Insight into the effect of nitrate on AGS granulation: Granular characteristics, microbial community and metabolomics response

As a promising wastewater treatment technology, aerobic granular sludge (AGS) process is still hindered by slow granule formation and easy disintegration in the application. While nitrate, one of the target pollutants in wastewater, showed a potential effect on AGS granulation process. Herein, this study attempted to reveal the role of nitrate in AGS granulation. By adding exogenous nitrate (10 mg L-1), the AGS formation was markedly improved and accomplished at 63 d, while the control group achieved AGS formation at 87 d. However, a disintegration was observed under a long-term nitrate feeding. A positive correlation was observed among granule size, extracellular polymeric substances (EPS) and intracellular c-di-GMP level in both formation and disintegration phases. The subsequent static biofilm assays indicated that nitrate might upregulate c-di-GMP via denitrification-derived NO, and c-di-GMP further upregulated EPS, thereby promoting AGS formation. However, excessive NO probably caused disintegration by downregulating c-di-GMP and EPS. Microbial community showed that nitrate favored the enrichment of denitrifiers and EPS producing microbes, which were responsible for the regulation of NO, c-di-GMP and EPS. Metabolomics analysis showed that amino acid metabolism was the most affected metabolism by nitrate. Some amino acids, such as Arg, His and Asp, were upregulated in the granule formation phase and downregulated in the disintegration phase, indicating the potential contribution to EPS biosynthesis. This study provides metabolic insight into how nitrate promotes/inhibits granulation, which may contribute to unwrapping the mystery of granulation and overcoming the limitations of AGS application.