CAESAR · Capillary Electrophoretic Separation of dissolved carbohydrates of the Aquatic Realm

Final Activity Report Summary - CAESAR (Capillary Electrophoretic Separation of dissolved carbohydrates of the Aquatic Realm)

Dissolved organic matter (DOM) from the ocean represents one of the largest organic carbon reservoirs of the biosphere. It consists mainly of sugars and proteins released by phytoplankton, but also other planktonic groups of organisms contribute to the DOM pool. The only consumers of significance of the DOM pool are prokaryotic plankton, largely bacteria. Despite the global importance of the oceanic DOM pool, only about 20-40% has been chemically characterised due to the methodological and analytic problems associated with the high salt content of marine samples. The two main components of this DOM are carbohydrates and proteins. Capillary electrophoresis allows separating molecules (e.g., the carbohydrates or the proteins) depending on their molecular weight and/or mass/charge ratios. The individual sugar and protein molecules can then be detected fluorometrically or spectrophotometrically using specific dyes. We tried to use this method to characterise the carbohydrates and the proteins in marine waters. As the components under investigation are present in rather low concentrations in seawater, we used filters with a certain molecular cut off to concentrate and also to desalt the samples. Samples were collected over an annual cycle from the coastal North Sea and at seven stations of different trophic status during a cruise in the North Atlantic. The concentrated samples were subsequently used for the analysis of the molecular weight distribution of carbohydrates by capillary electrophoresis. Despite initial promising results, the available filters used for concentration did not allow the quantification of the different saccharides. However, concentrated samples were successfully used to develop a method for discrimination of different enzymes from the prokaryotic community using capillary electrophoresis with a newly-installed UV-laser which allows detecting the commonly used substrates (MUF-substrates). Actually, work has been focused on the possibility to determine the activity rates of the different enzymes. The prokaryotic transformation of the organic matter was assessed by studying both, the DOM composition and the prokaryotic community composition and activity. During the annual cycle on the coastal North Sea, a close relationship was found between protein concentrations and prokaryotic abundance during a phytoplankton bloom. This result could be due to the preferential use of proteins by bacteria, after proteins are released by the phytoplankton, as shown in other studies. The uptake of specific components by the prokaryotes was assessed mainly by microautoradiography combined with fluorescence in situ hybridisation (MICRO-CARD-FISH). This method allows to determine which specific organisms are taking up a specific substrate. For this approach some major further developments of an existing method have been made to allow the quantification of substrate uptake on a single cell level. This improved method was used to assess the specialisation of different prokaryotic communities to use different DOM components. The initial findings, obtained from mixing communities and DOM from different environments, showed that prokaryotes have a higher cell specific activity when growing on their indigenous DOM, which would be related to a specialisation of the bacterial community to utilise the DOM present.

Data: CORDIS, © European Union