ARE ALL CELLS IN BATCH CULTURE EQUAL? SINGLE-CELL HETEROGENEITY AND THE EVOLUTION OF CHLOROTIC SUB-POPULATIONS IN PROCHLOROCOCCUS

Dalit Roth-Rosenberg ¹ Dikla Aharonovich ¹ Tal Luzzatto-Knaan ¹ Luca Zoccarato ² Hans-Peter Grossart ² Angela Vogts ³ Maren Voss ³ Daniel Sher ¹

¹Department of Marine Biology, Leon H. Charney School of Marine Sciences, University of Haifa, Haifa, Israel
²Department of Experimental Limnology, Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Stechlin, Alte Fischerhuette 2, D-16775, Germany
³Biological Oceanography, Leibniz-Institute for Baltic Sea Research Institute, Rostock, Warnemuende, Germany

Every milliliter of seawater contains millions of microbial cells, however, to date, it is unclear what fraction of these cells is alive and active, and what fraction is senescent or dead. In the much more simple system of phytoplankton batch cultures, chlorotic cells appear as the cultures age, characterized as a sub-population of cells with lower chlorophyll autofluorescence identifiable by flow cytometry. These cells have been observed in cultures of both prokaryotic and eukaryotic algae under different growth conditions and nutrient depletions (Phosphorous and Nitrogen starvation; Light/dark cycles) and are typically considered to be severely stressed or dying.

We have characterized the dynamics of the emergence of these sub-populations in different Prochlorococcus strains and different growth conditions. Using FACS sorting followed by NanoSIMS (nano secondary ion mass spectrometry) analyses, we measured two aspects of the metabolic activity of these cells, namely photosynthesis (¹³C-bicarbonate incorporation) and inorganic nutrient assimilation (¹⁵N ammonium uptake).

Our results show that semi-chlorotic Prochlorococcus cells, from ageing cultures, still photosynthesize and take up nutrients (~20% of the semi-chlorotic cells are active with ¹³C/¹²C ratio above 1.25%, half of which also have ¹⁵N/¹⁴N ratios above 5.5%), while fully-chlorotic cells are mostly metabolically inactive (~97% of the fully-chlorotic cells are inactive with ¹³C /¹²C and ¹⁵N/¹⁴N ratio lower than 1.25% and 1.2%, respectively). Thus, the interpretation of chlorotic cells as dying should be questioned. We also observed high variability in the photosynthesis and uptake rates of single cells within each FACS-defined population. We are now working on determining whether populations of Prochlorococcus (and other phytoplankton) in nature are as variable as those seen in lab cultures, including the presence of chlorotic cells. If so, the relative contribution of individual cells to oceanic primary productivity and export production might need to be re-assessed.