The objective of this study was to investigate the effects of the cultivation time, temperature, and pH value on the yield and composition of extracellular polymeric substances (EPS) from Enterobacter sp. FM-1 (FM-1) and to analyze the Pb 2+ adsorption behavior of soluble EPS (S-EPS), loosely bound EPS (LB-EPS), and tightly bound EPS (TB-EPS). Maximum EPS production was obtained when the cultivation time, temperature, and pH value were 24 h, 30 °C, and 8.0, respectively. The main components of EPS were proteins, polysaccharides, and nucleic acids, but the different EPS types contained different proportions and specific components. The Pb 2+ adsorption capacity of LB-EPS was 2.23 and 1.50 times higher than that of S-EPS and TB-EPS, respectively. After Pb 2+ adsorption by LB-EPS, the pH value of the reaction system decreased to the lowest of 5.23, which indicated that LB-EPS contained more functional groups that could release H + , which will help to better adsorb Pb 2+ through ion exchange. The three-dimensional excitation–emission matrix fluorescence spectroscopy (3D-EEM) analysis showed that the fluorescence intensity of tryptophan-containing substances decreased by 85.5% after Pb 2+ adsorption by LB-EPS, which indicated the complexation of tryptophan-containing substances with Pb 2+ . Fourier transform infrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS) O spectra indicated that the C=O peak from protein amide I of tryptophan-containing substances in LB-EPS was mainly responsible for the complexation of Pb 2+ . After the adsorption of Pb 2+ , the proportion of the C=O peak in LB-EPS increased by 33.89%, indicating that the complexation of LB-EPS with Pb 2+ was mainly attributed to the O atom in the C=O terminus of protein amide I.

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