Nitrogen starvation coupled with extended anoxic time induces rapid growth of ammonia oxidizers and enrichment of anammox bacteria in low-ammonium wastewater.

Partial nitrification-anammox (PNA) is an energy-efficient autotrophic nitrogen removal process, yet its application to low-ammonium wastewater remains challenging due to the difficulty in selectively suppressing nitrite-oxidizing bacteria (NOB) while maintaining ammonia-oxidizing bacteria (AOB) and anaerobic ammonium oxidation (anammox) bacteria. Here, we developed a chemical-free strategy based on sequential nitrogen starvation and extended anoxic operation to successfully establish one-stage PNA. Long-term reactor operation and cyclic nitrogen conversion performance demonstrated that repeated nitrogen starvation shifted the system from complete nitrification toward partial nitrification, while subsequent extension of the anoxic period promoted anammox activity and finally reached PNA performance. Batch activity tests revealed differential kinetic responses between nitrifiers, with faster AOB activity recovery rates, whereas repeated nitrogen starvation caused greater activity declines in NOB. Community-level microbial composition and functional gene analyses revealed a clear transition from a Nitrospira-dominated community toward an Nitrosomonas-Candidatus Brocadia consortium, accompanied by rapid increase of amoA gene abundance following starvation and enrichment of anammox populations. Genome-resolved metagenomics further revealed distinct succession trajectories among nitrifier populations, with some Nitrospira lineages persisting throughout the operation. Comparative genomic analyses incorporating global wastewater-treatment-plant metagenomes demonstrated that Nitrosomonas possessed greater genomic potential for reactive oxygen species (ROS) defense and redox maintenance than Nitrospira, suggesting a potential mechanism underlying their differential resilience to metabolic stress induced by the imbalance between nitrogen availability and oxygen supply. Nitrogen starvation also selectively reduced non-denitrifying heterotrophic organisms and enhanced the genomic potential for denitrification. Overall, this study demonstrates that a combined nitrogen starvation and oxygen availability strategy can effectively drive PNA establishment in low-strength ammonium wastewater, and provides new insights into stress-induced recovery and ecological selection of nitrifying communities.