Strategies of the ubiquitous freshwater diatom Achnanthidium minutissimum in response to phosphate limitation

Abstract

Diatoms are primary producers found in both marine and freshwater environments and are the most diverse group of microalgae. They contribute to ca. 20% of annual global carbon fixation and contribute to the biogeochemical cycles. Diatoms occupy a variety of environments, including the benthic habitat where they are often the most abundant eukaryotes. A crucial part of the biological success of the diatoms is their ability to form biofilm by excreting adhesive extracellular polymeric substances (EPS) structures, allowing them to attach to surfaces. Such a strategy allows the diatom and other microorganisms (eukaryotes, prokaryotes) to stay fixed on a substratum exposed to sufficient nutrients and light. Together, the consortium of microorganisms is encased in a polysaccharide matrix, which exerts considerable control over the movement of nutrients from the water column to the biofilm. A nutrient limitation may occur as the biofilm get denser, resulting in competition between microorganisms for nutrients. Phosphorus (P) is an essential nutrient utilized by the whole life kingdom for growth, energy transport and membrane synthesis. Despite the dramatic consequences of P starvation due to its scarcity in some aquatic environments, many microalgae can thrive in P-limited environments, including the benthic diatom A. minutissimum. Initially isolated from an oligotrophic lake (Lake Constance, Germany), the diatom can form a biofilm by producing a stalk made of polysaccharides in the presence of the bacterium Dyadobacter sp. 32. Works in laboratory cultures showed that the diatom in axenic or biofilm form can continue to divide for several days (ca. 2 weeks), after a transfer in a P-deplete cultivation medium. Such resistance implies the presence of P reserves within the diatom, sustaining the cell in P for several new generations. Several strategies are known in diatoms to cope with P-limited conditions, but most research to date has focused on the adaptations in marine phytoplankton and biogeochemical cycles. However, freshwater benthic diatoms such as A. minutissimum likely have developed strategies of survival in such environments. This thesis has the aim to understand the strategies of the ubiquitous diatom A. minutissimum for adapting under P limitation, in its planktonic as well as its biofilm form. Herein, we identified intracellular granules of polyphosphate (polyP), a variable-length chain of inorganic phosphate units linked by phosphoanhydride bonds. The identification was unambiguously supported via a combination of stimulated Raman spectroscopy as well as elemental electron microscopy analyses. We developed an easy-to-use method for extracting the polyP in diatoms based on gel filtration. We investigated the physiology of the axenic diatom under variable exogenous P availability. We discovered that the diatom can thrive in fluctuating P conditions by rapidly accumulating polyP when extracellular P is available. The polyP reserves in diatoms were rapidly declining when cells were subjected to exogenous P depletion, suggesting the use of polyP for buffering cells in P. The biofilm formation was of particular interest as we investigated the biochemical composition of the stalk produced by the diatom. Using a combination of microscopy techniques, we revealed that the stalk of A. minutissimum is composed of sulphated polysaccharides, probably cross-linked by metal ions such as magnesium and iron. We identified further adaptations developed by the diatom in biofilm form under P-limited conditions. Notably, the ability of the cell to produce surface-associated alkaline phosphatase (AP), allowing the scavenging of organic P forms. The enzyme was suggested to be an indicator of the intracellular P stress of the diatom, as it was displayed by cells subjected to P limitation. Diatoms were able to elongate their stalk only in cultures where higher amounts of cells were displaying AP activity, suggesting an advanced depletion of their intracellular P reserved. This elongation was suggested to elevate the cells from the substratum, allowing better nutrient access than in a dense biofilm. Therefore, intracellular P resources, and notably polyP are proposed to play an important role in the adaptations of the benthic diatom to fluctuating P conditions.