A New Method to Measure Prokaryote Respiration at the Single Cell Level by Flow Cytometry
In the ocean, microbial respiration is considered the major process representative of organic matter’s biological oxidation. The corresponding metabolic CO2 production was estimated to reach about 22 Pg C year−1 (1 Pg = 1015 g).
The balance between primary production and respiration determines if the biological system (i.e., the biological pump) acts as a source or a sink of carbon, a very important topic in the framework of the global warming. However, there is still some uncertainty about the importance of the respiration because the in situ respiration rate is generally too low (by several orders of magnitude) to be accessible to the available direct measurement methods. Some indirect methods were therefore applied, such as the measure of O2 consumption by the Winkler chemical method or the CO2 metabolic production by coulometry. There is also an enzymatic method (Electron Transport System test from Packard) based on the enzymatic activity of dehydrogenases, but it addresses only a potential activity.
All these methods are global and thus address the community level. Therefore, they do not take into consideration the heterogeneity of microbial assemblages.
Some fluorescent dyes, such as DiOC6(3) (Molecular Probes, USA), have been shown to be very sensitive to changes in the proton electrochemical potential difference (ΔμH+), characterizing mitochondrial and plasmic membranes bearing the cell respiratory system in eukaryotic and prokaryotic cells, respectively. In mitochondria, ΔμH+ 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. Our team 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 a fluorescent dye called DiOC6(3).
The accumulation of the dye is sensitive to the membrane potential of the mitochondria. We found and standardized a linear relationship between oxygen uptake by D. tertiolecta and its green fluorescence induced by DiOC6, enabling the determination by flow cytometry of the respiration rate of D. tertiolecta.
After this promising result, we are trying to extend the method to other microorganisms representative of the microbial assemblages. This study aims to continue this work and to apply it to aerobic heterotrophic prokaryotes, which are the main mineralizers of the organic matter in the ocean.
KeywordsExponential Growth Phase Microbial Respiration Single Cell Level Electron Transport System Dark Respiration Rate
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