In order to study the chemical composition of aquatic microbes it is necessary to obtain completely separated fractions of subpopulations. Size separation by filtration is usually unsuccessful because the smaller group of organisms contaminates the larger fractions due to being trapped on filter surfaces of nominally much larger pore sizes. Here we demonstrate that a simple sucrose density separation method allowed us to separate microorganisms of even subtle size differences and to determine their bulk biochemical composition (proteins, polysaccharides+nucleic acids, and lipids). Both autotrophs and heterotrophs (through anaplerotic pathways) were labeled with (14)C-bicarbonate for biochemical fractionation. We provided proof of concept that eukaryotic microbes could be cleanly separated from prokaryotes in cultures and in field samples, enabling detection of differences in their biochemical makeup. We explored methodological issues regarding separation mechanisms, fixation, and pre-concentration via tangential flow filtration of oligotrophic marine waters where abundances of microorganisms are comparably low. By selecting an appropriate centrifugal force, two processes (i.e., isopycnal and rate-zonal separation) can be exploited simultaneously resulting in finely-separated density fractions, which also resulted in size separation. Future applications of this method include exploration of the stoichiometric, biochemical and genetic differences among subpopulations of microbes in a wide variety of aquatic environments.

 

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