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1 微生物学通报 Microbiology China 生物实验室 Dec. 20, 2014, 41(12): by Institute of Microbiology, CAS DOI: /j.microbiol.china 磷脂脂肪酸分析中内标物的使用及数据处理建模 * 李范周星梅蒋海波李娜 陈建中 ( ) 摘要 目的 评估未知样品内标进样量的参考范围, 优化数据处理方法 方法 采用 C19 为内标, 不同进样量进行分析 ; 编写数据分析程序优化数据处理模型 结果 随着内标进样量的增加, 检测到的 PLFA 数量和响应值等指标均有增加, 但进样量超过 16 nmol/g 时反而下降 ;25 组数据显示采用响应值计算 PLFA 含量比百分比值计算所得含量略高 ; 采用 Dot Net C# 语言编写数据分析模型 结论 初步确定了未知样品内标进样量的参考范围, 得出最优处理方法 ; 采用响应值计算 PLFA 含量可避免由于百分比值缺失带来的误差 ; 校准系数的引入可减少仪器参数条件改变等因素带来的误差 ; 通过编写谱图数据分析程序获得一定自动化操作, 提高了数据分析效率和准确性 关键词 磷脂脂肪酸, 内标物, 数据处理 Using of internal standard and modeling the data processing of phospholipid fatty acids LI Fan ZHOU Xing-Mei JIANG Hai-Bo LI Na * CHEN Jian-Zhong (Key Laboratory of Mountain Ecological Restoration and Bioresource Utilization & Ecological Restoration Biodiversity Conservation Key Laboratory of Sichuan Province, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, Sichuan , China) Abstract: [Objective] To estimate the reference range of internal standard amount of unknown samples and optimize the method of data processing. [Methods] The different amount use of Methyl Nonadecanoate as internal standard was studied and discussed. The data analysis model of fatty acids was established to optimize the data processing of PLFA. [Results] The number and response values of detected PLFA were advanced with the increased of amount of internal standard, but both reduced when the amount over 16 nmol/g. Twenty-five sets of PLFA data showed that the PLFA contents calculated by response value were higher than by percentage value. A data analysis model of PLFA was established using Dot Net C# language. [Conclusion] The reference range of internal standard amount of unknown samples was preliminary estimated and optimized. Calculation of PLFA content by response value can avoid the error caused by the lack of percentage value. The use of calibration coefficient can reduce the system error. Data processing was automated by the data analysis mode. It 基金项目 : (No. Y2C Y3C ) * 通讯作者 :Tel: ; :lina@cib.ac.cn 收稿日期 : 接受日期 : 优先数字出版日期 (

2 : 2575 improves the efficiency and accuracy of data analysis. Keywords: PLFA, Internal standard, Data processing (Phospholipid fatty acids PLFAs) PLFA [1-4] Microbial Identification System (MIS) C9:0 C20:0 (PLFA) PLFA PLFA [5] PLFA Eco Sherlock Perl Perl R MySQL Windows XP Vista 2012 MIDI Sherlock PLFA (1) (2) Sherlock 1 材料与方法 1.1 材料 (103 53'E 31 41'N) 8.9 C mm 0 20 cm 4 2 mm 70 C ( Sigma) ( Sigma) C9 C20 ( MIDI) ( ) MIDI Sherlock Microbial Identification System (MIS) Agilent 方法 70 C 5 g 50 ml - - (1:2:0.8 ) [6-7] N μl (25 mg/l ) Agilent 19091B-102E (25 m 0.2 mm 0.33 μm) MIDI Sherlock (MIDI Inc. Newark DE) 250 C 9.5 Psi

3 2576 微生物学通报 Microbiol. China 2014, Vol.41, No C 300 C 5 C/min 260 C 40 C/min 310 C 1.5 min N 2 (30 ml/min) 2 μl 100:1 MIDI Sherlock System 6.1 MIDI MIDI 1.3 内标样的选择 C19 ( ) ( ) C19 2 结果与分析 C 内标进样量分析 25 mg/l C19 C μl 5 1 表 1 不同内标进样量对磷脂脂肪酸含量的影响 Table 1 Effect of different injection volume of internal standard on the concentration of PLFA PLFA Injection volume of internal (μl) The concentration of PLFA (nmol/g) :0 iso 3OH 0.282± ± ± ± ±0.083 Unknown ND 0.151± ±0.016 ND ND i14: ± ± ±0.023 ND ND 14:1 w5c 0.155± ± ±0.019 ND ND 14: ± ± ± ±0.043 ND Unknown ± ± ±0.023 ND ND i15: ± ± ± ± ±0.087 a15: ± ± ± ± ± : ± ± ± ±0.029 ND i16: ± ± ± ± ±0.063 a16: ± ± ± ±0.041 ND 16: ± ± ± ± ± :1 iso w9c 1.890± ± ± ± ± :0 3OH 0.520± ± ± ± ±0.079 i17: ± ± ± ± ±0.063 a17: ± ± ± ± ±0.087 cy17: ± ± ± ± ± : ± ± ± ±0.015 ND 16:1 2OH 0.729± ± ± ± ± Me17: ± ± ± ±0.037 ND 18:2 w6, 9c/18:0 ante 2.125± ± ± ± ± : ± ± ± ± ± Me18: ± ± ± ± ±0.062 i19: ± ± ± ± ± :0 cyclo w8c 4.511± ± ± ± ± :1 2OH 2.833± ± ± ± ±0.211 ND. Note: ND means not detected.

4 : C μl Unknown i14:0 4:1 w5c Unknown PLFA 240 μl 14:0 15:0 a16:0 17:0 10Me17:0 PLFA PLFA C19 PLFA PLFA 120 μl PLFA PLFA ( FID ) D=2NW/A ( D g/s N A A As W g) [8-9] PLFA 16:0 2 PLFA PLFA PLFA μl PLFA C PLFA nmol/g PLFA 数据处理分析 百分比与响应值计算 PLFA 含量比较 : PLFA (1) (2) [10-11] C k P k R (2) x x (1) x Cx Pi Ri k P x PLFA P i C19 R x PLFA R i C μl 25 mg/l 25 PLFA (1) (2) PLFA 1 表 2 不同内标进样量对检测值及 16:0 的响应值的影响 Table 2 Effect of different injection volume of internal standard on the detection value and response value of 16:0 Injection volume of internal standard (μl) Detection value (nmol/g) The total concentration of PLFA (nmol/g) 16:0 The response value of 16:0 16:0 The concentration of 16:0 (nmol/g) C19 The response value of C ± ± ± ± ± ± ± ± ± ± Table 3 The minimum response 表 3 对于未知样品内标使用量的估定 The assessed for amount of internal standard of the unknown sample C19 The response of C PLFA The detection of PLFA (nmol/g) The amount of internal standard (nmol/g)

5 2578 微生物学通报 Microbiol. China 2014, Vol.41, No.12 图 1 PLFA 含量计算结果 Figure 1 The results of PLFA calculated by percentage and response value 1 PLFA PLFA comments>max ar/ht comments<min ar/ht Peak name unknown PLFA 采用校正系数计算 PLFA 含量比较 : 16 PLFA (3) (2) 2 r x PLFA r i C19 (2) 2 PLFA PLFA k Rx rx Cx (3) R r 2.3 微生物群落脂肪酸数据生态学数据分析模型系统建立 工作流程 : Windows (Excel) 图 2 校正系数对 PLFA 计算结果的影响 Figure 2 Impact of correction factor to the results of PLFA i i Figure 3 图 3 脂肪酸数据分析模型主界面 The main interface of the data analysis model of fatty acids

6 : 2579 图 4 脂肪酸数据分析模型流程示意图 Figure 4 The flow diagram of the data analysis model of fatty acids Excel Excel (Peak Name) (Percent) (Peak Name Percent )/(19:0 Percent ) Peak Name Peak Name Peak Name - Excel 数据结构 : XML Excel XML Peak Name 程序应用实例 : Sherlock 3 PLFA 3 6 图 5 菌种数据库 Figure 5 The strain database

7 2580 微生物学通报 Microbiol. China 2014, Vol.41, No.12 图 6 脂肪酸数据分析模型输出结果示例 Figure 6 The output of the data analysis model of fatty acids (Peak Name) 33 3 (Peak Name) nmol/g 6 3 讨论 MIDI Sherlock PLFA C PLFA nmol/g 3 10/6 20 nmol/g PLFA PLFA PLFA r

8 : 2581 [5],,,. PLFA Eco[J]., 2009, 25(14): [6] Bligh EG, Dyer WJ. A rapid method of total lipid 参考文献 extraction and purification[j]. Canadian Journal of Biochemistry and Physiology, 1959, 37(8): [1] Xue D, Yao HY, Ge DY, et al. Soil microbial community [7],. structure in diverse land use systems: A comparative study [J]., 2004, 31(1): using Biolog, DGGE, and PLFA analyses[j]. Pedosphere, [8],,. 2008, 18(5): [2],,,. [J]., 2011, 29(3): PLFA [J]., 2012, [9],,,. 18(5): [J]., 2010(1): [3]. (PLFA) [10],,,. [D]. : PLFA [J]., 2009., 2008, 45(2): [11] Wang M, Qu LY, Ma KM, et al. Soil microbial properties [4],,,. under different vegetation types on Mountain Han[J]. [J]., 2010, 37(3): Science China: Life Sciences, 2013, 56(6): 一次亲吻, 交换 万口腔细菌 科技信息摘录 (Microbiome) (Remco Kort) (TNO) 90% (Lactobacillus) (Bifidobacteria) 3 10 s