Comammox Nitrospira Bacteria Are Dominant Ammonia Oxidizers in Mainstream Nitrification Bioreactors Emended with Sponge Carriers

Complete ammonia oxidation (i.e., comammox) is a newly discovered microbial process performed by a subset of the Nitrospira genus, and this unique microbial process has been ubiquitously detected in various wastewater treatment units. However, the operational conditions favoring comammox prevalence remain unclear. In this study, the dominance of comammox Nitrospira in four sponge biofilm reactors fed with low-strength ammonium (NH₄⁺ = 23 ± 3 mg N/L) wastewater was proved by coupling 16S rRNA gene amplicon sequencing, quantitative polymerase chain reaction (qPCR), and metagenomic sequencing. The results showed that comammox Nitrospira dominated in the nitrifying guild over canonical ammonia-oxidizing bacteria (AOB) constantly, despite the significant variation in the residual ammonium concentration (0.01-15 mg N/L) under different sets of operating conditions. This result indicates that sponge biofilms greatly favor retaining comammox Nitrospira in wastewater treatment and highlights an essential role of biomass retention in the comammox prevalence. Moreover, analyses of the assembled metagenomic sequences revealed that the retrieved amoA gene sequences affiliated with comammox Nitrospira (53.9-66.0% read counts of total amoA gene reads) were always higher than those (28.4-43.4%) related to β-proteobacterial AOB taxa. The comammox Nitrospira bacteria detected in the present biofilm systems were close to clade A Candidatus Nitrospira nitrosa.