<p>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.</p><p>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.</p><p>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 the¹⁴C 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.</p><p>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.</p><p>A detailed study was made of the production of carbohydrates and amino acids by <i>S. costatum </i>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.</p><p>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.</p>

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 &lt;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.

The dissolved silicon (DSi) has a crucial role for growth of a large group of primary producers – diatoms and, hence, impact on functioning of the aquatic food web. This thesis contributes to an increased understanding of the modifications of the DSi cycling in the Baltic Sea. The results provide new information about spatial and temporal changes in DSi concentrations and nutrient ratios for the period 1970-2001 as well as during the 20th century. For the period 1970-2001, the declining DSi trends were found at the majority of monitoring stations all over the Baltic Sea. This decrease is assumed to be mainly due to the ongoing eutrophication. It is supported by the increasing trends of inorganic nitrogen and phosphorus. The trends have implications for the nutrient ratios, DSi:DIN and DSi:DIP, which are important indicators of the state of an ecosystem. The long-term retrospective DSi budget has shown that the DSi concentrations before major hydrological alterations and eutrophication were about twice the present ones. This decrease is related to both eutrophication and anthropogenic perturbations in the catchment. The occurrence of DSi concentrations close to the potentially limiting levels has been also analysed. While DSi concentrations are still high in the northern regions of the Baltic, other areas may be at risk of developing Si limitation if the decrease in DSi concentrations persists. The results depict the Baltic Sea journey from being water body with DSi levels sufficient to support diatom production to one that may experience Si limitation and its adverse ecological consequences. / Löst kisel (DSi) har en viktig roll för tillväxten av en stor grupp av primärproducenter – kiselalger, och därmed även påverkar hela den akvatiska näringskedjan. Denna avhandling bidrar till en ökad förståelse av förändringarna i DSi kretsloppet i Östersjön. Resultaten tillhandahåller ny information om rumsliga och tidsmässiga förändringar i DSi koncentrationer såväl för perioden 1970-2001 som för hela 1900-talet. För perioden 1970-2001 återfanns minskade DSi koncentrationer på mätstationer över hela Östersjön. Minskningen antas främst bero på den pågående övergödningen. Detta antagande stöds av stigande halter av oorganiskt kväve och fosfor. Sammantaget har dessa trender en inverkan på ekosystemets tillstånd och näringsämnenas kvoter, DSi: DIN och DSi: DIP. Ur ett längre tidsperspektiv kan man se att innan övergödningen och de stora hydrologiska ombildningar i Östersjöområdet var DSi koncentrationerna ungefär dubbelt så höga som idag. Dagens förekomst av DSi koncentrationer som ligger nära de potentiellt begränsande nivåerna har också analyserats. DSi koncentrationerna är fortfarande höga i norra delar av Östersjön, men är i andra områden i riskzonen för att utveckla Si begränsning om minskningen av DSi koncentrationer fortsätter. Resultaten skildrar Östersjöns resa från att vara ett havsområde med DSi halter som är tillräckliga för att understödja kiselalgernas produktion till ett sådant som kan uppleva Si begränsning och dess negativa ekologiska konsekvenser.

Målet med den här studien var att undersöka kiselalger och dess eventuella missbildningar i tre vattendrag i södra Sverige - Höje å, Skivarpsån och M42. Kiselalger används som bioindikator för vattenkvalitet i hela Europa, men de befintliga kiselalgsindexen visar inte eventuell förekomst av miljöfarliga ämnen. Det har dock visats att en förekomst &gt; 1 % av missbildade kiselalger indikerar påverkan av miljöfarliga ämnen, såsom pesticider och tungmetaller. Denna studie är en av få som har undersökt kiselalgers missbildningar i vattendrag som är mer eller mindre påverkade av herbicider. Sex prov från varje vattendrag analyserades - kiselalger räknades och identifierades till artnivå och missbildningar dokumenterades och kategoriserades. Vattendragen delades in efter PTI (Pesticide Toxicity Index), där Höje å hade lägst PTI och ansågs minst påverkad av herbicider. Skivarpsån och M42 hade högre PTI, och ansågs därför ha en högre herbicidpåverkan. Det fanns en signifikant skillnad i missbildningsfrekvens vattendragen emellan, den kunde dock inte kopplas till PTI. Både Höje å och M42 hade &gt; 1 % missbildningar. Skivarpsåns missbildningsfrekvens var &lt; 1 % trots vattendragets relativt höga PTI. Det är möjligt att missbildningsfrekvensen bättre hade reflekterat PTI om herbicidprovtagningen skett någon månad tidigare, då herbicidhalter kan variera och kiselalger har visat sig kunna spegla ett vattendrags mående upp till tre månader bakåt i tiden. En annan förklaring till en varierande missbildningsfrekvens kan vara en lika varierande artsammansättning. Vissa arter är mindre “benägna” att missbildas än andra - ett prov som domineras av sådana skulle därför kunna innehålla få missbildningar trots eventuell miljögiftspåverkan. Det krävs dock ytterligare studier för att bättre förstå kiselalgers missbildningar i förhållande till herbicider.