Purpose: To improve the production of a novel exopolysaccharide (EPS) by Bacillus mucilaginosus CGMCC5766.

Methods: The culture medium for production of EPS was optimized using statistical experiment design. Sucrose, CaCO₃ and K₂HPO₄ were found to be the key factors based on the results obtained from Plackett–Burman experimental design. The optimal concentration range of the three factors was examined by the steepest ascent path, and then the optimum concentration was further investigated through response surface methodology. Validation experiments were also carried out to establish the adequacy and accuracy of the model obtained.

Results: Under optimal conditions (sucrose 61.31 g/l, CaCO₃ 5.06 g/l and K₂HPO₄ 0.17 g/l), EPS concentration achieved 22.04 g/l, which was about 1.6-fold increase compared with that using the original medium. EPS concentration in 5-l fermenter reached 23.46 ± 0.76 g/l.

Conclusion: The optimization medium is considered fundamental and useful for the development of B. mucilaginosus CGMCC5766 cultivation process for efficient production of EPS on a large scale.