Comammox Nitrospira are the dominant ammonia oxidizers in a mainstream low dissolved oxygen nitrification reactor

Recent findings show that a subset of bacteria affiliated with Nitrospira, a genus known for its importance in nitrite oxidation for biological nutrient removal applications, are capable of complete ammonia oxidation (comammox) to nitrate. Early reports suggested that they were absent or present in low abundance in most activated sludge processes, and thus likely functionally irrelevant. Here we show the accumulation of comammox Nitrospira in a nitrifying sequencing batch reactor operated at low dissolved oxygen (DO) concentrations. Actual mainstream wastewater was used as influent after primary settling and an upstream pre-treatment process for carbon and phosphorus removal. The ammonia removal rate was stable and exceeded that of the treatment plant's parallel full-scale high DO nitrifying activated sludge reactor. 16S rRNA gene sequencing showed a steady accumulation of Nitrospira to 53% total abundance and a decline in conventional ammonia oxidizing bacteria to <1% total abundance over 400 + days of operation. After ruling out other known ammonia oxidizers, qPCR confirmed the accumulation of comammox Nitrospira beginning around day 200, to eventually comprise 94% of all detected amoA and 4% of total bacteria by day 407. Quantitative fluorescence in-situ hybridization confirmed the increasing trend and high relative abundance of Nitrospira. These results demonstrate that comammox can be metabolically relevant to nitrogen transformation in wastewater treatment, and can even dominate the ammonia oxidizing community. Our results suggest that comammox may be an important functional group in energy efficient nitrification systems designed to operate at low DO levels.