Studies on the Development of Real-time PCR Assay for Monitoring Phytoplankton and Characterization of Dissolved Organic Matter in Lake Biwa and the Surrounding Rivers

リアルタイムPCRを用いる藻類モニタリング法の開発と琵琶湖・流域河川における溶存有機物質の特性評価に関する研究

2018-03-26

2018-03-26

Lake Biwa is an irreplaceable water source not only for tap water but also for industry, agriculture, and fishery. As the chemical oxygen demand (COD) in the northern basin of Lake Biwa has increased since 1984 in spite of a decrease in organic loadings from the watershed, refractory dissolved organic matter (DOM), which is not easily decomposed by microorganisms, may have gradually accumulated in Lake Biwa. In order to clarify the characteristics of algal DOM and its contribution to the DOM in Lake Biwa, we developed phytoplankton-monitoring methods using a SYBR Green real-time PCR assay with specific primer sets for each species. The real-time PCR assay was applied to the determination of cell densities of algal species during cultivation and in environmental samples, and analytical results by real-time PCR assay were compared with those by microscopy. The linear relationships between the threshold cycle (Ct) values and cell densities were obtained in the range of 2.7x102-2.7x107, 8.2x10-8.2x104, 2.1x10-2.1x104, and 4.6x103-4.6x105 cells mL-1 for M. aeruginosa, S. dorsidentiferum, C. ovata, and F. capucina, respectively. The PCR efficiency values were 117, 87, 66, and 84% for M. aeruginosa, S. dorsidentiferum, C. ovata, and F. capucina, respectively. Furthermore, the effects of the coexistence of other algal species and substances such as suspended solids (SS) in lake water were small in the real-time PCR assay. Fractionation of hydrophobic acids (humic substances) with DAX-8 resin is time consuming and cannot be applied to the water samples whose DOC values are low. Then, the rapid analysis of humic substances in waters using three dimensional excitation emission matrix (3-DEEM) and DOC was investigated comparing with fractionation analysis, and applied to the dynamics of humic substances in Lake Biwa and its surrounding rivers. The results of humic substances in Yodo rivers by rapid analysis using soil fulvic acid (Dando FA) as a standard were in relatively good agreement with those by fractionation analysis except for Uji River where the waters of Lake Biwa affect through Seta River. This rapid analysis was also applied to the dynamics of humic substances in the rivers flowing into Lake Biwa. In the case of rapid analysis of humic substances in Lake Biwa, Biwako FA was used as a standard instead of Dando FA.　The effects of photoirradiation on changes in the fluorescence intensities, DOC concentrations, and molecular weights of DOM　in Lake Biwa and its surrounding rivers were evaluated and compared with the results of humic substances and algal DOM. During the completely stratified period (summer), the fluorescence intensities of fulvic-like fluorophores in the surface water in the northern basin of Lake Biwa decreased and were lower than those in other months and in bottom water. The fluorescence quenching and degradation of high-molecular substances by further solar irradiation were hardly observed in the surface water samples but were significantly observed in bottom water samples. On the other hand, changes in the DOC concentrations in all samples were relatively small with solar irradiation. These results suggest that in the northern basin of Lake Biwa, the susceptibility of fulvic-like fluorophores to degradation by further solar irradiation is dependent on the water depth collected during the stratified period (summer), but the rest of fulvic-like fluorophores might be resistant to further photochemical degradation regardless of the water depth. Furthermore, the effects of the wavelength region on the characteristics of DOM and fluorophores in Lake Biwa and its surrounding rivers were examined by Xe lamp irradiation using two kinds of wavelength cut filters. From these results, it is considered that wavelengths between 290 and 495 nm and below 290 nm might largely affect the characteristics of fulvic-like fluorophores and protein-like fluorophores, respectively.

博士（工学）

京都工芸繊維大学

14303甲第869号

http://hdl.handle.net/10212/2464

eng

ETD

Thesis or Dissertation

Thesis