Single-Cell Profiling of Compositional and Functional Shifts in the Activated Sludge Microbiome during Wastewater Treatment using Flow Cytometric Fingerprinting

Understanding the dynamics of microbial community composition, function, and corresponding assembly mechanisms is crucial for sustaining water treatment performance in activated sludge systems. In this study, various cell staining techniques were applied to flow cytometric (FCM) fingerprinting for profiling microbial community assembly patterns in a sequencing batch activated sludge system. Samples were collected every 30 min during a complete reaction cycle at a full-scale wastewater treatment plant in northern Minnesota, United States, in the spring. Staining methods include SYTO9, carboxyfluorescein diacetate (CFDA), and Bioorthogonal Non-Canonical Amino Acid Tagging (BONCAT). The compositional assembly results were similar for SYTO9 FCM fingerprinting and 16S rRNA gene sequence data, with both methods indicating assembly processes primarily governed by extreme homogenizing dispersal (>72.22%). BONCAT FCM fingerprinting allowed for depicting variations in microbial community activities more effectively than CFDA, revealing that extreme homogenizing dispersal dominated the static fill (100.00%) and aerobic react phases (96.30%), while dispersal limitation was the dominant process for the mixed fill phase (100.00%). During the settling phase, the two deterministic processes, extreme homogenizing dispersal (55.56%) and dispersal limitation (33.33%), dominated community assembly. Our findings demonstrate the value of FCM fingerprinting as a diagnostic tool for the instant performance monitoring of activated sludge systems.