Quantification of heterotrophic bacteria during the growth of Synechocystis sp PCC 6803 using fluorescence activated cell sorting and microscopy

The presence of heterotrophic bacteria in microalgal cultures can dilute the microalgal content of the harvested biomass, compete for nutrients, and be associated with culture crashes. Being able to detect and quantify heterotrophic bacteria would be of high value for monitoring culture health and reducing deleterious effects. Here, we developed and applied a new method that combines flow cytometry (FC) and fluorescence activated cell sorting (FACS) for the quantification of heterotrophic bacteria in cultures of the cyanobacterium Synechocystis sp. PCC 6803. Particles not containing chlorophyll - heterotrophic bacteria and cell debris - were separated from mixed cultures using FACS based on autofluorescence of Synechocystis. Heterotrophic bacteria were differentiated from cell debris using FC with SYTOX green fluorescence. Using microscopy, we verified that FACS was able to quantify heterotrophic bacteria in Synechocystis cultures effectively. Applying these methods to batch cultures of Synechocystis showed that the count proportions of heterotrophic bacteria were significant (3-13%) and that depletion of inorganic P in the culture favored Synechocystis over heterotrophic bacteria, but led to more cell lysis.