Towards the Chemical Analysis of Diatoms’ Silicon Storage Pools: A Differential Centrifugation-Based Separation Approach

Abstract

Diatoms are unicellular algae and occur ubiquitously in almost every marine and freshwater habitat on earth. They produce intricately structured cell walls, which mainly consist of amorphous silica. To synthesize their cell walls, diatoms take up monosilicic acid from the environment and store it. These silicon storage pools (SSPs) can exceed the solubility of silicic acid by one to two orders of magnitude, as observed in various diatom species. However, their chemical composition and cellular localization has not yet been elucidated. It is suggested that SSPs may consist of stabilized aggregates such as pre-condensed silica particles or silica-containing vesicles. Isolation protocols for SSPs without significant chemical modification are required to prove such hypotheses. A critical issue is the efficient separation of components of the SSPs from cell wall fragments or artefacts, which may interfere with analytical methods targeting silicon. To this end, a comparative study was performed on exponentially grown cells and extracted, purified cell walls (biosilica) to observe the sedimentation behavior after lysis. Cell cultures were lysed by bead beating and then fractionated by differential centrifugation. The obtained fractions were analyzed for total silicon content (tSi) using molybdenum blue assay (MBA) after alkaline treatment. It was revealed that cell wall fragments are almost absent in fractions above 1000 × g. Compared with biosilica, a significantly higher silicon concentration is found in lysed cell pellets after centrifugation at moderately high forces. The differences correspond to a few percent of total cellular silicon, which are assumed to be part of SSPs. Only relatively low amounts of silica/silicic acid remain in the supernatant at high centrifugal forces. This indicates that SSPs are mainly present in larger aggregates that sediment at lower centrifugal forces. According to Stokes’ law, only silica particles below ca. 25 nm radius would remain in the final supernatant. This leads to the conclusion that SSPs must mainly consist of larger silica particles and/or are associated with larger compartments/aggregates.