11:20 AM - 11:40 AM
[AHW23-03] Cellular level elemental analysis of freshwater diatom using microbeam X-ray fluorescence spectrometry
★Invited Papers
Keywords:μ-XRF, diatom, elemental quota, single cell, freshwater
It is now widely recognized that trace metals such as Fe, Cu, Zn, and Co play an important role as limiting factors for biological production in the aquatic environment. Although culture experiment is direct way to evaluate species specific elemental quota, this method can apply only limited species and environmental parameter. In order to assess the species specific elemental quota through variable environmental condition, cellular level analytical method should be established. Here we show a case study on diatom from three Japanese eutrophic lakes using synchrotron microbeam X-ray fluorescence spectrometry (μ-XRF).
Surface water samples were collected from Kasumigaura, Ushiku-numa, and Teganuma lakes in June and August 2020. The dissolved and particulate concentrations of dissolved and suspended metals in each lake were measured by ICP-MS. The metal free glutaraldehyde was spiked to water sample on site to a final concentration of 1 %. On the day of collection, the samples were centrifuged at 1000 rpm for 5 min, and microbe-enriched phase was dropped onto a silicon nitride grid of electron microscopy then allowed to air dry. The dried samples were observed with a benchtop SEM and a 3D digital microscope, to pick up position of target cell. The μ-XRF analysis was performed at the BL-4A (beam size: 5×5 μm) in the Photon Factory, KEK. The excitation energy was set at 12 keV (6×108 photons/sec). The XRF spectra were acquired by integrating 50-120s at representative spots and analyzed using pyMCA (Sole et al., 2007). The cellular level of elements was computed by fundamental parameter (FP) method considering the absorption efficiency of photons, incident light intensity, detection efficiency, and absorption by the matrix. The accuracy of FP method was validated using pellet of biological certified materials for trace element (CRMs).
For natural samples, the high density of diatoms were observed (>103/ml) in all lakes with predominance of cyclotella, fragilaria, and Aulacoseira (10 to 50 µm). The XRF spectra of the each diatom showed significant peaks for Ca, Mn, Fe, Cu, and Zn with limits of quantification being 1-10 ppm. When comparing at same environmental setting, there were no significant difference of element level among three species. Dissolved Mn, Fe, Cu, and Zn were generally higher in June than August since higher pH in August induced by active photosynthesis suppress available metal fraction. Positive correlations were observed between dissolved and cellular level of each element. This correlation becomes stronger when dissolved metal level was divided by cell density suggesting that cellular elemental level is consistent with available element level for unit cell. Despite highly variable water quality of freshwater than seawater, application of cellular level analysis has been limited. The present study enables the analysis of individual trace elements in nanoplankton in freshwater system, and this method may be useful for the understanding of elemental partitioning between water and plankton, such as the determination of bioaccumulation factors of various elements.
Surface water samples were collected from Kasumigaura, Ushiku-numa, and Teganuma lakes in June and August 2020. The dissolved and particulate concentrations of dissolved and suspended metals in each lake were measured by ICP-MS. The metal free glutaraldehyde was spiked to water sample on site to a final concentration of 1 %. On the day of collection, the samples were centrifuged at 1000 rpm for 5 min, and microbe-enriched phase was dropped onto a silicon nitride grid of electron microscopy then allowed to air dry. The dried samples were observed with a benchtop SEM and a 3D digital microscope, to pick up position of target cell. The μ-XRF analysis was performed at the BL-4A (beam size: 5×5 μm) in the Photon Factory, KEK. The excitation energy was set at 12 keV (6×108 photons/sec). The XRF spectra were acquired by integrating 50-120s at representative spots and analyzed using pyMCA (Sole et al., 2007). The cellular level of elements was computed by fundamental parameter (FP) method considering the absorption efficiency of photons, incident light intensity, detection efficiency, and absorption by the matrix. The accuracy of FP method was validated using pellet of biological certified materials for trace element (CRMs).
For natural samples, the high density of diatoms were observed (>103/ml) in all lakes with predominance of cyclotella, fragilaria, and Aulacoseira (10 to 50 µm). The XRF spectra of the each diatom showed significant peaks for Ca, Mn, Fe, Cu, and Zn with limits of quantification being 1-10 ppm. When comparing at same environmental setting, there were no significant difference of element level among three species. Dissolved Mn, Fe, Cu, and Zn were generally higher in June than August since higher pH in August induced by active photosynthesis suppress available metal fraction. Positive correlations were observed between dissolved and cellular level of each element. This correlation becomes stronger when dissolved metal level was divided by cell density suggesting that cellular elemental level is consistent with available element level for unit cell. Despite highly variable water quality of freshwater than seawater, application of cellular level analysis has been limited. The present study enables the analysis of individual trace elements in nanoplankton in freshwater system, and this method may be useful for the understanding of elemental partitioning between water and plankton, such as the determination of bioaccumulation factors of various elements.