Microbial density-dependent viral dynamics and low activity of temperate phages in the activated sludge process

The ecological behavior of bacteriophages (phages), the most abundant biological entity in wastewater treatment systems, is poorly understood, especially that of temperate phages. Here, the temporal dynamics of lytic and temperate phages in a laboratory-scale activated sludge reactor with a sludge bulking issue was investigated using coupled sludge metagenomic and viromic analyses. The lysogenic fragments (prophages) identified were widely distributed in the reconstructed metagenome-assembled genomes (61.7%, n = 227). However, only 12.3% of the identified prophages experienced lysogenic-lytic switching, and the abundance contribution of prophages to free virus communities was only 0.02-0.3%, indicating low activity of temperate phages. Although the sludge community changed dramatically during reactor operation, no massive prophage induction events were detected. Statistical analyses showed strong correlations between sludge concentration and free virus and temperate phage communities, suggesting microbial density-dependent virus dynamics in the sludge microbiota.