浮游植物光合速率荧光动力学测量技术研究

发布时间：2019-04-29 17:52

【摘要】：浮游植物作为水体中最主要的初级生产者、食物链源头,是水体健康状况指示剂,从根本上影响着全球生物地球化学循环与气候变化。快速准确测量浮游植物光合速率对水生态环境监测、水华和赤潮灾害预防、渔业资源评估、全球气候变化预测等具有重要科学意义和应用价值。黑白瓶、14C和18O示踪等传统光合速率测量方法需要“现场采样-温育培养-离线分析”,存在测量时间长、手续繁琐、效率低,难以满足现场快速测量需求。叶绿素荧光法是一种快速高效测量方法,但目前的叶绿素荧光测量技术主要集中在叶绿素浓度和光合作用参数测量方面,缺乏对光合速率的分析,且存在测量系统复杂、信噪比低等问题。针对浮游植物光合速率现场快速测量需求以及叶绿素荧光测量技术不足,论文研究了一种基于荧光动力学的浮游植物光合速率测量技术。首先,研究并建立了浮游植物荧光动力学曲线测量系统。在分析浮游植物荧光动力学信号获取中激发光干扰抑制、光合电子传递链阻塞位点的精确调控、荧光信号的快速高灵敏检测等关键技术基础上,从激发与发射光路、激发光强调控以及荧光信号检测三个方面进行测量系统总体分析和设计。研究了激发光强自适应调控技术,设计了可变光脉冲调控电路,实现了不同浮游植物测量过程中的光强自适应调控,解决了光合电子传递链阻塞位点的精确调控问题;研究了快相荧光信号的快速获取和弛豫荧光的高灵敏度检测技术,设计了低噪声光电信号转换与基于时间交替的多路采样电路,实现了微秒量级快相荧光动力学曲线精确采样;针对弛豫荧光低信噪比微弱信号,设计了基于同步积分技术的豫荧光高灵敏检测电路。在此基础上,建立了高灵敏和微秒量级的荧光动力学测量系统。不同叶绿素浓度蛋白核小球藻测试结果表明,系统对准确测量快相与弛豫荧光动力曲线,对200μ叫快相荧光进行高分辨率16位采样,采样率达1.50MHz; 0.125μg/L叶绿素浓度的浮游植物弛豫荧光信噪比达11.2dB以上。其次,研究了浮游植物光合速率荧光动力学曲线反演方法。基于建立的浮游植物荧光动力学测量系统获得的快相与弛豫荧光动力学曲线,研究了浮游植物光合作用参数反演算法,包括最大荧光产率滑动窗口斜率判定方法,光化学量子效率和功能吸收截面线性最小二乘算法,以及质体醌平均还原时间常数离散迭代算法,实现浮游植物光合作用参数的准确反演;基于光合作用电子传递能流过程,以QA和PQ作为节点,建立了基于光合作用参数的浮游植物光合速率分析方法,并利用Megard模型对光合速率的光响应曲线进行分析,进一步获得了表征光合速率的生物学参数最大光合速率PMax、初始斜率《、最大光强IMax。最后,开展了基于荧光动力学的浮游植物光合速率对比分析实验。通过测量不同光照、营养盐和铜离子胁迫条件下蛋白核小球藻的荧光动力学曲线,反演获得光化学量子效率、功能吸收截面和质体醌平均还原时间常数等光合作用参数,其中光化学量子效率与Water-PAM荧光仪具有良好的一致性,相关系数达0.95以上,其它参数的变化规律也与理论分析相符;通过对DCMU、盐浓度胁迫及不同光照、不同营养盐培养条件下蛋白核小球藻光合速率对比分析测试,结果表明荧光动力学法测量得的光合电子传递通量与Chlorolab2液相氧电极测量的光合放氧量具有良好的一致性,最小线性相关系数R2分别为0.876、0.889、0.968、0.948。
[Abstract]:Phytoplankton as the main primary producer in the water body, the source of the food chain, is the indicator of the health status of the water body, which fundamentally affects the global biogeochemical cycle and the climate change. The rapid and accurate measurement of the photosynthetic rate of phytoplankton has important scientific significance and application value for water ecological environment monitoring, water bloom and red tide disaster prevention, fishery resource assessment and global climate change prediction. The conventional photosynthetic rate measurement methods such as black and white bottles, 14C and 18O tracers require "On-site sampling-incubation culture-off-line analysis". The measurement time is long, the procedures are complicated, the efficiency is low, and the rapid measurement requirements on the site are difficult to meet. Chlorophyll fluorescence method is a kind of rapid and efficient measuring method, but the present method of chlorophyll fluorescence measurement is mainly focused on the measurement of chlorophyll concentration and photosynthesis parameters, and the analysis of the photosynthetic rate is lacking, and the problems of complex measuring system, low signal-to-noise ratio and the like exist. In view of the demand of the on-site rapid measurement of the photosynthetic rate of phytoplankton and the shortage of the measurement of chlorophyll fluorescence, a technique for measuring the photosynthetic rate of phytoplankton based on the fluorescence dynamics was studied. First, a system for measuring the fluorescence of phytoplankton was studied and established. on the basis of analyzing the key technologies such as the excitation light interference suppression, the accurate control of the blocking site of the photosynthetic electron transport chain, the rapid and high-sensitivity detection of the fluorescent signal, and the like in the analysis of the fluorescence dynamic signal acquisition of the phytoplankton, And the overall analysis and design of the measurement system are carried out in three aspects of excitation light intensity regulation and fluorescence signal detection. The self-adaptive control technology of excitation light intensity was studied, and the variable light pulse control circuit was designed, and the self-adaptive control of light intensity in different phytoplankton measurement was realized, and the problem of precise control of the blocking site of the photosynthetic electron transport chain was solved. The rapid acquisition of fast-phase fluorescence signal and the high-sensitivity detection of the relaxation fluorescence are studied. The low-noise photoelectric signal conversion and the time-based multi-path sampling circuit are designed, and the accurate sampling of the fast-phase fluorescence dynamic curve on the order of microseconds is realized. In that light of the low signal-to-noise ratio weak signal of the relaxation fluorescence, a high-sensitivity detection circuit based on the synchronous integration technique is designed. On this basis, a high-sensitivity and microsecond-order fluorescence dynamic measurement system is established. The results of the test of Chlorella pyrenoidosa with different chlorophyll concentrations show that the system can measure the fast phase and the relaxation fluorescence power curve accurately, carry out high-resolution 16-bit sampling on 200 mu m fast-phase fluorescence, the sample rate is 1.50 MHz, and the signal-to-noise ratio of the phytoplankton with the concentration of 0.125. mu.g/ L is 11.2 dB or more. Secondly, the method of inversion of the photosynthetic rate of phytoplankton was studied. Based on the established fast phase and relaxation fluorescence dynamic curve obtained by a phytoplankton fluorescence dynamic measurement system, a method for determining the photosynthesis parameter of phytoplankton is studied, which comprises a method for judging the slope of the maximum fluorescence yield sliding window, The invention relates to a photochemical quantum efficiency and a function absorption cross-section linear least square algorithm, as well as an average reduction time constant discrete iterative algorithm of a plastid, to realize the accurate inversion of the photosynthesis parameters of the phytoplankton, The photosynthetic rate analysis method of phytoplankton was established based on the parameters of photosynthesis, and the light response curve of the photosynthetic rate was analyzed by the Meard model, and the maximum photosynthetic rate, Pmax, initial slope and maximum light intensity, were obtained. Finally, a comparative analysis of the photosynthetic rate of phytoplankton based on the fluorescence dynamics was carried out. by measuring the fluorescence dynamic curve of the protein nucleus chlorella under different illumination, nutrient salt and copper ion stress condition, the photosynthesis parameters such as the photochemical quantum efficiency, the function absorption cross section and the average reduction time constant of the plastid are obtained, in which the photochemical quantum efficiency and the water-PAM fluorescence instrument have good consistency, the correlation coefficient is more than 0.95, and the variation law of other parameters is consistent with the theoretical analysis; and through the treatment of the DCMU, the salt concentration stress and the different illumination, The photosynthetic rate of Chlorella pyrenoidosa under different nutrient culture conditions was compared and analyzed. The results showed that the photosynthetic electron transfer flux measured by the fluorescence method had good agreement with the measured photosynthetic oxygen content of the chlorolab2 liquid-phase oxygen electrode, and the minimum linear correlation coefficient (R2) was 0.876, 0.889, 0.968, and 0.948, respectively.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q945.11;O657.3

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