Genome-resolved metatranscriptomics unveils distinct microbial functionalities across aggregate sizes in aerobic granular sludge

Microbial aggregates of different sizes in aerobic granular sludge (AGS) systems have been shown to exhibit distinct microbial community compositions. However, studies comparing the microbial activities of different-sized aggregates in AGS systems remain limited. In this study, genome-resolved metatranscriptomics was used to investigate microbial activity patterns within differently sized aggregates in a full-scale AGS plant. Our analysis revealed a weak correlation between the relative abundance of metagenome-assembled genomes (MAGs) and their transcriptomic activity, indicating that microbial abundance does not directly correspond to metabolic activity within the system. Flocculent sludge (FL; <0.2 mm) predominantly featured active nitrifiers and fermentative polyphosphate-accumulating organisms (PAOs) from <i>Candidatus</i> Phosphoribacter, while small granules (SG; 0.2-1.0 mm) and large granules (LG; >1.0 mm) hosted more metabolically active PAOs affiliated with <i>Ca.</i> Accumulibacter. Differential gene expression analysis further supported these findings, demonstrating significantly higher expression levels of key phosphorus uptake genes associated with <i>Ca.</i> Accumulibacter in granular sludge (SG and LG) compared to flocculent sludge. Conversely, <i>Ca.</i> Phosphoribacter showed higher expression of these genes in the FL fraction. This study highlights distinct functional roles and metabolic activities of crucial microbial communities depending on aggregate size within AGS systems, offering new insights into optimizing wastewater treatment processes.