Exploring the effects of operational mode and microbial interactions on bacterial community assembly in a one-stage partial-nitritation anammox reactor using integrated multi-omics

Background: The metabolic capacities of anammox bacteria and associated microbial community interactions in partial-nitritation anammox (PNA) reactors have received considerable attention for their crucial roles in energy-efficient nitrogen removal from wastewater. However, a comprehensive understanding of how abiotic and biotic factors shape bacterial community assembly in PNA reactors is not well reported. Results: Here, we used integrated multi-omics (i.e., high-throughput 16S rRNA gene, metagenomic, metatranscriptomic, and metaproteomic sequencing) to reveal how abiotic and biotic factors shape the bacterial community assembly in a lab-scale one-stage PNA reactor treating synthetic wastewater. Analysis results of amplicon sequences (16S rRNA gene) from a time-series revealed distinct relative abundance patterns of the key autotrophic bacteria, i.e., anammox bacteria and ammonia-oxidizing bacteria (AOB), and the associated heterotrophic populations in the seed sludge and the sludge at the new stable state after deterioration. Using shotgun metagenomic sequences of anammox sludge, we recovered 58 metagenome-assembled genomes (MAGs), including 3 MAGs of anammox bacteria and 3 MAGs of AOB. The integrated metagenomic, metatranscriptomic, and metaproteomic data revealed that nitrogen metabolism is the most active process in the studied PNA reactor. The abundant heterotrophs contribute to the reduction of nitrate to nitrite/ammonium for autotrophic bacteria (anammox bacteria and AOB). Genomic and transcriptomic data revealed that the preference for electron donors of the dominant heterotrophs in different bacterial assemblages (seed and new stable state) varied along with the shift in anammox bacteria that have different metabolic features in terms of EPS composition. Notably, the most abundant heterotrophic bacteria in the reactor were more auxotrophic than the less abundant heterotrophs, regarding the syntheses of amino acids and vitamins. In addition, one of the abundant bacteria observed in the bacterial community exhibited highly transcribed secretion systems (type VI). Conclusions: These findings provide the first insight that the bacterial communities in the PNA reactor are defined by not only abiotic factors (operating mode) but also metabolic interactions, such as nitrogen metabolism, exchange of electron donors, and auxotrophies.