Evaluation of genomic DNA preparation for sequencing translationally active subcommunities of activated sludge microbiome using BONCAT-FACS + 16S rRNA gene sequencing

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. Different genomic DNA preparation approaches were evaluated in this study including cell lysis, DNA extraction, and multiple displacement amplification for sequencing low biomass samples after BONCAT-FACS. 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.