The recovery mechanism of granular sludge fragmentation and re-granulation caused by long-term high-concentration organic matter erosion in the SAD process: performance, sludge evolution, and metagenomic sequencing.

The Simultaneous Anammox and Denitrification (SAD) process effectively removes organic carbon sources, the impact of high-concentration carbon sources on the SAD process remains unclear. This study investigated the performance, sludge characteristics, microbial community correlations, and metagenomic sequencing of the SAD system under conditions of excessive organic matter exposure. The results showed that the organic matter metabolism ability of SAD granular sludge increased from 90.16 ± 1.16 % to 95.2 ± 2.3 %. The Mantel test revealed that Anaerobic Ammonium Oxidation Bacteria (AnAOB) (Candidatus_Kuenenia) were positively correlated with VSS/SS and instability coefficient, while Denitrifying Bacteria (DNB) (Truepera, Ottowia, Deniratisoma, Arenimonas) were negatively correlated with wet density, settling velocity, granule size, protein/polysaccharides (PN/PS) ratio, and the complete coefficient. Following the recovery of the SAD system, the bacterial community correlations increased, and the stability and mechanical strength of the granular sludge were enhanced. Metagenomic sequencing showed that a decrease of Quorum sensing (QS) and the increase of c-di-GMP levels led to up-regulation of exopolysaccharide and extracellular protein expression, resulting in the disintegration of SAD granular sludge. As the sludge aggregated, recombined, and re-granulated, the system up-regulated the expression of the hzs gene in AnAOB nitrogen metabolism via c-di-GMP and QS signals. It also up-regulated the expression of genes such as Dissimilatory Nitrate Reduction to Ammonium (DNRA) and potential denitrification pathways, enhancing the metabolism of AnAOB and symbiotic bacteria.