液氮冻融前处理优化测定浮游植物叶绿素a的初步研究

Determination of Phytoplankton Chlorophyll-a Concentration Using a Liquid Nitrogen Freeze-Thaw Pretreatment

叶绿素a是水环境评价和水生态研究中的一项重要指标。针对叶绿素a测定中前处理耗时长、提取不完全等问题,研究了液氮冻融前处理对叶绿素a测定效率的影响。以实验室培养的小球藻(Chlorella pyrenoidosa)和微囊藻(Microcystis aeruginosa)藻液为实验材料,通过单因素实验和正交实验设计,探究速冻时间、冻融次数、浸提时间等主要参数对提取效率的影响,并确定液氮冻融前处理测定叶绿素a的最优条件。结果表明,液氮冻融前处理测定叶绿素a的最优条件为速冻30 s、冻融4次、90%的丙酮浸提4 h,在此条件下,小球藻和微囊藻叶绿素a浓度测定相对标准偏差分别为0.57%和2.25%,变异系数CV分别为0.46%和1.84%。野外水样测定结果显示,与丙酮-研磨法、分光光度法(SL 88-2012)、分光光度法(HJ 897-2017)和反复冻融浸提法相比,液氮冻融前处理法的提取效率高于其他方法且测定结果相对偏差更小,浸提时间较其他4种方法缩短50%以上,其浸提效率达93%以上。液氮冻融前处理法对叶绿素a的提取效率明显高于其他方法且数据重现性较好,具有操作简单、结果准确、操作耗时短等特点。

Eutrophication of water bodies is a worldwide environmental problem, and assessing eutrophication status supports efforts to prevent and remediate eutrophication. Chlorophyll-a is an important indicator of water quality and used to indicate the degree of eutrophication and to monitor harmful algal blooms(HABs). Extraction of chlorophyll-a from the algae sample is time-consuming and often incomplete, and is the most problematic step in chlorophyll-a analysis. The objective of this study was to increase the chlorophyll-a extraction efficiency by liquid nitrogen freeze-thaw pretreatment. Single-factor and orthogonal experiments were carried out to optimize the extraction efficiency of chlorophyll-a in pure cultured Chlorella pyrenoidosa and Microcystis aeruginosa. Each step was considered: the quick freeze time, the number of freeze-thaw cycles, and the extraction time. Furthermore, we compared the chlorophyll-a concentrations determined in C. pyrenoidosa and M. aeruginosa cultures, and in field samples, using the liquid nitrogen freeze-thaw method(M1) and other four methods: two spectrophotometric methods(M2, M3), a repeated freeze-thaw method(M4) and the acetone/grinding method(M5). The optimal conditions for pretreatment with liquid nitrogen were found to be as follows: quick freeze for 30 s, use four freeze-thaw cycles, and extract with 90% acetone for 4 h. Under these conditions, the relative standard deviations of chlorophyll-a concentration in the C. pyrenoidosa and M. aeruginosa cultures were 0.57% and 2.25%, and the coefficients of variation were 0.46% and 1.84%. The liquid nitrogen freeze-thaw pretreatment increased chlorophyll-a extraction efficiency(>93%), and decreased the relative deviation and analysis time. Compared to M2, M3, M4 and M5, respectively, pretreatment of algae cultures increased extraction efficiency 5.2%, 3.1%, 3.0% and 8.4%, and those of field samples by 8.0%, 1.7%, 7.4% and 11.3%. The liquid nitrogen freeze-thaw pretreatment significantly increased the extraction efficiency of chlorophyll-a, shortened analysis time and can be used in the determination of chlorophyll-a concentration in different water bodies.