Seasonal dynamics of the activated sludge microbiome in sequencing batch reactors, assessed using 16s rRNA transcript amplicon sequencing

Activated sludge is comprised of diverse microorganisms which remediate wastewater. Previous research has characterized activated sludge using 16S rRNA gene amplicon sequencing, which can help to address questions on the relative abundance of microorganisms. In this study, we used 16S rRNA transcript sequencing in order to characterize "active" populations (via protein synthesis potential) and gain a deeper understanding of microbial activity patterns within activated sludge. Seasonal abundances of individual populations in activated sludge change over time, yet a persistent group of core microorganisms remains throughout the year which are traditionally classified on presence or absence without monitoring of their activity or growth. The goal of this study was to further our understanding of how the activated sludge microbiome changes between seasons with respect to population abundance, activity, and growth. Triplicate sequencing batch reactors were sampled at 10-min intervals throughout reaction cycles during all four seasons. We quantified the gene and transcript copy numbers of 16S rRNA amplicons using realtime PCR and sequenced the products to reveal community abundance and activity changes. We identified 108 operational taxonomic units (OTUs) with stable abundance, activity, and growth throughout the year. Nonproliferating OTUs were commonly human health related, while OTUs that showed seasonal abundance changes have previously been identified as being associated with floc formation and bulking. We observed significant differences in 16S rRNA transcript copy numbers, particularly at lower temperatures in winter and spring. The study provides an analysis of the seasonal dynamics of microbial activity variations in activated sludge based on quantifying and sequencing 16S rRNA transcripts. IMPORTANCE Sequencing batch reactors are a common design for wastewater treatment plants, particularly in smaller municipalities, due to their low footprint and ease of operations. However, like for most treatment plants in temperate/continental climates, the microbial community involved in water treatment is highly seasonal and its biological processes can be sensitive to cold temperatures. The seasonality of these microbial communities has been explored primarily in conventional treatment plants and not in sequencing batch reactors. Furthermore, most studies often only address which organisms are present. However, the activated sludge microbial community is very diverse, and it is often hard to discern which organisms are active and which organisms are simply present. In this study, we applied additional sequencing techniques to also address the issues of which organisms are active and which organisms are growing. By addressing these issues, we gained new insights into seasonal microbial populations dynamics and activity patterns affecting wastewater treatment.