Anammox Granule Enlargement by Heterogenous Granule Self-assembly

Expansion in the size is an indispensable stage in the granular sludge life cycle, but little attention has been payed to the enlargement mechanism of granular sludge. Here, we propose a novel anammox granule enlargement mechanism by the self-assembly of heterogenous granules. Two different colors of anammox granules, dark-red granules (DR-Granules) and bright-red granules (BR-Granules) were found in an expanded granular sludge bed reactor. These two heterogenous granules were not isolated but were assembled into granules with a larger DR-Granule in the center and many smaller BR-Granules aggregated on the surface, increasing the overall granular size. Their physiochemical characteristics in terms of EPS, adherence, rheological properties, and microbial compositions, were identified and compared to elucidate the interaction between the different colors of granules. The BR-Granules created 92% more extracellular polymeric substances than the DR-Granules. This material blocked the passage of gas and substrate, leading to BR-Granules smaller size and a yield stress approximately 48% lower than that of the DR-Granules. Nevertheless, the BR-Granules had compact extracellular protein secondary structures and a high adherence rate to the surface of the DR-Granules, upon which they formed a compact adhered layer. These unique features enabled them to directionally adhere to DR-Granules in the core, that is, two heterogenous colors of granules self-assembled into large anammox granules. The enlargement mechanism was further supported by the abundance of K-strategy Ca. Kuenenia in the DR-Granules (inner layer) being higher than in the BR-Granules (outer layer; 2.9 ± 0.4% vs. 0.4 ± 0.1%; p = 0.0003) and by visualized confirmation that the larger BR-Granules wrapped around smaller DR-Granules inside. This demonstrates that heterogenous anammox granules actively self-assemble into large granules, which is an important step in the lifecycle of anammox granules.