of strins. Frswtr Biology Assoition, Amlsi, 164 pp. Tripti, U., Sr, R. & Rvisnr, G. A Efft of ultur onitions on growt of grn lg: Hmtoous pluvilis n stxntin proution. At Pysiol. Plnt. 24: Wn, Z. -Y. & Cn, F Htrotropi proution of iospntnoi i y mirolg. Biotnol. Av. 21: Wu, Z. & Si, X Optimiztion for ig-nsity ultivtion of trotropi Clorll s on yri nurl ntwor mol. Ltt. Appl. Miroiol. 44: Xiong, W., Li, X., Xing, J. & Wu, Q Hig-nsity frmnttion of mirolg Clorll prototois in iortor for miroio-isl proution. Appl. Miroiol. Biotnol. 78: Yooi, T., Hon, D., Higsir, T. & Nr, T Optimiztion of oosxnoi i proution y Sizoytrium liminum SR21. Appl. Miroiol. Biotnol. 49: ttp://-lg.r