Tracking de novo protein synthesis in the activated sludge microbiome using BONCAT-FACS.

In order to ensure stable performance of engineered biotechnologies that rely on mixed microbial community systems, it is important to identify process-specific microbial traits and study their in-situ activity and responses to changing environmental conditions and system operational parameters. We used BioOrthogonal Non-Canonical Amino acid Tagging (BONCAT) in combination with Fluorescence-Activated Cell Sorting (FACS) and 16S rRNA gene amplicon sequencing to identify translationally active cells in activated sludge. We found that only a subset of the activated sludge microbiome is translationally active during the aerobic treatment phase of a full-scale sequencing batch reactor designed to enhance biological phosphorus removal from municipal wastewater. Relative abundance of amplicon sequence variants was not a reliable predictor of species activity. BONCAT-positive and -negative cells revealed a broad range of population-wide and taxa-specific translational heterogeneity. BONCAT-FACS in combination with amplicon sequencing can provide new insights into the ecophysiology of highly dynamic microbiomes in activated sludge systems.