Colony formation is crucial for the development of Microcystis blooms, which pose a significant ecological challenge on a global scale. Previous studies have demonstrated that bound extracellular polymeric substances (EPS) play a vital role in the colony formation process. However, the influence of the coexisting soluble EPS (S-EPS) on Microcystis colony formation remains unclear. This study investigated the effect of S-EPS on the colony formation of Microcystis induced by high concentrations of Ca2+. The results indicated that the combined treatment of S-EPS (100 mg/L) and Ca2+ (200 mg/L) resulted in loosely bound Microcystis colonies, in contrast to the dense colonies induced by 200 mg/L Ca2+. In contrast, no colonies were formed when the Microcystis cells were treated with S-EPS alone. In addition, the incorporation of 100 mg/L of S-EPS into 200 mg/L Ca2+-treated Microcystis cells reduced the cell zeta potential by 2.09 mV and decreased surface hydrophobicity, as indicated by a 1.60° increase in the water contact angle that increased the interaction energy among Microcystis cells. The energy barrier increased from 293.32 KT in the 200 mg/L Ca2+ treatment groups to 538.10 KT following the introduction of 100 mg/L S-EPS, indicating a reduced propensity for colony formation after S-EPS addition. Further studies indicated that Ca2+ in the inoculum exhibited a strong binding capacity with the O
H groups of polysaccharides (KM = 0.032 ± 0.004), C
C stretching of aromatics (KM = 0.022 ± 0.003) and tryptophan-like proteins (KM = 0.013 ± 0.001) from S-EPS. This resulted in extensive networks and lower free Ca2+ concentrations that reduced the bound EPS secretion by approximately 0.15 pg/cell compared to that with a Ca2+ concentration of 200 mg/L. This study provides new insights into the mechanism of colony formation in Microcystis.
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