浮游植物光化学量子产率双波段可变脉冲光诱导荧光测量技术

Dual-Band Tunable Pulse Light Induced Fluorescence Measurement Technology for Photochemical Quantum Yield of Phytoplankton

以蓝藻门铜绿微囊藻和绿藻门蛋白核小球藻为研究对象,提出了浮游植物光化学量子产率双波段TPLIF测量技术。采用红蓝双波段脉冲激发,以反应中心关闭比例达到99%为判断条件,自适应调整饱和激发光波长和光强度,通过拟合饱和激发下单周转荧光动力学曲线获得光化学量子产率。结果表明:双波段激发模式能够同时饱和激发不同门类的浮游植物,光化学量子产率测量误差显著低于单波长模式,在双波段激发模式下,测量纯种藻的光化学量子产率最大误差由单波段激发模式的26.50%降低至1.12%,测量混合藻的光化学量子产率最大误差由14.02%降低至0.94%。该研究为不同门类浮游植物光化学量子产率的准确测量提供了一种重要的技术手段。

Objective The quantum yield of photosynthesis is a crucial parameter that reflects the efficiency of utilizing absorbed light quanta in initial photochemical reactions.It plays a pivotal role in assessing the strength of plant photosynthesis and primary productivity,with extensive applications in plant physiology,pathology,and toxicology.Various technologys have been developed for measuring photochemical quantum yield(FV/FM)since the advent of fluorescence kinetics.The pulse amplitude modulation(PAM)technology,proposed by Schreiber et al.,involves inducing fluorescence dynamics using saturating light exceeding 10000μmol/(m2·s),which fully reduces all PSII reaction centers in a short duration.This approach allows chlorophyll fluorescence to peak before measuring photochemical quantum yield with weak modulated light.While widely applied in higher plant research,PAM suffers from a low signal-to-noise ratio due to the low intensity of measuring light,making it challenging for low-chlorophyll environments,and unsuitable for phytoplankton monitoring.Kolber et al.introduce the fast repetition rate(FRR)fluorescence measurement technology,utilizing rapid,repeated saturation pulses to block the photosynthetic electron transfer chain and modulate chlorophyll fluorescence.By fitting the fluorescence dynamics curve using the exponential curve,FV/FM can be obtained.FRR employs high-frequency sequences(up to 250 kHz)of narrow light pulses(0.3 to 2μs full width at half maximum)as the excitation light source,providing a high signal-to-noise ratio for measuring FV/FM in phytoplankton.However,it poses high demands on the excitation light source driving circuit and high-speed data acquisition circuit design,increasing the system design complexity and cost.Building upon FRR,Shi et al.propose the tunable pulse light induced fluorescence(TPLIF)technology.The TPLIF technology changes the fast-repeated pulse light excitation in the single turnover mode to single pulse light excitation,reducing the requirements for signal sampling rate and simplifying system design complexity.Based on this,Wang et al.study an adaptive excitation light intensity method.By regulating the saturation excitation light to block the photosynthetic electron transfer chain based on fluorescence saturation parameters,they accurately obtain photochemical quantum yield for different growth stages of phytoplankton.However,phytoplankton classes vary significantly in their light-harvesting pigment compositions and characteristic absorption bands.Eukaryotic algae cells like green algae,diatoms,dinoflagellates,and coccolithophores mainly concentrate their characteristic absorption of light-harvesting pigments in the blue-green light region,with lower absorption in the longer wavelength range.Cyanobacteria,as large single-celled prokaryotic organisms,concentrate their characteristic absorption in the orange-red light region,with lower absorption in the shorter wavelength range.Therefore,when measuring photochemical quantum yield for different classes of phytoplankton,the TPLIF technology using single-wavelength excitation faces challenges in simultaneously saturating different classes of phytoplankton,leading to large errors in the measurement of photochemical quantum yield.Methods Based on the light absorption characteristics of different classes of phytoplankton,focusing on Microcystis aeruginosa(belonging to phylum Cyanophyta)and Chlorella pyrenoidosa(belonging to phylum Chlorophyta),we employ a dual-band pulse excitation comprising red and blue light.Saturation conditions are optimized to achieve 99%closure of PSII photosynthetic reaction centers within a single turnover cycle,with adaptive adjustments made to excitation wavelength and intensity.Fluorescence kinetics curves are fitted under saturation excitation to accurately measure photochemical quantum yield across phytoplankton classes.Results and Discussions For Microcystis aeruginosa,errors in 10 repeated FV/FM measurements compared to FastOcean sensor results are 26.50%,1.58%,and 1.12%under blue light,red light,and combined light excitation modes,respectively(Fig.3).Similarly,for Chlorella pyrenoidosa,errors are 1.12%,8.99%,and 0.53%under the respective excitation modes(Fig.4).Measurements of mixed algae with varying volume ratios show errors of 11.95%,14.02%,and 0.94%,under blue light,red light,and combined light excitation modes compared to FastOcean sensor results(Fig.6).Conclusions To address the limitations of the single-wavelength TPLIF technology,which fails to simultaneously saturate different classes of phytoplankton and leads to significant errors in measuring photochemical quantum yield,we propose a dual-band TPLIF technology.This method is designed for the accurate measurement of the photochemical quantum yield of photosynthesis based on the light absorption characteristics of various classes of phytoplankton.Our measurements of pure algae demonstrate that the error in measuring photochemical quantum yield using the characteristic absorption of the excitation wavelength is similar to that of the dual-band excitation mode.However,the error is significantly higher when using non-characteristic absorption of the excitation wavelength compared to the dual-band excitation mode.Under blue light,red light,and dual-band excitation modes,the measurement errors of photochemical quantum yield for Microcystis aeruginosa are 26.50%,1.58%,and 1.12%,respectively,in comparison with FastOcean sensor measurements.For Chlorella pyrenoidosa,the measurement errors are 1.12%,8.99%,and 0.53%,respectively.In addition,measurements of mixed algae show that the error in measuring the photochemical quantum yield using the dual-band excitation mode is significantly lower than that using the single wavelength excitation mode.Relative errors for measuring the photochemical quantum yield of mixed algae are 11.95%,14.02%,and 0.94%,respectively,when compare to FastOcean sensor results.The dual-band excitation mode effectively saturates and excites different phyla of phytoplankton simultaneously,leading to high accuracy in measuring photochemical quantum yield.We introduce a dual-band TPLIF technology for the measurement of phytoplankton quantum yield of photosynthesis,enabling precise measurement of the quantum yield of photosynthesis across different classes of phytoplankton.This technology offers a significant advancement for assessing photosynthetic strength and calculating primary productivity.