Response of extracellular polymeric substances to high load shock in an aerobic granular sludge reactor performing simultaneous heterotrophic nitrification-aerobic denitrification

Extracellular polymeric substances (EPS) are secreted by microbes in aerobic granular sludge (AGS) to resist environmental stresses. However, the response mechanism of EPS to high load shock remains incompletely understood due to a knowledge gap regarding the correlations between EPS components, influent loads, and AGS performance. In this study, the properties of EPS and their role in maintaining AGS stability were investigated in detail as influent loads increased from Stage 1 (6.5 kg COD/m 3 /d and 0.255 kg NH 4 + -N/m 3 /d) to Stage 4 (10.1 kg COD/m 3 /d and 0.455 kg NH 4 + -N/m 3 /d). AGS demonstrated excellent removal efficiencies for COD (> 96.5%) and NH 4 + -N (> 82%). From Stage 1 to Stage 3, a higher proportion of proteins (PN) were found in tightly bound-EPS (TB-EPS), both PN content and PN/PS ratio increased. In Stage 4, there was a significant increase in LB/TB ratio along with polysaccharide (PS) content surpassing PN content within loosely bound-EPS (LB-EPS), accompanied by the increase of flocs; these observations potentially indicated impending instability of aerobic granules. Additionally, three-dimensional excitation–emission matrix (3D-EEM) revealed that hydrophobic proteins such as tyrosine and tryptophan contributed to AGS stability while humic acid-like substances present within EPS facilitated floc re-granulation. Therefore, both the contents and compositions of EPS changed in response to stress induced by influent COD and NH 4 + -N load. The TB-EPS, and PN and humic acid-like substances within EPS played crucial roles in maintaining AGS stability.