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Biodemulsifiers are environmentally friendly agents used in recycling oil or purifying water from emulsion, yet the demulsifying feature of cell-surface composition remains unclear. In this study, potentiometric titration, attenuated total reflectance-Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry were combined to characterize cell-surface chemical composition of demulsifying strain Alcaligenes sp. S-XJ-1 cultivated with different carbon sources. Cells cultivated with alkane contained abundant elemental nitrogen and basic functional groups, indicating that their surface was rich in proteins or peptides, which contributed to their highest demulsifying efficiency. For cells cultivated with fatty acid ester, the relatively abundant surface lipid contributed to a 50% demulsification ratio owing to the presence of more acidic functional group. The cells cultivated with glucose exhibited a high oxygen concentration (O/C ~0.28), which indicated the presence of more polysaccharides on the cell surface. This induced the lowest demulsification ratio of 30%. It can be concluded that cell surface-associated proteins or lipids other than the polysaccharide of the demulsifying strain played a positive role in the demulsification activity. In addition, the cell-surface oligoglutamate compounds identified in situ were crucial to the demulsifying capability. [ABSTRACT FROM AUTHOR]