Spatiotemporal Assembly and Immigration of Heterotrophic and Anammox Bacteria Allow a Robust Synergy for High-Rate Nitrogen Removal

The novel partial denitrification-driven anammox (PD/A) is an energy-efficient method for nitrogen removal from wastewater. However, its stability and efficiency are impeded by the competition between heterotrophic denitrifying bacteria and relatively slow-growing anammox bacteria. In this study, a PD/A granular sludge system was developed, which achieved a nitrogen removal efficiency of 94% with 98% anammox contribution, even as the temperature dropped to 9.6 °C. Analysis of bacterial activity in aggregates of different sizes revealed that the largest granules (>2.0 mm) exhibited the highest anammox activity, 2.8 times that of flocs (<0.2 mm), while the flocs showed significantly higher nitrite production rates of PD, more than six times that of the largest granules. Interestingly, fluorescent in situ hybridization (FISH) combined with confocal laser scanning microscopy (CLSM) revealed a nest-shaped structure of PD/A granules. The Thauera genus, a key contributor to PD, was highly enriched at the outer edge, providing substrate nitrite for anammox bacteria inside the granules. As temperature decreased, the flocs transformed into small granules to efficiently retain anammox bacteria. This study provides multidimensional insights into the spatiotemporal assembly and immigration of heterotrophic and autotrophic bacteria for stable and high-rate nitrogen removal.