藻类叶绿素a提取的优化研究

doi:10.11949/0438-1157.20190149

摘要/Abstract

摘要：

叶绿素a是参与光合作用的基础物质，是衡量水体藻类生物量以及评价湖泊富营养化的重要指标之一。利用90%丙酮、无水乙醇、无水乙醇∶90%丙酮（1∶1）、无水乙醇∶90%丙酮（1∶2）、无水乙醇∶90%丙酮（2∶1）五种有机溶剂萃取已知浓度标准叶绿素a和叶绿素b，对传统萃取叶绿素a的计算公式加以修正，并且在超声辅助破碎藻细胞的前提下分析了该五种有机溶剂对普通小球藻和铜绿微囊藻中叶绿素a的萃取效果。确定了无水乙醇∶90%丙酮（2∶1）的萃取效果最好，而无水乙醇从效果、安全、环保等角度考虑为最优溶剂。通过多参数水质监测仪对不同时间段的浅水型湖库水体中的叶绿素a进行监测，验证了在超声条件下无水乙醇测定结果的准确性并利用正交实验确定出了无水乙醇萃取叶绿素a的最佳条件。对于叶绿素a测定的优化研究使得以后野外水体大量测量叶绿素a的过程变得更为简便、快捷、准确。

关键词: 叶绿素a, 计算公式, 无水乙醇, 多参数水质监测仪, 正交实验

Abstract:

Chlorophyll a is a basic substance involved in photosynthesis, and is one of the important indicators for measuring algae biomass in water and evaluating eutrophication of lakes. The traditional calculation formula for extracting chlorophyll a was modified when the standard chlorophyll a and chlorophyll b was extracted by these organic solvents including 90% acetone, anhydrous ethanol, anhydrous ethanol∶acetone (1∶1), anhydrous ethanol∶acetone (1∶2) and anhydrous ethanol∶acetone (2∶1). The effects of these five organic solvents on Chlorella vulgaris and Microcystis aeruginosa were analyzed under the ultrasonic treatment. It was determined that anhydrous ethanol∶acetone (2∶1) was most effective for extraction and anhydrous ethanol was considered as the best solvent in terms of efficiency, safety and environmental protection. The chlorophyll a in shallow water reservoir was monitored by multi-parameter water quality monitor at different times. The accuracy of the determination results of anhydrous ethanol under ultrasonic treatment was verified. The optimum extraction conditions of chlorophyll a by anhydrous ethanol were determined by orthogonal experiment. The optimization of chlorophyll a measurement makes the process of measuring chlorophyll a for large quantities in water more simple, fast and accurate.

Key words: chlorophyll a, calculation formula, anhydrous ethanol, multi-parameter water quality monitor, orthogonal experiment

中图分类号:

TQ 611.5

李鹏飞, 孙昕, 杨娌, 何飞飞, 王垿. 藻类叶绿素a提取的优化研究[J]. 化工学报, 2019, 70(9): 3421-3429.

Pengfei LI, Xin SUN, Li YANG, Feifei HE, Xu WANG. Optimization of extraction protocol of chlorophyll a from algae[J]. CIESC Journal, 2019, 70(9): 3421-3429.

图/表 10

表1 各溶剂萃取纯叶绿素的吸光度

Table 1 Extraction of pure chlorophyll by solvent

纯叶绿素	波长/nm	丙酮（90%）	乙醇（99.7%）	1∶1（乙醇∶丙酮）	1∶2（乙醇∶丙酮）	2∶1（乙醇∶丙酮）
a	663 645 750	0.105 0.024 0.002	0.099 0.023 0.001	0.102 0.025 0.002	0.103 0.024 0.002	0.096 0.025 0.001
a
a
b	645 750	0.003 -0.005	0.002 -0.001	0.002 -0.004	0.004 0.002	0.004 0.001
b	645 750	0.003 -0.005	0.002 -0.001	0.002 -0.004	0.004 0.002	0.004 0.001

表2 各溶剂对应的比例系数

Table 2 Correspondence ratio of each solvent

纯叶绿素	丙酮（90%）	乙醇（99.7%）	1∶1（乙醇∶丙酮）	1∶2（乙醇∶丙酮）	2∶1（乙醇∶丙酮）
a	1	0.951	0.971	0.981	0.922
b	1	0.500	0.750	0.750	0.625

表3 各溶剂的校正系数

Table 3 Corresponding correction coefficients for each solvent

$K E 663$

$K E 645$

$K E 663'$

$K E 645'$

11.640

2.160

0.869

10.115

1.877

12.234

4.320

0.906

11.084

3.914

11.989

2.880

0.883

10.586

2.543

11.870

2.880

0.881

10.457

2.537

12.620

3.456

0.896

11.308

3.097

表3 各溶剂的校正系数

Table 3 Corresponding correction coefficients for each solvent

系数

丙酮（90%）

乙醇（99.7%）

1∶1（乙醇∶丙酮）

1∶2（乙醇∶丙酮）

2∶1（乙醇∶丙酮）

$K E 663$

$K E 645$

$K E 663'$

$K E 645'$

11.640

2.160

0.869

10.115

1.877

12.234

4.320

0.906

11.084

3.914

11.989

2.880

0.883

10.586

2.543

11.870

2.880

0.881

10.457

2.537

12.620

3.456

0.896

11.308

3.097

表4 各溶剂对脱镁叶绿素a的萃取效果

Table 4 Extraction of pheophytin a by solvent

100

1.000

1.019

0.989

0.867

0.788

0.652

1.000

1.033

0.996

0.862

0.757

0.638

1.000

1.016

0.9801

0.840

0.709

0.622

1.000

1.023

0.979

0.838

0.707

0.620

1.000

1.031

0.985

0.853

0.719

0.631

图1 小球藻叶绿素a提取效果对比

Fig.1 Extraction of chlorophyll a from Chlorella vulgaris under different solvents

图2 铜绿微囊藻叶绿素a提取效果对比

Fig.2 Extraction of chlorophyll a from Microcystis aeruginosa under different solvents

图3 丙酮研磨与乙醇超声测定叶绿素a对比

Fig.3 Comparison of acetone with grinding and ethanol with ultrasound on extraction of chlorophyll a

图4 浅水型湖库中藻类叶绿素a的测定

Fig.4 Chlorophyll a of microalgae in shallow lakes and reservoirs

表5 乙醇的正交实验（3水平4因素）

Table 5 Ethanol orthogonal experiment

离心转速（D）/

(r·min^-1)

表6 乙醇正交实验数据表

Table 6 Ethanol orthogonal test data

K ₁

K ₂

K ₃

k ₁

k ₂

k ₃

100

3.4201

3.7730

3.7367

1.1400

1.2577

1.2456

0.1176

3.5212

3.3854

4.0233

1.1737

1.1285

1.3411

0.2126

3.5800

3.6792

3.6707

1.1933

1.2264

1.2236

0.0331

6000

8000

10000

6000

8000

10000

6000

3.4323

3.5994

3.8982

1.1441

1.1998

1.2994

0.1553

1.008

1.051

1.361

1.314

1.111

1.349

1.199

1.224

1.314

—

参考文献 35

1	丛海兵, 黄廷林, 周真明, 等 . 藻类叶绿素测试新方法[J]. 给水排水, 2007, 33(6): 28-32.
	Cong H B , Huang T L , Zhou Z M , et al . A new measurement method for chlorophylls[J]. Water & Wastewater Engineering, 2007, 33(6): 28-32.
2	Catherine A , Escoffier N , Belhocine A , et al . On the use of the FluoroProbe^®, a phytoplankton quantification method based on fluorescence excitation spectra for large-scale surveys of lakes and reservoirs[J]. Water Research, 2012, 46(6): 1771-1784.
3	国家环境保护总局 . 水和废水监测分析方法[M]. 4版. 北京: 中国环境科学出版社, 2012.
	State Environmental Protection Administration of China . Inspects and Analysis Methods of Water and Wastewater[M]. 4th ed. Beijing: China Environmental Science Press, 2012.
4	Qiu N , Wang X , Zhou F . A new method for fast extraction and determination of chlorophylls in natural water[J]. Zeitschrift Für Naturforschung C, 2018, 73(1/2): 77-86.
5	Derrien M , Aghabararnejad M , Gosselin A , et al . Optimization of supercritical carbon dioxide extraction of lutein and chlorophyll from spinach by-products using response surface methodology[J]. LWT, 2018, 93: 79-87.
6	Petruş-Vancea A . Cell ultrastructure and chlorophyll pigments in hyperhydric and non-hyperhydric Beta vulgaris, var. Conditiva plantlets, treated with deuterium depleted water[J]. Plant Cell, Tissue and Organ Culture(PCTOC), 2018, 135(1): 13–21.
7	徐彩平, 刘霞, 陈宇炜 . 浮游植物叶绿素a浓度测定方法的比较研究[J]. 生态与农村环境学报, 2013, 29(4): 438-442.
	Xu C P , Liu X , Chen Y W . Comparison of methods for determination of phytoplankton chlorophyll-a[J]. Journal of Ecology and Rural Environment, 2013, 29(4): 438-442.
8	Humphrey G F , Wootton M . Comparison of the techniques used in the determination of phytoplankton pigments[R]. Paris: SCOR-UNESCO, 1966.
9	林罗敏, 唐鹊辉, 彭亮, 等 . 浮游植物叶绿素a的微波法研究及其与反复冻融法的比较[J]. 湖泊科学, 2016, 28(5): 1148-1152.
	Lin L M , Tang Q H , Peng L , et al . Microwave-assisted method for extracting chlorophyll-a in phytoplankton and its comparison with freezing-thawing extraction method[J]. Journal of Lake Sciences, 2016, 28(5): 1148-1152.
10	Macíassánchez M D , Mantell C , Rodríguez M , et al . Comparison of supercritical fluid and ultrasound-assisted extraction of carotenoids and chlorophyll a from Dunaliella salina [J]. Talanta, 2009, 77(3): 948-952.
11	张琛, 孙丰婷, 胡舰, 等 . 小麦苗叶绿素在食品加工因素中的稳定性分析[J]. 食品研究与开发, 2018, (5): 7-11.
	Zhang C , Sun F T , Hu J , et al . Analysis of the stability of wheat seedling chlorophyll in food processing[J]. Food Research and Development, 2018, (5): 7-11.
12	王晓飞, 郭海蓉, 李志春, 等 . 微波辅助提取甘蔗糖厂滤泥中叶绿素的初探[J]. 食品科学, 2011, 24(16): 201-205.
	Wang X F , Guo H R , Li Z C , et al . Optimization of microwave-assisted extraction process for chlorophyll from filter mud in sugar cane mill factories [J]. Food Science, 2011, 24(16): 201-205.
13	鲁静, 牛晓静 . 均匀设计法优化附子中多糖的超声提取工艺[J]. 中国药房, 2017, (13): 1834-1836.
	Lu J , Niu X J . Optimization of ultrasonic extraction technology for polysaccharide from aconiti lateralis radix praeparata by uniform design method[J]. China Pharmacy, 2017, (13): 1834-1836.
14	张佰清, 公谱 . 超声波辅助提取红树莓籽油工艺优化[J]. 食品科学, 2010, 31(2): 67-69.
	Zhang B Q , Gong P . Optimization of ultrasonic-assisted extraction of red raspberry seed pil by prthogonal array design[J]. Food Science, 2010, 31(2): 67-69.
15	章莹颖, 邓春芳, 崔岩, 等 . 不同方法对微藻细胞破碎及油脂提取效果的影响[J]. 中国油脂, 2016, 41(3): 61-65.
	Zhang Y Y , Deng C F , Cui Y , et al . Effect of different methods on cell disruption and oil extraction of microalgae[J]. China Oils and Fats, 2016, 41(3): 61-65.
16	曹治国, 徐杰, 刘静玲, 等 . 淡水湖泊营养状态监测新方法——叶绿素比值模型[J]. 环境科学学报, 2010, 30(2): 275-280.
	Cao Z G , Xu J , Liu J L , et al . A new trophic status monitoring method for freshwater lakes—the chlorophyll ratio model[J]. Acta Scientiae Circumstantiae, 2010, 30(2): 275-280.
17	Latasa M , Bidigare R R , Ondrusek M E , et al . HPLC analysis of algal pigments: a comparison exercise among laboratories and recommendations for improved analytical performance[J]. Marine Chemistry, 1996, 51(4): 315-324.
18	袁方, 李鑫, 余君萍, 等 . 分光光度法测定叶绿素含量及其比值问题的探讨[J]. 植物生理学通讯, 2009, (1): 63-66.
	Yuan F , Li X , Yu J P , et al . Methodological study on determination of chlorophyll contents and ratio of chl a/chl b by spectrophotometry[J]. Plant Physiology Communications, 2009, (1): 63-66.
19	刘国红, 林营志, 刘波, 等 . 芽胞杆菌菌体浓度的快速检测方法研究[J]. 中国农学通报, 2012, 28(24): 213-217.
	Liu G H , Lin Y Z , Liu B , et al . Study on rapid detection method of genus Bacillus cell concentration[J]. Chinese Agricultural Science Bulletin, 2012, 28(24): 213-217.
20	邓祥元, 高坤, 张丽娟 . 不同溶剂萃取桑叶叶绿素效率的初步研究[J]. 中国农学通报, 2011, 27(2): 389-394.
	Deng X Y , Gao K , Zhang L J . Preliminary study on extraction efficiency of chlorophyll by different organic solvents in mulberry leaves[J]. Chinese Agricultural Science Bulletin, 2011, 27(2): 389-394.
21	李得孝, 员海燕, 郭月霞, 等 . 混合液浸提法测定玉米叶绿素含量的研究[J]. 玉米科学, 2006, 14(1): 117-119.
	Li D X , Yuan H Y , Guo Y X , et al . Mixture solution soaking extraction efficiencies of chlorophyll from maize[J]. Journal of Maize Sciences, 2006, 14(1): 117-119.
22	童洋, 肖国民, 潘晓梅 . 响应面法优化螺旋藻中叶绿素的超声提取工艺[J]. 化工学报, 2009, 60(11): 2813-2819.
	Tong Y , Xiao G M , Pan X M . Optimization of ultrasonic extraction of chlorophylls from spirulina platenis by response surface methodology[J]. CIESC Journal, 2009, 60(11): 2813-2819.
23	李灿良, 钟敏, 胡雪琼, 等 . 从富油新绿藻提取叶绿素的研究[J]. 食品工业科技, 2015, 36(4): 199-203.
	Li C L , Zhong M , Hu X Q , et al . Study on the extraction of chlorophyll from a green microalga[J]. Science and Technology of Food Industry, 2015, 36(4): 199-203.
24	白飞妮, 穆富香, 杨代宇, 等 . 亚心形扁藻中叶绿素优化提取条件[J]. 安徽农业科学, 2017, (3): 108-110.
	Bai F N , Mu F X , Yang D Y , et al . Optimum conditions of extracting chlorophyll from Platymonas subcordiformis [J]. Journal of Anhui Agricultural Sciences, 2017, (3): 108-110.
25	Ave G D , Adams T A . Techno-economic comparison of acetone-butanol-ethanol fermentation using various extractants[J]. Energy Conversion & Management, 2018, 156: 288-300.
26	Meriluoto J , Spoof L , Codd G A . Chlorophyll a extraction and determination[M]// Handbook of Cyanobacterial Monitoring and Cyanotoxin Analysis. John Wiley & Sons, Ltd, 2017.
27	王玉芳, 杨士斌, 刘得银, 等 . 浮游植物叶绿素a含量不同方法测定比较——以白洋淀区域水体为例[J]. 水文, 2014, 34(5): 57-60.
	Wang Y F , Yang S B , Liu D Y , et al . Comparison of different methods for phytoplankton chlorophyll-a concentration measurement: a study case of water in Baiyangdian area[J]. Journal of China Hydrology, 2014, 34(5): 57-60.
28	Gerde J A , Montalbo-Lomboy M , Yao L , et al . Evaluation of microalgae cell disruption by ultrasonic treatment[J]. Bioresource Technology, 2012, 125(12): 175-181.
29	马卫星 . 暴雨径流潜流对峡谷分层型水源水库水质影响与水质原位改善[D]. 西安: 西安建筑科技大学, 2015.
	Ma W X . The effects of rainstorm flow undercurrent on water quality and the in-situ water quality improvement of a canyon-shaped, stratified, source water reservoir[D]. Xi’an: Xi’an University of Architecture and Technology, 2015.
30	宋挺, 朱冰川, 严飞, 等 . YSI6600V2水质多参数仪现场快速法与实验室分光光度法测定湖泊藻类叶绿素的比较[J]. 环境监控与预警, 2016, (5): 14-18.
	Song T , Zhu B C , Yan F , et al . Comparison of onsite YSI6600V2 multi-parameter water quality meter and laboratory spectrophotometry for the determination of lake algal chlorophyll[J]. Environmental Monitoring and Forewarning, 2016, (5): 14-18.
31	Zhang Y Q , Zhang Q , Luo J X , et al . Acaricidal active fractions from acetone extract of Aloe vera L. against Tetranychus cinnabarinus and Panonychus citri [J]. Acta Physiologiae Plantarum, 2017, 39(9): 195.
32	Rahayuningsih E , Pamungkas M S , Olvianas M , et al . Chlorophyll extraction from suji leaf (Pleomele angustifolia Roxb.) with ZnCl₂ stabilizer[J]. Journal of Food Science and Technology, 2018, 55(3): 1028-1036.
33	林少君, 贺立静, 黄沛生, 等 . 浮游植物中叶绿素a提取方法的比较与改进[J]. 生态科学, 2005, 24(1): 9-11.
	Lin S J , He L J , Huang P S , et al . Comparison and improvement on the extraction method for chlorophyll a in phytoplankton[J]. Ecological Science, 2005, 24(1): 9-11.
34	吕鹏翼, 崔建升 . 温度对现场荧光法测定水体中叶绿素a的影响[J]. 中国环境监测, 2015, 31(2): 130-134.
	Lyu P Y , Cui J S . Temperature effect on the determination of in situ fluorescence of chlorophyll-a in water [J]. Environmental Monitoring in China, 2015, 31(2): 130-134.
35	梁兴飞, 郭宗楼 . 超声辅助热乙醇提取法测定浮游植物叶绿素a的方法优化[J]. 水生生物学报, 2010, 34(4): 856-861.
	Liang X F , Guo Z L . Optimization of the method on determination of phytoplankton chlorophll a by using ultrasound-assisted hot-ethanol extration[J]. Acta Hydrobiologica Sinica, 2010, 34(4): 856-861.

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