Study On Quantitative Method Of Chlorophyll In Vivo Planktonic Algae Based On Three-dimensional Fluorescence Spectrum

Accurate and rapid acquisition of chlorophyll a content of planktonic algae in natural waters can not only provide basic data for water quality monitoring and early warning,water ecological environment protection and management,but also provide ground calibration means for large-scale water quality remote sensing monitoring.As a fast and real-time analysis tool for algae chlorophyll a concentration,fluorescence method is confronted with the difficult problem of accurate measurement in practical application due to the difference of fluorescence yield efficiency of different algae,instability of algae fluorescence spectrum in vivo,interference of dissolved organic matter in water and suspended particulates,etc.Based on the abundant characteristic information of three-dimensional fluorescence spectroscopy,an experimental study on accurate measurement of algae chlorophyll a concentration was carried out based on three-dimensional fluorescence spectroscopy.The main achievements are as follows:The classified quantitative analysis method of algae chlorophyll a concentration based on three-dimensional fluorescence spectroscopy was studied.The relationship between the three-dimensional fluorescence spectra and the composition of photosynthetic pigments of different species of algal in vivo was measured and analyzed.Based on spectral similarity analysis,the three-dimensional fluorescence spectra of different algae were divided into five spectral groups:Cyanophyta,Chlorophyta,Bacillariophyta,Pyrrophyta,and Cryptophyta.The method of three-dimensional fluorescence spectra analysis of algal based on non-negative weighted least squares algorithm was studied.The quantitative analysis of chlorophyll a concentration of algae was realized,and the problem of accurate measurement of chlorophyll a concentration caused by the difference of fluorescence yield efficiency of different algae was solved.The results of comparative analysis showed that the total chlorophyll a concentration of pure and mixed algae samples measured by three-dimensional fluorescence spectrometry was in good agreement with that measured by spectrophotometry.The correlation coefficients were 0.972 and 0.943,and the average recall rates of chlorophyll a concentration were 102.9%and 89.7%,respectively.The recognition accuracy of Cyanophyta,Chlorophyta,Pyrrophyta,and Cryptophyta was 100%,and the classification accuracy was all less than 9.6%.The average recognition accuracy,average recall rate and average classification accuracy of Bacillariophyta were 75.6%,43.8%and 56.3%respectively.Compared with the results of excitation fluorescence spectroscopy,the recall rates and classification accuracies of Cyanophyta,Chlorophyta,Pyrrophyta,and Cryptophyta have been improved in varying degrees;the recognition accuracy of Bacillariophyta has been improved by 17.1%,the average recall rate has been increased by 29.1%,and the average classification error has been reduced by 32.4%.The stability of algal fluorescence spectra in vivo and the method of constructing fluorescence standard spectra were studied.Based on the method of regional integration,spectral envelope and pigmentation ratio,the differences of pigmentation composition and fluorescence efficiency of different algae species in different growth periods were analyzed,and the stability of fluorescence spectra of living algae under different conditions was quantitatively characterized.The method of constructing standard spectra of algae in vivo based on weighted average method was studied with the standard deviation of spectra as the weighting factor.Three-dimensional fluorescence standard spectra of algal were established and applied to algae chlorophyll a concentration classification measurement algorithm.The results show that the relative errors of three-dimensional fluorescence standard spectroscopy measurement range from 0.1%to 30.4%,and the average relative error is 12.8%.Compared with the other 12 groups of reference spectroscopy measurements representing different species and different growth periods,the average relative error was reduced by 13.3%,and the measurement accuracy of Anabaena can be improved by 104.1%in maximum.Colored dissolved organic matter(CDOM)and turbidity interference correction methods in algae chlorophyll a concentration measurement were studied.Similarity indices of three-dimensional fluorescence spectra of endogenous CDOM,exogenous CDOM,and humic acid(HA)were analyzed.Using HA three-dimensional fluorescence spectra as independent spectral group and representing different sources of CDOM,the interference correction algorithm of CDOM in algae chlorophyll concentration measurement was studied.The results showed that the recall rates of algae samples were significantly improved after correction.The average recall rates of Cryptophyta Pyrrophyta,Bacillariophyta and Chlorophyta increased by 162.7%,50.3%,106.4%and 19.1%,respectively.The average classification errors of Pyrrophyta,Bacillariophyta,Cyanophyta,and Chlorophyta were decreased by 83.9%,100%,38.2%and 48.0%,respectively.The turbidity compensation method for measuring chlorophyll a concentration of algae based on extinction coefficient at 850 nm was studied.The average recall rates of Cyanophyta,Chlorophyta,Bacillariophyta,and Pyrrophyta were increased by 1897.4%,61.0%,78.0%and 15.8%respectively after correction.The classification errors of Chlorophyta,Bacillariophyta,and Pyrrophyta were reduced by 94.6%,90.1%and 61.4%,respectively.The research method of this paper has been applied to the independent developed AFA,and the field measurement demonstration application has been completed in Yangzhou,Taihu and Xiangshan Port.The results showed that the chlorophyll a content of planktonic algae in vivo was measured rapidly and accurately based on the three-dimensional fluorescence spectrometry,which was in good agreement with spectrophotometry.