Initial colonization advantage facilitates rapid cultivation of highly enriched planktonic anammox bacteria via active degranulation.

Anammox bacteria play a pivotal role in natural and engineered ecosystems, yet their physiological characterization remains severely limited by the lack of pure cultures. Highly enriched planktonic anammox cultures are essential for physiological studies, but conventional cultivation is extremely time-consuming (200-500 d) and hampered by uncontrolled cell aggregation and persistent non-anammox contaminants. Here, we developed a novel two-stage strategy for the rapid cultivation of planktonic anammox bacteria. Anammox granules were first subjected to magnetic-mechanical hybrid dispersion to slowly release and preliminarily enrich planktonic cells (Stage I for approximately 50 days), which were then selectively enriched in a membrane bioreactor under optimized conditions (Stage II). Within only 78 days, we obtained a 99.6% (FISH-based) planktonic culture dominated by monodisperse single cells (∼1.0 μm, stable suspension >48 h). The culture also showed exceptional characteristics, i.e., the specific anammox activity of 7.28 g-N g-VSS⁻¹ d⁻¹ (>8-fold higher than initial biomass) and an apparent maximum specific growth rate of 0.25 d⁻¹ (apparent doubling time of 2.8 d vs. 3-8 d previously reported). This strategy is rapid, simple and reproducible, requiring neither additional supplements (e.g., vitamins) nor complex purification steps (e.g., density-gradient centrifugation). The early establishment of an anammox-rich planktonic founder population during Stage I (FISH-based: 90.3% vs. 71.9% in control), together with the subsequent selective enrichment conditions, contributed to rapid proliferation and functional dominance during Stage II. This work provides a cellular material basis for deepening understanding of anammox physiology, enabling precise microbial manipulation toward more stable and highly efficient anammox processes.