Seasonal Variations In The Activated Sludge Microbiome With Respect To Seasonal Nitrification Failure

University of Minnesota Ph.D. dissertation. July 2020. Major: Civil Engineering. Advisor: Sebastian Behrens. 1 computer file (PDF); xiii, 232 pages.Activated sludge consists of a diverse microbial community that is used by wastewater engineers to metabolize excessive nutrients in domestic wastewater so that these excessive nutrients do not impact downstream waters. While most biological contaminant removal processes, such as carbon (measured as Biological Oxygen Demand) and phosphorous removal are performed consistently year-round, nitrification performance significantly declines in cold temperatures. The seasonal decline in nitrification performance is known as seasonal nitrification failure. To understand seasonal nitrification failure, this thesis analyzed triplicate, full-scale, sequencing batch reactors throughout several years to investigate seasonal variations in the activated sludge microbiome with respect to community composition (16S rRNA gene), the metabolically active composition (16S rRNA transcript), and expression of amoA (ammonia monooxygenase) which is a key-nitrification functional gene. There were 114 OTUs (operational taxonomic units), which were consistently present in all three reactors, every week, for an entire year and together comprise 74.3% - 84.0% of the entire community. The changes in abundances of these OTUs and other seasonally present OTUs make each season’s community significantly distinct from each other. The community composition was also significantly distinct from the protein-synthesis composition throughout the entire year. While the entire activated sludge community and protein-synthesis compositions fluctuated, the ammonia-oxidizing community was at a constant abundance throughout the year based on tracking known ammonia oxidizers and the amoA functional gene despite seasonal nitrification failure. While the amoA transcripts declined with the seasonally cold temperatures, which explain the seasonal nitrification failure’s decline in activity, the known-ammonia oxidizer protein-synthesis potential measured by Nitrosomonas sp. 16S rRNA transcripts did not significantly decline with temperature. This suggests there are other metabolic activities performed by the known ammonia oxidizing community to maintain stable community abundance and protein synthesis potential when ammonia oxidization is no longer the most thermodynamically favorable metabolism. This result changes the narrative that seasonal nitrification failure occurs due to declining abundances of ammonia oxidizing organisms in cold temperatures, and instead provides insight as to how amoA expression seasonally changes with the complex and seasonally dynamic microbial ecology of the activated sludge community. Additionally, this research provides the most comprehensive baseline of the activated sludge communities seasonal composition, protein-synthesis potential and amoA expression to date. Future researchers can use these results to investigate specific highlighted seasonally variant OTUs which may influent the activated sludge microbiome, as well as explore the additional roles known ammonia oxidizers play in this complex microbial system