Three-dimensional excitation emission matrix (EEM) fluorescence spectroscopy and gel-permeating chromatography (GPC) were employed to characterize the extracellular polymeric substances (EPS) in aerobic granulation. EPS matrix in this study was stratified into four fractions: (1) supernatant, (2) slime, (3) loosely bound EPS (LB-EPS), and (4) tightly bound EPS (TB-EPS). The results showed that the dissolved organic carbon was mainly distributed in TB-EPS fraction, and increased with increasing the operating time. The supernatant, slime, and LB-EPS fractions exhibited four fluorescence peaks, an autochthonous signature, unimodal MW distribution and lower molecular weight (MW) (3 < log [MW]<5), whereas the TB-EPS fraction only had two peaks, an allochthonous signature, multiple peaks and higher MW (5 < log [MW]<7). It was deemed that the formation of aerobic granules was correlated with the accumulation of proteins in the TB-EPS fraction. EEM spectroscopy and GPC profiles could be used as appropriate and effective methods to characterize the EPS in aerobic granulation from a micro-view level.

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