The effect of three Group IV metals (titanium, zirconium and tin) on the growth, morphology and chemical composition of the freshwater diatom Synedra acus subsp. radians (Kützing) Skabichevsky was studied and compared with germanium. The elements in their highest oxidation states were introduced into the culture medium in the form of hydroxides. Germanium was found to be toxic at ≥5 mol. % of the total Ge-Si content in the culture medium. In the presence of other elements, a slight decrease in the cell division rate was observed independent of the element within 1-15% content interval. The analysis of the obtained biomass and silica valves revealed the presence of all the added elements within the cells. However, only germanium was incorporated into the valves in considerable amounts. S. acus cultivation with the addition of 5% Group IV elements resulted in cells having the following aberrations in the structure of the silica valves: changes in valve shape, thickening of valves, alterations of the areolae rows, irregularity or absence of the areolae and a decrease in the mechanical strength of valves. Moreover, the effect of Group IV elements on silica formation was simulated in vitro using a synthetic polymer bearing polyamine and phosphate groups found in silaffines (proteins from Electronic supplementary material The online version of this article (diatom frustules). The studied elements were observed to provoke the formation of unstable silica particles in solution. We propose that the observed effects of germanium, titanium, zirconium and tin on diatom growth and structure are due to uncontrollable silica condensation.

We studied the growth of the araphid pennate diatom Synedra acus subsp. radians (Kützing) Skabichevskii using a fluorescent dye N(1),N(3)-dimethyl-N(1)-(7-nitro-2,1,3-benzoxadiazol-4-yl)propane-1,3-diamine (NBD-N2), which stains growing siliceous frustules but does not stain other subcellular organelles. We used a clonal culture of S. acus that was synchronized by silicon starvation. Epifluorescence microscopy was performed in two different ways with cells stained by the addition of silicic acid and the dye. Individual cells immobilized on glass were observed during the first 15-20 min following the replenishment of silicic acid after silicon starvation. Alternatively, we examined cells of a batch culture at time intervals during 36 h after the replenishment of silicic acid using fluorescence and confocal microscopy. The addition of silicic acid and NBD-N2 resulted in the rapid (1-2 min) formation of several dozen green fluorescent submicrometer particles (GFSPs) in the cytoplasm, which was accompanied by the accumulation of fluorescent silica inside silica deposition vesicles (SDVs) along their full length. In 5-15 min, GFSPs disappeared from the cytoplasm. Mature siliceous valves were formed within the SDVs during the subsequent 14-16 h. In the next 8-10 h, GFSPs appeared again in the cytoplasm of daughter cells. The data obtained confirm observations about the two-stage mechanism of silicon assimilation, which includes rapid silicon uptake (surge uptake) followed by slow silica deposition. It is likely that the observed GFSPs are silicon transport vesicles, which were first proposed by Schmid and Schulz in (Protoplasma 100:267-288, 1979).

Diatom algae realize highly intriguing processes of biosynthesis of siliceous structures in living cells under moderate conditions. Investigation of diatom physiology is complicated by frustule (siliceous exoskeleton). Frustules consist of valves and girdle bands which are adhered to each other by means of organic substances. Removal of the frustule from the lipid membrane of diatom cells would open new possibilities for study of silicon metabolism in diatoms. We found that submillimeter laser irradiation produced by a free-electron laser causes splitting of diatom frustules without destruction of cell content. This finding opens the way to direct study of diatom cell membrane and to isolation of cell organelles, including silica deposition vesicles. We suppose that the dissection action of the submillimeter irradiation results from unusual ultrasonic waves produced by the short (30-100 ps) but high-power (1 MW) terahertz laser impulses at 5.6 MHz frequency.