126 微生物学通报 2012, Vol.39, No.1 (1. Third Institute of Oceanography, State Oceanic Administration, Xiamen, Fujian , China) (2. School of Life Scie

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1 微生物学通报 Microbiology China JAN 20, 2012, 39(1): by Institute of Microbiology, CAS 生物实验室尼罗红染色法筛选产油酵母及定量检测胞内油脂含量的研究林义 1,2 钟添华 1* 骆祝华 1 黄翔玲 1 叶德赞 ( ) ( ) 1 摘要 : 目的建立产油酵母筛选以及胞内油脂含量测定的简便方法方法利用尼罗红与胞内油脂成分结合后在紫外光照射下发出荧光且荧光强弱与油脂含量相关的原理通过在添加尼罗红的培养基中培养酵母, 并观察菌落荧光的方法对 385 株深海酵母进行产油脂菌株筛选, 利用 26S rdna D1/D2 区序列分析方法对筛选获得的产油酵母菌株进行鉴定, 并以其中的一株高产油脂酵母 (2A00015) 为试验菌株, 建立了一套尼罗红染色快速测定油脂含量的方法结果获得 22 株产油酵母, 其中油脂含量最高可达 62.9%, 经分子鉴定后显示这 22 株酵母分别属于 (Candida viswanathii) 近平滑假丝酵母(Candida parapsilosis) 粘质红酵母(Rhodotorula mucilaginosa) 汉逊德巴利酵母(Debaryomyces hansenii) 季也蒙毕赤酵母(Pichia guilliermondii) 以及 Rhodosporidium paludigenum 酵母尼罗红染色快速测定油脂含量方法的最佳检测条件为 : 菌悬液 OD 600 小于 1.2, 尼罗红浓度 0.5 mg/l, 染色时间 5 min, 激发波长 488 nm, 发射波长 570 nm 该测定方法得到相对荧光强度与称重法得到油脂含量呈正相关性, R 2 = 关键词 : 尼罗红染色法, 产油酵母, 26S rdna D1/D2, 荧光, 油脂含量 Study on screening of oleaginous yeast and determination of intracellular lipid content by nile red dyeing LIN Yi 1,2 ZHONG Tian-Hua 1* LUO Zhu-Hua 1 HUANG Xiang-Ling 1 YE De-Zan 1 基金项目 : (No ) * 通讯作者 :Tel: ; : zth @163.com 收稿日期 : ; 接受日期 :

2 126 微生物学通报 2012, Vol.39, No.1 (1. Third Institute of Oceanography, State Oceanic Administration, Xiamen, Fujian , China) (2. School of Life Sciences, Xiamen University, Xiamen, Fujian , China) Abstract: [Objective] To establish a simple method to screen oleaginous yeast and determine the intracellular lipid content. [Methods] The study is based on the theory that the combination of nile red and intracellular oil will emit fluorescence when induced by UV light and the fluoresence indensity is associated with the lipid content. We cultivate yeast in the culture medium added with nile red, and screen the oleaginous yeast strains from the 385 deep-sea yeasts by measns of obeserving the fluorescence of the yeast colony. We have identified the screened oleaginous yeast strains by the 26S rdna D1/D2 series analysis method. Designating one of the oleaginous yeasts (2A00015) as the test strain, the lipid content rapid determination method by nile red dyeing was established. [Results] 22 oleaginous yeasts were obtained with the lipid content reaching up to 62.9%. Based on the molecular identification, it showed that the 22 yeasts are separately belong to Candida viswanathii, Candida parapailosis, Rhodotorla mucilaginosa, Debaryomyces hansenii, Pichia guilliermondii and Rhodosporidium paludigenum. The optimum condition for lipid content determination by nile red dyeing is: bacterium suspension OD 600 lower than 1.2, nile red concentration 0.5 mg/l, dyeing time 5 min, exciting wavelength 488 nm, emission wavelength 570 nm. The relative fluorescence intensity obtained by this method exhibits a positive association with the lipid content obtained by weighing method, which can be explained as R 2 = Keywords: Nile red dyeing, Oleaginous yeast, 26S rdna D1/D2, Fluorescence, Lipid content, 20%,, [1 2], C16 C18, [3],,,, [4 5],, -A, [6], ( -A),,,, Spiekermann, (DMSO) [7],,, Greenspan [8], [9 11],,

3 : nm,,,, 385, 2A00015,, 1 材料与方法 1.1 仪器 (Nikon Eclipse 50i), Nikon ; (SpectraMax M5), Molecular Devices ; (J2-MI), Beckman;, ZEALWAY ;, ;, ;, 1.2 菌种与试剂 DY DY (Trichosporn cutaneum) ( 65%) [12], (Sigma ) DMSO ( ) () ( ) ( ) 1.3 培养基 (PDA ): 200 g,, ml, 1 h, 10 g, ml, 2%, ph PYG : 20 g 10 g 5.0 g 15 g, ml, ph : 20 g 10 g 5 g ml, ph : 70 g (NH 4 ) 2 SO 4 1 g KH 2 PO 4 1 g MgSO 4 7H 2 O 1 g 0.5 g, ml, ph 产油酵母的筛选及鉴定平板培养基添加尼罗红筛选产油酵母 : PYG 60 C 1.0 g/l (Nile/DMSO) [13], 0.5 mg/l,,, Trichosporn cutaneum C 3 4 d nm [14],, [15] 产油酵母分子鉴定 : DNA [3] NL1 (5 -T ATCAATAAGCGGAGGAAAAG-3 ) NL4 (5 -G GTTCCGTGTTTCAAGACGG-3 ) 26S rdna D1/D2 PCR : 95 C 5 min; 94 C 1 min, 52 C 1 min, 72 C 1 min 20 s, 36 ; 72 C 8 min [16 18] PCR ( OMEGA PCR ), 产脂摇瓶培养 : PYG 3 d,, 27 C 160 r/min 24 h; 10%

4 128 微生物学通报 2012, Vol.39, No.1 (V/V) (100 ml/ 500 ml), 27 C 150 r/min 72 h 菌体生物量测定 : 250 ml, r/min 5 min, 2,, 80 C, 带 (g/l) = 1000 样积油脂提取方法 ( 索氏提取法 ): 100 C 105 C, 5 g,,, 1:1 2/3,,, 6 12 h,, 80 C 2 h, 0.5 h [19] : ( ) = 100% 残糖测定 : [20] 1.5 荧光检测酵母油脂相对含量方法的建立酵母 2A00015 培养及处理 : 1.4.3, 10 ml, g 5 min,,, g 5 min, 2, 酵母 2A00015 细胞密度检测 : 600 nm, 尼罗红染色后荧光特性检测 : 2A00015, ( 0.1 g/l Nile red/dmso),, 200 μl 96, 3 SpectraMax M5 (Molecular Devices ), 96, 2 结果 2.1 产油酵母简便初筛结果,,, 1 1,,, A 07Y76 07Y144 07Y147 07Y162 B 07Y74 2A A00015, Trichosporn cutaneum 2.571, 07Y162 07Y147 2A00015, 07Y15,, (0.5 mg/l Nile/DMSO) nm, 2,, 07Y126, 07Y162 07Y147 07Y15,,,

5 : 129 Fig. 1 图 1 紫外光下菌落荧光情况 The fluorescence of yeast colony in the UV light 图 2 荧光显微镜下酵母细胞 Fig. 2 Yeast cells under the fluorescence microscope Note: A: Yeasts cells under the ordinary light; B: Yeasts cells under the blue light (Wavelength nm); 1, 2, 3, 4: Respectively represent yeasts of 07Y126, 07Y162, 07Y147, 07Y15. The microscope magnification , 22, (20 ) (2 ), 1,, 07Y162, 62.92%, g/l, 10.32, 07Y147, 50.81%, g/l, Y12 07Y15 07Y144 2A A %,, 6, 20%,,,,, 2.2 产油酵母酵母分子鉴定 [21] 26S rdna D1/D2 22, Kuttzman Robnett

6 130 微生物学通报 2012, Vol.39, No.1 Strain serial number Table 1 Sample source 表 1 产油酵母筛选结果 The result of oleaginous yeasts screening Biomass (g/l) Lipid content (%) (g /100g) Lipid coefficient (g lipid/100g substrate utilized) 07Y12 WPA 9 12 cm ( m) Y15 WPA cm ( m) Y29 WPA 200 m Y30 WPA 200 m Y31 WPA 800 m Y35 WPA m Y42 WPA 500 m ( m) Y43 WPA 200 m ( m) Y46 WPA 50 m ( m) Y52 WPA m Y53 WPA m Y74 WPA 800 m Y76 WPA 500 m ( m) Y87 WPA Y116 WPA m Y141 WPA 150 m Y144 WPA Y147 WPA 15 m Y152 WPA 500 m Y162 WPA 500 m A00011 EPA 1.6 m ( m) A00015 EPA 鲀 : EPA: ; WPA:. Note: EPA: Eastern Pacific Ocean; WPA: West Pacific Ocean. 1% [18],, 3, 22 4, 6 ( Candida parapsilosis Candida viswanathii Pichia guilliermondii Debaryomyces hansenii Rhodotorula mucilaginosa Rhodosporidium paludigenum),,

7 : 131 图 3 基于 26S rdna D1/D2 区域序列和 Neighbor-Joining 分析绘制的系统树 Fig. 3 Phylogenetic tree drawn from neighbor-joining analysis based on the 26S rdna D1/D2 domain sequence alignment Note: Numbers in parentheses represent the sequences accession number in GenBank. The number at each branch points is the percentage supported by bootstrap. Bar: 5% sequence divergence. 2.3 尼罗红染色检测酵母油脂相对含量,, - - [22],,,, 2A00015, 波长选择 :, nm, 488 nm, nm, 4,, [23] 4A B, 488 nm,, nm,, nm

8 132 微生物学通报 2012, Vol.39, No.1 图 nm 激发波长下发射光光谱扫描 Fig. 4 Emission spectrum light scanning with excitation wavelength at 488 nm Note: A: Emission spectra of cell suspension; B: Emission spectra of Nile red-stained cell suspension; C: The corrected spectra. A B, nm, 570 nm, 4C, 488 nm, 570 nm, 染色时间 : 1.67, 0.5 mg/l 2.0 mg/l,, 1 min, 5 5,, 3 min,, 80% 90%, 5 min 10 min,, 1.59% 0.86%, 4 9 min,, 5 min

9 : 133 图 5 尼罗红染料与胞内脂类结合后荧光强度随时间的变化 Fig. 5 The changes of fluorescence intensity with time 细胞密度 : 2A ml,, 600 nm ( 6A), OD (R 2 =0.9927), 6B, 7,,,,,, 0 1.2, R ,,, 600 nm 1.2,, 尼罗红浓度 : 3 (OD ),,,, 8, 8,,, OD mg/l, ;, OD , 0.5 mg/l 1.5 mg/l,,,,,, (OD ), 0.5 mg/l

10 134 微生物学通报 2012, Vol.39, No.1 图 6 细胞密度与 600 nm 波长下光密度值的关系 Fig. 6 The relationship between cell density and OD 600 图 7 相对荧光强度与 600 nm 波长下光密度值的关系 Fig. 7 The relationship between relative fluorescence and OD 600 Fig. 8 图 8 尼罗红浓度与相对荧光强度关系 The relationship between relative fluorescence and nile red concentration

11 : 荧光检测油脂含量方法的可行性分析 :, 2A00015, 1.20, 0.5 mg/l, 5 min 488 nm 570 nm, 9 9, (R 2 =0.9637), 3 讨论 385,, 22, 26S rdna,,,,,,,,, [24 25],,, 2A00015,, (R 2 = ) 96, 3, 32,,,,,, Fig. 9 图 9 产油率与相对荧光强度关系 The relationship between relative fluorescence and lipid content

12 136 微生物学通报 2012, Vol.39, No.1 参考文献 [1] Ratledge C, Wynn JP. The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms[j]. Advances in Applied Microbiology, 2002, 51: [2] Papanikolaou S, Komaitis M, Aggelis G. Single cell oil (SCO) production by Mortierella isabellina grown on high-sugar content media[j]. Bioresource Technology, 2004, 95(3): [3],. [J]., 2006, 11(2): [4] Lambert MA, Moss CW. Comparison of the effects of acid and base hydrolyses on hydroxy and cyclopropane fatty acids in bacteria[j]. Journal of Clinical Microbiology, 1983, 18(6): [5] Diaz G, Melis M, Batetta B, et al. Hydrophobic characterization of intracellular lipids in situ by Nile Red red/yellow emission ratio[j]. Micron, 2008, 39(7): [6] Kranz RG, Gabbert KK, Madigan MT. Positive selection systems for discovery of novel polyester biosynthesis genes based on fatty acid detoxification[j]. Applied and Environmental Microbiology, 1997, 63(8): [7] Spiekermann P, Rehm BHA, Kalscheuer R, et al. A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds[j]. Archives of Microbiology, 1999, 171(2): [8] Greenspan P, Mayer EP, Fowler SD. Nile red: a selective fluorescent stain for intracellular lipid droplets[j]. The Journal of Cell Biology, 1985, 100(3): [9] Genicot G, Leroy JLMR, van Soom A, et al. The use of a fluorescent dye, Nile red, to evaluate the lipid content of single mammalian oocytes[j]. Theriogenology, 2005, 63(4): [10] Kimura K, Yamaoka M, Kamisaka Y. Rapid estimation of lipids in oleaginous fungi and yeasts using Nile red fluorescence[j]. Journal of Microbiological Methods, 2004, 56(3): [11] Huang GH, Chen G, Chen F. Rapid screening method for lipid production in alga based on Nile red fluorescence[j]. Biomass and Bioenergy, 2009, 33(10): [12],,,. [J]., 2005, 25(12): [13],. [J]., 2010, 29(1): [14]. -β- [J]., 1994, 34(1): [15] Berlanga M, Montero MT, Fernández-Borrel J, et al. Rapid spectrofluorometric screening of poly-hydroxyalkanoate-producing bacteria from microbial mats[j]. International Microbiology, 2006, 9(2): [16],,. rdna D1/D2 [J]., 2002, 21(1): [17] Kurtzman CP, Robnett CJ. Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequence[j]. Antonie Van Leeuwenhoek, 1986, 73(4): [18] Kurtzman CP, Robnett CJ. Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5' end of the large-subunit (26S) ribosomal DNA gene[j]. Journal of Clinical Microbiology, 1997, 35(5): [19],,,. Mucor circinelloides [J]., 2004, 27(4): [20],,,. - [J]., 2003, 7(1): [21] Certik M. Fermentation physiology and regulation of microbial polyunsaturated fatty acid biosynthesis[j]. Research in Advanced Bioscience and Bioengineering, 2000(1): [22] Bligh EG, Dyer WJ. A rapid method of total lipid

13 : 137 extraction and purification[j]. Canadian Journal of Biochemistry and Physiology, 1959, 37(8): [23] Sackett DL, Wolff J. Nile red as a polarity-sensitive fluorescent probe of hydrophobic protein surfaces[j]. Analytical Biochemistry, 1987, 167(2): [24],,,. [J]., 2006, 46(4): [25],,,. [J]., 2008, 35(8): 关于微生物学通报专题刊申请的通知编辑部公告,,,,,,,,, 2008,,,,,,,, : 1., ; 2. : () ;, tongbao@im.ac.cn, : ; 3.,, : Tel: ; tongbao@im.ac.cn

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Microsoft Word tb 林义微生物学通报 Microbiology China tongbao@im.ac.cn JAN 20, 2012, 39(1): 125 137 2012 by Institute of Microbiology, CAS 生物实验室尼罗红染色法筛选产油酵母及定量检测胞内油脂含量的研究林义 1,2 钟添华 1* 骆祝华 1 黄翔玲 1 叶德赞 (1. 国家海洋局第三海洋研究所福建厦门 361005)