Optimization of extraction protocol of chlorophyll a from algae

doi:10.11949/0438-1157.20190149

Abstract

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

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

CLC Number:

TQ 611.5

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.

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.

Figures/Tables 10

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

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

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

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

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

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

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

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

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

Table 5 Ethanol orthogonal experiment

离心转速（D）/

(r·min^-1)

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

—

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