Protozoan Communities and Their Contribution to Predation on <i>E. coli</i> in Aerobic Granular Sludge

Protozoa contribute to water purification through predation in wastewater treatment systems. Full-scale aerobic granular sludge (AGS) reactors treating municipal wastewater contain AGS of varying sizes, with those larger than 2 mm dominating. These size fractions exhibit different sludge morphologies and microbial communities. To date, little is known about protozoan communities and their role in the removal of human-associated bacteria (like pathogens) in AGS plants, particularly across different size fractions. This study conducted uptake experiments with fluorescent <i>Escherichia coli</i>, as a model for human-associated bacteria, followed by microscopic observation to investigate protozoan communities and their predatory behavior in six AGS size fractions and activated sludge collected from full-scale municipal wastewater treatment plants. Sessile ciliates, particularly <i>Epistylis</i> and <i>Vorticella</i>, dominated protozoan populations across six AGS size fractions, with <i>Epistylis</i> being more abundant in larger AGS fractions (>1 mm) and <i>Vorticella</i> in smaller fractions (<1 mm). Additionally, microcosm experiments under aerobic (including predation) and anoxic conditions (excluding predation) revealed that predation was likely to be the main <i>E. coli</i> removal pathway, contributing an additional 0.5 to 2.5 log₁₀ CFU mL^-1 reduction over a combination of non-predatory biological and abiotic processes. Larger AGS fractions showed greater predation capacity, linked to higher <i>Epistylis</i> abundance, while activated sludge, dominated by <i>Vorticella</i>, resembled smaller AGS fractions with lower predation capacity. These findings advance the understanding of the distribution of protozoan communities and their contribution to <i>E. coli</i> removal by predation in AGS wastewater treatment.