Exopolysaccharides in activated sludge: from genetic potential to full-scale expression

In wastewater treatment plants, extracellular polymeric substances are responsible for the flocculation of activated sludge. These biopolymers also show a great upcycling potential, for use in plastics, flocculants and flame-retardant materials. However, little is known about the taxonomy and expression patterns of biopolymer producers in activated sludge.

The aim of this study was to find out which exopolysaccharide gene clusters are encoded by individual activated sludge species, and at which stage of the wastewater treatment process these gene clusters are most highly expressed.

To uncover the genetic potential of individual bacterial species in a typical Danish wastewater treatment plant, state-of-the-art long-read sequencing with short-read polishing was performed, followed by metagenomic binning. Exopolysaccharide gene clusters were detected using a bioinformatics pipeline from a previous paper, which was improved and expanded to search for 26 additional gene clusters. Metatranscriptomic reads were recovered from multiple samples across the full-scale treatment process.

Of the 214 high- and 229 medium-quality genomes, 162 contained at least one exopolysaccharide gene cluster. The most highly expressed gene clusters were associated with production of galactoglucan, hyaluronic acid, stewartan, and emulsan. These were found in highly abundant species which are members of the polyphosphate-accumulating, glycogen-accumulating and commamox functional groups. Gene cluster expression was highest in the anoxic and aerobic stages of the system, and lowest in the anaerobic sidestream tanks.

Since several of these species are part of the core wastewater treatment species as defined by the Microbial Database for Activated Sludge, results from this study can be extrapolated globally.