36 3 Vol. 36, No.3 2012 5 ACTA HYDROBIOLOGICA SINICA May, 2 0 1 2 DOI: 10.3724/SP.J.1035.2012.00426 徐 1, 2 进 1 徐旭东 1 方仙桃 1 胡晗华 (1., 430072; 2., 100049) :,,,, C16 C18,, 21, Chlorella sp. NMX37N 15 35, 25, 0.53/d, 250 μmol photons/(m 2 s),,, Chlorella sp. NMX37N 40d, 33%,, ( 20d) 48h,,,, C16 0 C18 2, 70%, C20, : ; ; ; 中图分类号 : Q819 文献标识码 : A 文章编号 : 1000-3207(2012)03-0426-07,, [1],,,,, [2, 3],,,, 2 12 μm, [4] 9, 4 [5],, 5% 63%( ) [6 8], C16 0 C18 1 C18 2 C18 3 [2], (15 ),, Chlorella sp. NMX37N,,, 收稿日期 : 2011-04-02; 修订日期 : 2012-01-07 基金项目 : (2011CB200901); (KSCX2-YW-G-060) 作者简介 : (1986 ),, ; ; E-mail: xjlf198624@sina.com 通讯作者 :, E-mail: hanhuahu@ihb.ac.cn
3 : 427 1 1.1 BG11 [9] 1.5% BG11, 25 30 50 μmol photons/(m 2 s) 1.5% BG11,, 20 ml BG11 50 ml, 25 50 70 μmol photons/(m 2 s) 20d,, Chlorella sp. NMX37N, 208 (41 29 06 N, 113 22 21 E),, 1.2 18S rdna, Leica CRT5000, DNA (Fermentas, Vilnius, Lithuania), 18S-1: 5 -tggttgatcctgcca gtagtc-3 18S-2: 5 -tgatccttctgcaggttcacc-3 PCR 18S rdna pmd18-t(takara), 18S rdna 18S rdna MEGA 4.0,, 1.7 kb MEGA 4.0 Phylipwx 1.3 / [10] 6000 r/min (1 1), 1 2min, 3/10 1 mol/l 0.2 mol/l, 12000 r/min 5min, EP,, 20 [11] Silica gel 60 F254 (Merk KgaA Darmstadt, Ger- many), (70 30 1, v/ v/ v),, 37 5 10 min [12] sigma 1.4 Chlorella sp. NMX37N Chlorella sp. NMX37N 50 ml BG11 100 ml, 100 μmol photons/(m 2 s), A 750 15 20 25 30 35 40, A 750 =0.05 [13] Chlorella sp. NMX37N 25 100 μmol photons/(m 2 s), 6000 r/min,, A 750 =1.0, 2 ml Oxy Lab (Hansatech), 100 250 500 1000 2000 μmol photons/(m 2 s), 25 1.5 Chlorella sp. NMX37N 1.5 L BG11 2 L, 25 250 μmol photons/(m 2 s),,,, A 750 (OD 220 ) NO 3 [14] 1.6 Chlorella sp. NMX37N 1.5 L BG11 2 L, 25 150 μmol photons/(m 2 s), 6000 r/min, ddh 2 O,, [10] 100 mg (1 2), ddh 2 O,,, N 2,, 1.7
428 36 1.5 ml Aglient, 1 ml 1 mol/l, N 2, 100 1h, 200 μl,, 200 μl,, N 2 100 μl, 1 μl Ultra Trace (Thermo Scientific, United States) 2 2.1, BG11 18S rdna, BG11,, ( 1 ), 21, Chlorella sp. NMX37N,,, ; ; 4 6 μm 18S rdna Chlorella sp. NMX37N 18S rdna Chlorella sp. NDem 9/21 T-13d(AY197628), ; Chlorella sp. NJ-18(DQ377324), Chlorella sp. NMX37N, ( 2) 2.3 Chlorella sp. NMX37N 3 A Chlorella sp. NMX37N 15 35, 25, 0.53/d, 20 30, 0.51/d 0.52/d, 15 35 0.41/d, 40 Chlorella sp. NMX37N 5, 100 250 μmol photons/(m 2 s), 250 μmol photons/(m 2 s),, 2000 μmol photons/(m 2 s) 300 μmol O 2 mg/(chl h)( 3B), 2.4 Chlorella sp. NMX37N Chlorella sp. NMX37N 1.5 L 1/5 NaNO 3 BG11,,, NO 3 ( 4), 4, 10, 17, 1 ( ) Fig. 1 Comparison of triacylglycerol (indicated by the arrow) contents in Chlorella species by thin layer chromatography 1 7. ; 2. Chlorella sp. NMX37N; S. (0.02 mg); 8. 1 7. Other Chlorella species; 2. Chlorella sp. NMX37N; S. Triolein (0.02 mg); 8. Phaeodactylum tricornutum 2.2 Chlorella sp. NMX37N Chlorella sp. NMX37N, NO 3, 17, NO 3,,,,,, [15, 16],,,
3 : 429 2 18S rdna ( Chlorella sp. NMX37N ) Fig. 2 Maximum-likelihood tree of Chlorella species Sensu lato inferred from 18S rdna gene sequences (Inset: light microscopic image of Chlorella sp. NMX37N) (1000 ) (1000 ) (100 ), 50, 0.01 Bootstrap values are shown at the internal nodes for neighbor joining (1000 replications), maximum parsimony (1000 replications) and maximum likelihood (100 replications), respectively, if the node is supported by at least two bootstrap values of 50% or above. Branch lengths correspond to evolutionary distances. A distance of 0.01 is indicated by the scale Fig. 3 3 Chlorella sp. NMX37N (A) (B) Temperature (A) and irradiance (B) tolerance of Chlorella sp. NMX37N, [7, 8],, 2.5 Chlorella sp. NMX37N 5 Chlorella sp. NMX37N,
430 36 4 NO 3 Chlorella sp. NMX37N (A) (B) Fig. 4 NO 3 concentrations in medium, cell density (A) and oil content (B) of Chlorella sp. NMX37N cultured at different growth phase S. Triolein(0.02mg) 5 Chlorella sp. NMX37N (+N) ( N) Fig. 5 The dry biomass and total lipid contents of Chlorella sp. NMX37N cultured under nitrogen-replete (+N) or -deplete ( N) conditions BG11 ( 40d), 33%, 1.3 g/l 1/5 NaNO 3 BG11 ( 20d), 48h, (1.1 g/l) (1.3 g/l),, 36%,, 0.43 g/l,,,,,,,,,,,, 2.6 Chlorella sp. NMX37N Chlorella sp. NMX37N ( 1),, C16 0 C18 2, 70% 45%, C20 表 1 Chlorella sp. NMX37N 藻细胞总脂及三酰基甘油中脂肪酸组成与含量 ( 摩尔百分比 ) Tab. 1 Fatty acid composition (mol %) in total lipids and triacylglycerol of Chlorella sp. NMX37N Fatty acids Total lipids Triacylglycerol C14 0 1.3 ±0.05 1.6 ±0.12 C14 1 0.21±0.01 0.24±0.01 C16 0 31.05±1.23 30.66±0.71 C16 1 0.4 ±0.01 0.43±0 C18 0 1.87±0.04 1.74±0.07 C18 1 13.83±0.42 11.92±0.14 C18 2 40.22±1.2 40.08±0.48 γ-c18 3 0.13±0.08 0.12±0.05 α-c18 3 10.98±0.63 13.15±0.02, C16 C18 [17, 18],, [19]
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432 36 SCREENING AND LIPID ANALYSES OF HIGH OLEAGINOUS CHLORELLA SPECIES XU Jin 1, 2, XU Xu-Dong 1, FANG Xian-Tao 1 and HU Han-Hua 1 (1. Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China; 2. Graduate University of Chinese Academy of Sciences, Beijing 100049, China) Abstract: Chlorella species which have strong adaptability distributes widely in various habitats, especially in the freshwater. Their assimilation product is mainly starch, but environmental stress can trigger lipid accumulation within the cells. The predominant fatty acids are C16 and C18 in Chlorella species, which are suitable feedstocks for biodiesel production. A number of Chlorella strains were isolated from fresh water of China, and their triacylglycerol contents were analyzed by thin layer chromatography. Out of 21 oleaginous Chlorella strains, a high oleaginous stain, NMX37N, was identified. It grew at temperatures from 15 to 35 but not at 40, and the optimum conditions, at 25 and 250 μmol photons/m 2 s, it grew with a specific growth rate of 0.53/d. Batch culture experiments showed that the content of triacylglycerols in Chlorella sp. NMX37N increased with time and reached to the highest at the late stationary phase when the nitrate was exhausted. The total lipid content was 33% of dry cell weight when Chlorella sp. NMX37N grew under batch culture conditions for 40d. A two-step culture method, namely, transfer to the nitrogen deficiency condition at the late exponential phase (about 20d), gave a higher total lipid (36%) compared with the batch culture method. The main fatty acids (70%) in total lipids and triacylglycerols of Chlorella sp. NMX37N were C16 0 and C18 2. In addition, there were no long-chain fatty acids, such as C20, in Chlorella sp. NMX37N. The results indicated that the triacylglycerols in Chlorella sp. NMX37N may serve as excellent feedstocks for biodiesel production. Key words: Chlorella; Triacylglycerols; Fatty acids; Biodiesel