基于叶绿素荧光动力学的压载水活体藻细胞数表征参数选择研究

Study on Selecting Characterization Parameters of Viable Algae Cells Number in Ballast Water Based on Chlorophyll Fluorescence Kinetics

叶绿素荧光动力学过程与藻类光合作用过程密切相关,叶绿素荧光动力学法是探测活体藻类细胞的天然工具。与常用的压载水荧光素染色-显微镜检活体藻细胞计数相比,叶绿素荧光动力学法具有测量快速、灵敏度高、无需前处理等特点。海洋船舶压载水中存在大量死亡藻细胞和有色溶解有机物(CDOM),荧光背景复杂,因此,获得不受荧光背景影响且能够准确表征活体藻细胞数的光合荧光参数,是叶绿素荧光动力学法直接测量压载水活体藻细胞数的关键。本文以死细胞和CDOM溶液模拟压载水的复杂荧光背景,以二氯苯基二甲脲(DCMU)胁迫条件模拟实际应用,研究了不同荧光背景下Fm、F0、[RCII]和Fv等多个光合荧光参数与活体藻细胞数之间的关系。结果表明:Fm、F0、[RCII]对活体藻细胞数的表征都不同程度地受死细胞数和CDOM浓度的影响,仅有Fv不受荧光背景的影响,相对标准偏差小于5%;在不同稀释液稀释活体藻细胞溶液实验中,Fv与活体藻细胞数均具有良好的线性相关性,相关系数R2可达0.98以上;在DCMU胁迫作用下,有且仅有Fv与活体藻细胞数呈良好的正相关性,相关系数为0.986。本研究结果证明了光合荧光参数Fv不受荧光背景干扰,是表征压载水中活体藻细胞数的最佳光合荧光参数。

Chlorophyll fluorescence kinetics is closely related to the algae photosynthesis and is a natural tool to detect viable algae cells. Compared with the common method of combining fluorescein staining in ballast water with the microscopic examination of viable algae cells number, chlorophyll fluorescence kinetics has the characteristics of rapid measurement, high sensitivity, and no need for pretreatment. However, there are a large number of dead algae cells and colored dissolved organic matter(CDOM) in marine ballast water, and the fluorescence background is complex. Therefore, obtaining the photosynthetic fluorescence parameters that are not affected by the fluorescence background and can accurately characterize the viable algae cells number is the key to directly measuring the viable algae cells number in ballast water through chlorophyll fluorescence kinetics. In this paper, with the dead cells and CDOM solution simulating the complex fluorescence background of ballast water and the 3-(3 c, 4-(dichlorophenyl)-1,1-dimethylurea(DCMU) stress condition simulating the practical application, the relationship of various photosynthetic fluorescence parameters such as Fm, F0, [RCII], and Fv with the viable algae cells number was investigated at different fluorescence backgrounds. The results show that Fm, F0, and [RCII] are all affected by the dead cells number and the CDOM concentration to varying degrees, and only Fv is not affected by the fluorescence background, with a relative standard deviation being less than 5%. In an experiment of diluting the solution of viable algae cells with different diluents, Fv and the viable cells number have a good linear correlation, with the correlation coefficient R2 above 0.98. Under the stress of DCMU, only Fv displays an excellent positive correlation with the viable algae cells number, and the correlation coefficient is 0.986. These results demonstrate that Fv is not disturbed by the fluorescence background and is the best photosynthetic fluorescence parameter to characterize the number of viable algae cells in ballast water.