Microsoft Word - 06 徐进.doc

Size: px

Start display at page:

Download "Microsoft Word - 06 徐进.doc"

滋莘
6 years ago
Views:

1 36 3 Vol. 36, No ACTA HYDROBIOLOGICA SINICA May, DOI: /SP.J 徐 1, 2 进 1 徐旭东 1 方仙桃 1 胡晗华 (1., ; 2., ) :,,,, 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 : (2012) ,, [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 : ; 修订日期 : 基金项目 : (2011CB200901); (KSCX2-YW-G-060) 作者简介 : (1986 ),, ; ; xjlf198624@sina.com 通讯作者 :, hanhuahu@ihb.ac.cn

2 3 : BG11 [9] 1.5% BG11, μmol photons/(m 2 s) 1.5% BG11,, 20 ml BG11 50 ml, μmol photons/(m 2 s) 20d,, Chlorella sp. NMX37N, 208 ( N, E),, S 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, r/min 5min, EP,, 20 [11] Silica gel 60 F254 (Merk KgaA Darmstadt, Ger- many), ( , v/ v/ v),, min [12] sigma 1.4 Chlorella sp. NMX37N Chlorella sp. NMX37N 50 ml BG ml, 100 μmol photons/(m 2 s), A , A 750 =0.05 [13] Chlorella sp. NMX37N μmol photons/(m 2 s), 6000 r/min,, A 750 =1.0, 2 ml Oxy Lab (Hansatech), μmol photons/(m 2 s), Chlorella sp. NMX37N 1.5 L BG11 2 L, μmol photons/(m 2 s),,,, A 750 (OD 220 ) NO 3 [14] 1.6 Chlorella sp. NMX37N 1.5 L BG11 2 L, 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.

3 ml Aglient, 1 ml 1 mol/l, N 2, 100 1h, 200 μl,, 200 μl,, N 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, /d, 40 Chlorella sp. NMX37N 5, 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); 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],,,

4 3 : S 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.

5 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 C ± ±0.12 C ± ±0.01 C ± ±0.71 C ± ±0 C ± ±0.07 C ± ±0.14 C ± ±0.48 γ-c ± ±0.05 α-c ± ±0.02, C16 C18 [17, 18],, [19]

6 3 : 431 Chlorella sp. NMX37N, C16 C18, 98%, [1] Chisti Y. Biodiesel from microalgae beats bioethanol [J]. Trends Biotechnology, 2008, 26(3): [2] Hu Q, Smmerfeld M, Jarvis E, et al. Microalgal triacylglycerols as feedstock for biofuel production: perspectives and advances [J]. The Plant Journal, 2008, 54(4): [3] Huang G, Chen F, Wei D, et al. Biodiesel production by microalgal biotechnology [J]. Applied Energy, 2010, 87(1): [4] Hoek C V, Mann D G, Jahns H M. Algae: an Introduction to Phycology [M]. Cambridge University Press. 1995, 623 [5] Huss V A R, Frank C, Hartmann E C, et al. Biochemical taxonomy and molecular phylogeny of the genus Chlorella sensu lato (Chlorophyta) [J]. Journal of Phycology, 1999, 35(3): [6] Teresa M M, Antonio A M, Nidia S C. Microalgae for biodiesel production and other applications: A review [J]. Renewable and Sustainable Energy Reviews, 2010, 14(1): [7] Converti A, Casazza A A, Ortiz E Y, et al. Effect of temperature and nitrogen concentration on the growth and lipid content of Nannochloropsis oculata and Chlorella vulgaris for biodiesel production [J]. Chemical Engineering and Processing, 2009, 48(6): [8] Hsieh C H, Wu W T. Cultivation of microalgae for oil production with a cultivation strategy of urea limitation [J]. Bioresource Technology, 2009, 100(17): [9] Stanier R Y, Kunisawar M M, Mandel M, et al. Purification and properties of unicellular blue-green algae (Order Chroococcales) [J]. Bacteriological Reviews, 1971, 35(2): [10] Bligh E G, Dyer W J. A rapid method of lipid extraction and purification [J]. Canadian Journal Biochemistry and Physiology, 1959, 37(8): [11] Reiser S, Somerville C. Isolation of mutants of Acinetobacter calcoaceticus deficient in wax ester synthesis and complementation of one mutation with a gene encoding a fatty acyl-coenzyme A reductase [J]. Journal Bacteriology, 1997, 179(9): [12] Yu E T, Zendejas F J, Lane D P, et al. Triacylglycerol accumulation and profiling in the model diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum (Baccilariophyceae) during starvation [J]. Journal Applied Phycology, 2009, 21(6): [13] Shestakov S V, Anbudurai P R, Stanbekova G E, et al. Molecular cloning and characterization of the ctpa gene encoding a carboxyl-terminal processing protease. Analysis of a spontaneous photosystem II-deficient mutant strain of the Cyanobacterium Synechocystis sp. PCC 6803 [J]. Journal Biology Chemistry, 1994, 269(30): [14] Collos Y, Mornet F, Sciandra A, et al. An optical method for the rapid measurement of micromolar concentrations of nitrate in marine phytoplankton cultures [J]. Journal Applied Phycology, 1999, 11(2): [15] Bigogno C, Khozin G I, Boussiba S, et al. Lipid and fatty acid composition of the green oleaginous alga Parietochloris incisa, the richest plant source of arachidonic acid [J]. Phytochemistry, 2002, 60(5): [16] Boussiba S, Vonshak A, Cohen Z, et al. Lipid and biomass production by the halotolerant microalga Nannochloropsis salina [J]. Biomass, 1987, 12(1): [17] Ohlrogge J, Browse J. Lipid biosynthesis [J]. Plant Cell, 1995, 7: [18] Li X B, Xu X D, Kong R Q. Studies on the production of oil and polyunsaturated fatty acids in five species of Nannochloropsis [J]. Acta Hydrobiologica Sinica, 2010, 34(5): [,,.., 2010, 34(5): ] [19] Liu J, Huang, J C, Sun Z, et al. Differential lipid and fatty acid profiles of photoautotrophic and heterotrophic Chlorella zofingiensis: Assessment of algal oils for biodiesel production [J]. Bioresource Technology, 2011, 102(1):

7 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 , China; 2. Graduate University of Chinese Academy of Sciences, Beijing , 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

Microsoft Word - 142-147 tb20140320艾江宁-文献新.doc

Microsoft Word - 142-147 tb20140320艾江宁-文献新.doc 微生物学通报 Microbiology China tongbao@im.ac.cn 研究报告 Jan. 20, 2015, 42(1): 142 147 2015 by Institute of Microbiology, CAS DOI: 10.13344/j.microbiol.china.140320 生长速度快且油脂产率高的湛江等鞭金藻诱

Microsoft Word - 06 徐 进.doc

Microsoft Word - 06 徐进.doc