Two approaches for P-uptake measurements of specific osmotroph populations using cell-sorting flow cytometry - benefits and limitations

Larsen, A., T. Tanaka, M.V. Zubkov,F. Thingstad

Limnol. Oceanogr. Methods 6:355-363 (2008) | DOI: 10.4319/lom.2008.6.355

ABSTRACT: To elucidate the role that the marine microbes play in global nutrient cycling, it is necessary to recognize how various phyto- and bacterioplankton groups compete for limiting nutrients. Specific phosphate affinity describes an organism’s ability to harvest phosphate at low concentrations from the surrounding water. For the first time, we have taken advantage of cell-sorting flow cytometry in combination with radio-labeled phosphorus to measure this feature of specific osmotrophic groups in natural communities. Specific phosphate affinities for Synechococcus spp. and picoeukaryotes were measured using live, unstained cells. The results were always lower than theoretical calculated maximum values, corresponding well with observations of P-deficiency, or sub-optimal P supply for the osmotroph community, at the time of investigation. Fixing and staining cells before flow sorting offers the advantage of better separation of phytoplankton and showed high sorting reproducibility when applied to nonaxenic Synechococcus cultures. A subsequent investigation of P-leakage from isotopically labeled, fixed, stained cells in nonaxenic cultures of Synechococcus showed that it was only slightly larger than the loss of 17% found when uptake of new label was stopped with adding "cold" phosphate. Possible applications of the currently developed methodology for population specific P affinity measurements by flow sorting are discussed.