Wastewater from intensive aquaculture can contain nitrogen and phosphorus that contribute to water-quality problems if they are not adequately removed. Biological treatment is widely used, but nitrogen removal and phosphorus removal are often managed as separate processes. Microorganisms capable of denitrification and polyphosphate accumulation (DPA) can potentially carry out both functions in a coordinated way, creating opportunities for more efficient treatment.
In a study published in Acta Microbiologica Sinica, researchers examined Cereibacter azotoformans ATCC 17025, an anoxygenic phototrophic bacterium associated with beneficial functions in aquaculture. They tested the strain in a laboratory model of wastewater treatment, first under anaerobic conditions and then under anoxic conditions. During the anaerobic stage, the cells released phosphate into the surrounding medium. After the system shifted to anoxic conditions, the cells took up phosphate while reducing nitrate.
By the end of the 96-hour experiment, the nitrate removal rate had reached 95%, and phosphate removal exceeded 90%. Nitrite, an intermediate in denitrification, rose temporarily during the process and later fell to below 0.2 milligrams per liter. Fluorescence microscopy also showed intracellular polyphosphate accumulation during the phosphate-uptake stage. Together, these results demonstrated a typical DPA pattern in this strain under the tested laboratory conditions.
The team then investigated the role of the NtrX/Y two-component regulatory system, which helps bacteria sense signals and adjust gene expression. When the researchers deleted ntrY, the mutant strain no longer displayed the same coordinated nitrogen-and-phosphorus removal pattern. Phosphate concentration in the medium continued to increase rather than declining, nitrate reduction was impaired, and nitrite accumulation was more pronounced. The mutant also showed a marked reduction in the production of intracellular polyhydroxyalkanoates (PHAs), energy-rich storage compounds that can support microbial metabolism. Reintroducing ntrY restored the DPA phenotype, strengthening the evidence that this gene is important for the process.
To investigate the molecular basis of this effect, the researchers used DNA affinity purification sequencing (DAP-seq) to map DNA regions bound by NtrX. The analysis identified targets that include nirK and norB/C, which encode proteins involved in denitrification, as well as phaC, which is involved in PHA synthesis. NtrX also targeted nuoD, a gene associated with the respiratory electron-transport chain. The core polyphosphate metabolism genes ppk and ppx were not identified among the direct NtrX targets in this analysis. This suggests that the NtrX/Y system may influence polyphosphate accumulation indirectly, in part by coordinating denitrification with PHA production and energy metabolism, rather than by directly controlling the central polyphosphate genes.
The findings expand the range of microorganisms known to have DPA capabilities and provide new insight into how nitrogen metabolism, carbon and energy storage, and phosphorus accumulation can be linked. The strain may be a candidate for further research on biological treatment of aquaculture wastewater. However, the results were obtained in a controlled laboratory system. Further work will be needed to evaluate performance in real aquaculture effluent, determine how operating conditions affect removal efficiency, and assess the requirements for practical application at larger scales.
This work was supported by the Natural Science Foundation of Shandong Province (ZR2024MC069), the National Natural Science Foundation of China (31800108), and the Jinan City-University Integration Development Strategic Project (JNSX2025030).