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Abstract. Bacterial cellulose (BC), due to its high porosity, high tensile strength, biocompatibility and crystal structure, can be used as a value-added product in the pharmaceutical, food, and processing industries. Studies have been conducted aimed at enhancing BC production and physical properties. Among all the existing methods, polysaccharide addition is one of the most effective ways to increase the production and to alter the mechanical properties of BC. Agitated fermentation, compared to a conventional static cultivation method, is also reported to enhance the BC production. However, there is a lack of study regarding combining agitated fermentation and polysaccharide additive. Moreover, it might not be practical to use polysaccharides directly in industrial production due to cost considerations. This study evaluated an agitated fermentation with pure pullulan additives and a co-culturing system in which the BC-producing microorganisms (Gluconacetobacter hansenii) grow together with Aureobasidium pullulans, a fungus that produces pullulan as exopolysaccharide (EPS). The study included shake-flask studies, fermentation optimization using response surface methodology (RSM). The research has shown a maximum of 4.5-fold enhancement in the production of BC with pure pullulan addition and a maximum of 6-fold increment for Young‘s modulus with pullulan addition and co-culturing. The study provided a more in-depth understanding of the performance of G. hansenii in agitated and co-culture fermentation systems and could lead to further studies aimed at producing better quality BC products for a variety of applications.