A flow cytometric approach to assess phytoplankton respiration

Microbial respiration in the ocean is considered as the major process representative of the organic matter biological oxidation. The corresponding metabolic CO2 production was estimated to be about 22 Pg C y(-1). However, the in situ respiration rate is generally too low (by several orders of magnitude) to be accessible to the available direct measurement methods. Some fluorescent probes, such as DiOC6(3) (Molecular Probes, USA) have been shown to be very sensitive to changes in the proton electrochemical potential difference (DeltamuH+), characterising mitochondrial and plasmic membranes bearing the cell respiratory system in eukaryotic and prokaryotic cells respectively. In mitochondria, DeltamuH+ is linked to the flux of oxygen uptake by a linear relationship. To our knowledge, no such relationship has been established in the case of whole marine cells. In the present work, we addressed the dark respiration rate of the Chlorophyceae Dunaliella tertiolecta (Butcher) in axenic cultures, both directly by using a highly sensitive oxygraph (Oroboros) and by staining cells with DiOC6(3). We found and standardized a linear relationship between oxygen uptake by D. tertiolecta and its green fluorescence induced by DiOC6(3), enabling the determination by flow cytometry of the respiration rate of D. tertiolecta.