Wastewater treatment plants in temperate climate zones frequently undergo seasonal nitrification failure in the winter month yet maintain removal efficiency for other contaminants. We tested the hypothesis that nitrification failure can be correlated to shifts in the nitrifying microbial community. We monitored three parallel, full-scale sequencing batch reactors over the course of a year with respect to reactor performance, microbial community composition via 16S rRNA gene amplicon sequencing, and functional gene abundance using qPCR. All reactors demonstrated similar changes to their core microbiome, and only subtle variations among seasonal and transient taxa. We observed a decrease in species richness during the winter, with a slow recovery of the activated sludge community during spring. Despite the change in nitrification performance, ammonia monooxygenase gene abundances remained constant throughout the year, as did the relative sequence abundance of Nitrosomonadacae. This suggests that nitrification failure at colder temperatures might result from different reaction kinetics of nitrifying taxa, or that other organisms with strong seasonal shifts in population abundance, e.g. an uncultured lineage of Saprospiraceae, affect plant performance in the winter. This research is a comprehensive analysis of the seasonal microbial community dynamics in triplicate full-scale sequencing batch reactors and ultimately strengthens our basic understanding of the microbial ecology of activated sludge communities by revealing seasonal succession patterns of individual taxa that correlate with nutrient removal efficiency.

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 use 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 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 in 10-minute intervals throughout reaction cycles during all four seasons. We quantified the gene and transcript copy numbers of 16S rRNA amplicons using real-time PCR and sequenced the products to reveal community abundance and activity changes. We identified 108 OTUs with stable abundance, activity, and growth throughout the year. Non-proliferating 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 most treatment plants in temperate/continental climates, the microbial community involved in water treatment is highly seasonal and their biological processes can be sensitive to cold temperatures. The seasonality of these microbial communities has been explored primarily in conventional treatment plants, but not in sequencing batch reactors. Furthermore, most studies often only address “which organisms are present?”. However, the activated sludge microbial community is very diverse that it is often hard to discern which organisms are “active” and which organisms are simply “present”. In this study we are applying additional sequencing techniques to also address “which organisms are active?” and “which organisms are growing?” By addressing these three questions, we gain new insights into seasonal microbial populations dynamics and activity patterns affecting wastewater treatment.

The phylum Chloroflexota is ubiquitous; however, its biology and evolution are poorly understood due to limited cultivability. Here, we isolated two motile, thermophilic bacteria from hot spring sediments belonging to the class Dehalococcoidia within the phylum Chloroflexota. A combination of cryo-electron tomography, exometabolomics, and cultivation experiments using stable isotopes of carbon revealed three unusual traits: flagellar motility; a peptidoglycan-containing cell envelope; and heterotrophic activity on aromatics and plant-associated compounds. Although these traits are unusual among cultivated Chloroflexota and Dehalococcoidia, ancestral character state reconstructions showed flagellar motility and peptidoglycan-containing cell envelopes were ancestral within the Dehalococcoidia and subsequently lost prior to a major adaptive radiation of Dehalococcoidia into marine environments. This evolutionary past explains the widespread genomic potential to degrade terrestrial organic matter among marine Dehalococcoidia and raises new questions about the timing and selective forces driving their successful niche expansion into the global oceans.

Although the phylum Chloroflexota is ubiquitous, its biology and evolution are poorly understood due to limited cultivability. Here, we isolated two motile, thermophilic bacteria from hot spring sediments belonging to the genus Tepidiforma and class Dehalococcoidia within the phylum Chloroflexota. A combination of cryo-electron tomography, exometabolomics, and cultivation experiments using stable isotopes of carbon revealed three unusual traits: flagellar motility, a peptidoglycan-containing cell envelope, and heterotrophic activity on aromatics and plant-associated compounds. Outside of this genus, flagellar motility has not been observed in Chloroflexota, and peptidoglycan-containing cell envelopes have not been described in Dehalococcoidia. Although these traits are unusual among cultivated Chloroflexota and Dehalococcoidia, ancestral character state reconstructions showed flagellar motility and peptidoglycan-containing cell envelopes were ancestral within the Dehalococcoidia, and subsequently lost prior to a major adaptive radiation of Dehalococcoidia into marine environments. However, despite the predominantly vertical evolutionary histories of flagellar motility and peptidoglycan biosynthesis, the evolution of enzymes for degradation of aromatics and plant-associated compounds was predominantly horizontal and complex. Together, the presence of these unusual traits in Dehalococcoidia and their evolutionary histories raise new questions about the timing and selective forces driving their successful niche expansion into global oceans.

The diverse microbial community of activated sludge used in biological treatment systems exhibits dynamic seasonal shifts in community composition and activity. Many wastewater treatment plants in temperate/continental climates experience seasonal cold temperature nitrification failure. “Seasonal nitrification failure” is the discharge of elevated concentrations of ammonia (greater than 4 mg/liter) with treated wastewater during the winter (influent wastewater temperatures below 13°C).