16S rRNA Gene Amplicon Sequencing of Mainstream Partial Nitrification Activated Sludge Bioreactors

Side-stream treatment of activated sludge (AS) with free-ammonia (FA) has been proposed as a strategy to repress and wash-out (i.e. out-select) nitrite oxidizing bacteria (NOB) for attaining stable mainstream partial nitritation (PN), a critical component of energy-efficient biological nitrogen removal processes. The objective of this study was to assess the stability of AS treatment with FA as an NOB out-selection strategy for mainstream PN. We hypothesized that the metabolic and physiological diversity of NOB populations in AS could confer their resilience to FA-treatment. We applied time-series 16S rRNA gene amplicon sequencing to elucidate the impact of FA-based AS treatment on the structure of the AS microbiome in parallel experimental and control sequencing batch reactors (SBRs). We also applied bioorthogonal non-canonical amino acid tagging (BONCAT) coupled with fluorescence activated cell sorting (FACS) and 16S rRNA gene amplicon sequencing to investigate changes in the translational activity of nitrifying populations throughout sidestream exposure to FA.