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The mechanism of self-flocculation remains unclear, partially impeding its efficiency enhancement and commercial application of microalgae-based municipal wastewater (MW) bioremediation technology. This study revealed the contributions of exoproteins [PN, proteins in extracellular polymeric substances (EPS)] to the separation of indigenous microalgae from treated MW. Compared to the low light intensity group, the high light intensity (HL) group produced Chlorella sp. with 4.3-fold higher self-flocculation efficiencies (SE). This was attributed to the enriched biological functions and positional rearrangement of increased PN within 2.9-fold higher EPS. Specifically, a total of 75 PN was over-expressed in the HL group among the 129 PN identified through label-free proteomics. The algal cell-adhesion molecules (Algal-CAMs) and metal-ion-binding PN were demonstrated as two dominant contributors promoting cell adhesion and bridging, through function prediction based on the contained domains. The modeled 3D structure showed that Algal-CAMs presented less hydrophilic α-helix abundance and were distributed in the outermost position of the EPS matrix, further facilitating microalgal separation. Moreover, the 10.1% lower hydrophily degree value, negative interfacial free energy (−19.5 mJ/m2), and 6.8-fold lower energy barrier between cells also supported the observed higher SE. This finding is expected to further fill the knowledge gap of the role of PN in microalgal self-flocculation and promote the development of biomass recovery from the microalgae-wastewater system.

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