Cellular and extracellular production of carbohydrates and amino acids by the marine diatom Skeletonema costatum: diel variations and effects of N depletion

β-1,3-Glucan (chrysolaminaran) is the principal storage polysaccharide in diatoms (Bacillariophyceae), the major primary producers in the sea. The glucan generally contributes a substantial fraction of the algal biomass, but its level varies markedly in response to growth conditions. The scope of this work was to study the metabolism and function of the polysaccharide in marine diatoms. Axenic cultures of the marine planktonic diatom Skeletonema costatum (Grev.) Cleve were used in the experiments. Glucan metabolism was studied by growing the alga in batch culture, and measuring metabolite fluxes by chemical analyses as well as by 14C tracer technique using labeled bicarbonate. A photobioreactor was developed for strictly controlled growth of microalgae. Fine pH regulation was obtained by relay-activated titration with dilute acid (HCl) and base (NaOH). Irradiance and temperature were also carefully controlled. Batch cultures were grown with a 14:10 h light:dark cycle, and pH curves were recorded during different growth phases.A new method was developed for the combined determination of β-1,3-glucan and cell wall polysaccharides in diatoms, representing total cellular carbohydrate. The glucan is rapidly extracted by hot dilute H2SO4, and the cell wall polysaccharides are subsequently hydrolyzed by cold 80% H2SO4overnight. Each carbohydrate fraction is finally determined by the phenol-sulphuric acid method. This procedure is simple and rapid compared to previous methods, and applies well to laboratory cultures as well as natural phytoplankton populations dominated by diatoms.Synthesis and mobilization of β-1,3-glucan in N-limited S. costatum were studied by combined 14C tracer technique and chemical analyses. Radiolabeled bicarbonate was added to the cultures, and 14C incorporation in different metabolites was determined using biochemical fractionation. In a pulse phase, 14C label was mainly incorporated in the glucan fraction (85%) during photosynthesis under nitrogen limitation. Subsequently, a 14C chase was carried out by adding NH4+ and incubating the cells under different light conditions. Radiolabeled glucan decreased significantly (by 26% in the dark, and by 19% in low light) whereas radiolabeled amino acids, proteins and other polysaccharides increased significantly during NH4+ assimilation. Chemical analyses of β-1,3-glucan and cellular free amino acids supported the 14C measurements. Changes in amino acid composition strongly indicated that de novo biosynthesis took place, with a Gln/Glu ratio increasing from 0.4 to 10. This study provides new evidence of β-1,3-glucan supplying carbon skeletons for synthesis of amino acids and protein in diatoms. Mobilization of glucan yields glucose, which is further metabolized by the respiratory pathways to provide precursors as well as energy. The results from the 14C chase also indicated significant synthesis of other polysaccharides or possibly RNA from glucan.In a different study, dark carbon fixation in N-limited S. costatum was measured using 14C-bicarbonate. Addition of NH4+ resulted in 4-fold increase in carboxylation rate, and biochemical fractionation showed that mainly amino acids were radiolabeled. Chemical analyses confirmed that cellular free amino acids increased rapidly (with increasing Gln/Glu), and showed that cellular glucan decreased significantly (by 28%) during NH4+ assimilation. The results strongly indicate that β-carboxylation provides C4 precursors for amino acid synthesis, and β-1,3-glucan is likely to be the ultimate substrate for β-carboxylation. Moreover, a C/N uptake ratio of 0.33 indicated that β-carboxylation was related to protein synthesis.A detailed study was made of the production of carbohydrates and amino acids by S. costatum during different growth phases. During exponential growth under diel light conditions, the glucan level oscillated between 17% (end of scotophase) and 42% (end of photophase) of cellular organic carbon, and the corresponding protein/glucan ratio alternated between 2.3 and 0.7. Concurrently, the cellular free amino acid pool oscillated between 8% (end of scotophase) and 22% (end of photophase) of cellular organic nitrogen, and the corresponding Gln/Glu ratio alternated between 0.05 and 2. Depletion of nitrogen from the medium resulted in rapid accumulation of glucan, reaching 75-80% of cellular organic carbon, whereas the cellular nitrogenous components decreased significantly. Consequently, the protein/glucan ratio decreased to <0.1. This study indicates that β-1,3-glucan functions both as a short-term diurnal reserve and a long-term stockpile reserve.Field investigations by other workers suggest that glucan plays a very active role in the dynamics of natural diatom populations, and the protein/glucan ratio has been used as a sensitive parameter for nutrient status. The glucan dynamics may be involved in physiological control of buoyancy. Glucan accumulation by nutrient-deplete cells causes increased cellular density and sinking below the nutricline. Upon nutrient replenishment and mobilization of glucan, the cells rise toward the surface of the water column, thereby transporting deep nutrients to the euphotic zone. β-1,3-Glucan also seems to play an important role in the development of resting stages in diatoms.