New insights into floc morphology and its associations with PAOs in activated sludge via quantitative image analysis

Recent advancements in molecular biological techniques have enabled detailed investigations of functional microbes, including polyphosphate-accumulating organisms (PAOs), which play a crucial role in biological phosphorus removal. However, these techniques are time-consuming and costly, limiting their applicability for routine monitoring in wastewater treatment plants. In this study, as a simple alternative approach, we focused on microscopic images of activated sludge and conducted a proof-of-concept study to explore whether floc morphology could provide information related to the abundance of functional bacteria, particularly polyphosphate-accumulating organisms. We collected 16 activated sludge samples and acquired microscopic images after high-concentration DAPI staining. The images were segmented into whole floc regions and PAO-occupied regions, enabling linkage between floc morphological parameters and PAO abundance at the level of individual flocs. We showed that floc morphology is correlated with PAO abundance, and that PAO-dominated flocs exhibit high circularity and small equivalent diameters. We further implied that these findings may be driven by Ca . Accumulibacter. Furthermore, we attempted to predict the relative abundance of Ca . Accumulibacter from morphological parameters using quantitative image analysis (QIA). Our results suggest that morphological parameters derived from QIA can be used to qualitatively monitor changes in Ca Accumulibacter abundance and may also have potential for sample-level quantification within a treatment line. However, the model for predicting Ca . Accumulibacter abundance showed poor performance when samples from different treatment lines were included, indicating limited generalizability and the need for further assessments.