Bacteria and Photosynthetic Cells in a Photobioreactor Treating Real Municipal Wastewater: Analysis and Quantification Using Flow Cytometry

Wastewater born microalgal-bacterial consortia in closed photobioreactors (PBRs) have demonstrated high efficiency in pollutant removal, relying only on photosynthetic oxygenation. However, their qualitative and quantitative composition is still poorly understood. This paper describes the extension of flow cytometry in order to analyze a microalgal-bacterial consortium developed in a long-term PBR treating real wastewater. A multi-step procedure was proposed to obtain an accurate flow cytometry (FCM) analysis. Ultrasonication at specific applied energy of 90 kJ L−1 was identified as optimal for sample pre-treatment to recover the maximum number of bacteria and photosynthetic cells while avoiding their disruption. Fluorescence and scattering signals were used to distinguish and quantify microalgae and bacteria. In the tested consortium, bacteria (marked green with SYBR-Green I) were 98% of total cells (on average 1.2E +12 cells/L) while microalgae (having red autofluorescence) accounted for only 2% of total cells (2.8E+10 cells/L). Microalgae were characterized by a biovolume 2 orders of magnitude larger than bacteria, resulting in a photosynthetic biomass quantitatively comparable to bacteria. The proposed FCM approach represents a valuable tool with which to enhance knowledge about the composition and quantification of microorganisms in PBRs, which have not yet been fully understood.