Study On Fluorescence Spectra For Identifying Phytoplankton Community

Classifying and quantifying phytoplankton is an important task in marine red-tide monitoring. The in vivo fluorescence method can provide abundant information and avoid using organic solvent. With high sensitivity and selectivity,it has been widely used in scientific research. It is significant to find effective methods to detect components of phytoplankton community for preventing red-tide disasters.This paper aims to study the characteristics of phytoplankton chlorophyll fluorescence excitation spectrum and emission spectrum by means of Fourth-derivative analysis and Gauss decomposition,then establish phytoplankton community identifying technique with Non-negative Least Square(NNLS) based on Fisher analysis. Twelve common algae species which belongs to Bacillariophyta,Pyrrophyta,Chlorophyta and Cryptophyta were chosen and cultured under certain temperature(20℃) and three different illumination intensities(15000Lx,10000Lx,6000Lx). In the twelve species,ten is typical red-tide species of the East China Sea. Their three dimensional fluorescence spectra(EEM) were measured and the main research work is as follows:(1)The fluorescence information of phytoplankton is always covered up by the Rayleigh scatter in EEM. Delaunay triangulation interpolation was used to correct Rayleigh scatter. The R.S.D of EEM was between 5%~6% for Pyrrophyta except Al,and was less than 5% for other species except Oc. The elimination of scatter was helpful to improve spectrum precision.(2) In vivo chlorophyll fluorescence excitation spectra discriminating technique of phytoplankton community on division level was established. The fourth-derivative analysis was performed on fluorescence excitation spectra to separate overlapping fluorescence peaks. The results showed there were six fluorescence peaks in the fourth-derivative spectra between 350nm and 550nm,which were representative of non-photosynthetic pigments,chlorophylls and carotenoides respectively. Based on the presence of position and number of maxima on the fourth-derivative spectra,11 initial central wavelengths were set for Bacillariophyta,Pyrrophyta,Chlorophyta and 12 for Cryptophyta between 300nm and 600nm. With these wavelengths as centre,Gaussian decomposition and multi-peak fitting were done to the spectra. The characteristics of Gauss spectrum suggested that significant differences were presented between different divisions while similar characteristics were found in the same division. The R. S. D of Gaussian parameters U and C were less than 0.5% and 2.5% respectively,and R. S. D of parameter S was lower than 5% for Gauss peaks between 350 and 550nm. That showed high precision and good reproducibility. Using the intensity ratio of neighboring Gauss peaks could not only distinguish Bacillariophyta and Pyrrophyta,but also distinguish chlorophyll b and chlorophyll c-containing algae. Seven representative Gauss wavelengths were used as characteristic fluorescence excitation spectrum. Then differentiation and discrimination for algae test assemblages were studied with Fisher differentiation and NNLS discrimination. As a result,on the level of division,the differentiation accuracy for single algae and mixed algae were 96.06% and 89.53%. The discrimination accuracy for them is 92.71% and 81.4% respectively. This shows effective differentiability of phytoplankton fluorescence excitation spectrum when using 380nm,415nm,440nm,465nm,495nm,530nm,560nm as characteristic wavelengths. It can provide technical sustentation for invention of new auto-analysis fluorescence apparatus.(3) Single-excitation wavelength fluorescence emission spectrum discriminating technique of phytoplankton community on division level was established. The fluorescence emission spectrum under excitation wavelengths of 410nm,440nm,470nm,500nm and 530nm were compared among four divisions. The single-excitation wavelength fluorescence emission spectrum at 530nm most distinct among divisions was chosen to study by Fisher differentiation and NNLS discrimination. The classifying figure showed Bacillariophyta and Pyrrophyta were overlapped and all species can separated into three classes. When Bacillariophyta and Pyrrophyta were regarded as the same class,the differentiation accuracy and discrimination accuracy came to 94.68% and 87.50% for single algae test assemblages with Fisher differentiation and NNLS discrimination. It accounted for single-excitation wavelength fluorescence emission spectrum discriminating technique could detect three classes of Bacillariophyta-Pyrrophyta,Chlorophyta and Cryptophyta effectively. The wavelength of 530nm is always used as excitation wavelength for lidar fluorescence,so this technique can contribute to lidar fluorescence.(4) Multi-excitation wavelengths fluorescence emission spectrum discriminating technique of phytoplankton community on division level was established. Choose three Gauss excitation wavelengths of 440nm,470nm and 530nm to construct multi-wavelengths emission spectrum. The new spectra were also done with Fisher differentiation and NNLS discrimination for test assemblages. As a result,the differentiation accuracy in single algae was 98.15%,and the discrimination accuracy was 90.63%. For the mixed algae,the differentiation accuracy came to 92.57% and the discrimination accuracy was 87.07%. Thus , using the multi-excitation wavelengths fluorescence emission spectra could well discriminate different phytoplankton divisions.In all,this paper studies in vivo fluorescence spectra of phytoplankton. Using the Fourth-derivative analysis and Gauss decomposition can obtain characteristic information on different divisions. The fluorescence excitation spectra and emission spectra of different divisions are studied by differentiation and NNLS discrimination. Bacillariophyta,Pyrrophyta,Chlorophyta and Cryptophyta are well differentiated on division level.