Preparation Of Bacterial Cellulose Film By Gluconacetobacter Xylinus Fermentation And Its Modification

Bacterial cellulose（BC）is a microorganism-producing biopolymer with glucose as a repeating unit.Due to its unique nanostructures,high purity,high crystallinity,and biocompatibility,BC has been widely used in wound dressings,biosensors,flexible electronics,food additives,and skin care products.However,the low yield and the disequilibrium between the swelling and mechanical properties of native BC impede its further development and application.For this purpose,on the basis of medium optimization,the two-step fermentation including agitated incubation and static fermentation was constructed to improve the yield of native bacterial cellulose film（BCF）.Furthermore,chitosan oligosaccharide（COS）was in-situ added in the culture medium of two-step fermentation,and the addition protocol was optimized to explore the effect of COS on the yield,structure,and other properties of BCF.Finally,the carboxy-activatedγ-poly glutamic acid（γ-PGA）combined with the COS embedded in the BCF by covalent crosslinking to balance the swelling and the mechanical properties.The effect of the above modifications on the structural properties of BCFs was verified by loading and releasing Benzalkonium chloride（BAC）.The details are as follows:（1）The two-step fermentation for BCF production based on the component optimization:The optimal components were determined by one-factor optimization as follows:Yeast extract 14 g/L,Na₂HPO₄·12H₂O₄ g/L,glucose 70g/L,citric acid 0.5 g/L,and anhydrous magnesium sulfate 0.2 g/L.Furthermore,the relationship among BCF yield,agitation speed,and agitation time was addressed to achieve the model,Z=（-0.00102x+0.03053y-1.53378）³+（-0.00492x+0.03739y-2.43793）²（R²=0.759）.According to the model,the optimal two-step fermentation conditions were deduced as 220 rpm for 3 h（total fermentation time 10 d）,under which the top yield of BCF increased to 5.5 g/L,1.9-fold of the control in complete static fermentation.（2）In-situ addition of COS to prepare BC/COS composite film:Various polymers were in-situ embedded,and the results showed that the yield and mechanical property of BCF can be significantly improved through embedding COS.The effects of COS dosage,addition time,and addition mode on the yield,structure,and mechanical properties of BCF were further explored.It was found that BCF yield reached 9.75 g/L,and the breaking stress was 36.4 MPa,which were 1.78-fold and 9.38-fold higher than those of native BCF,respectively,while2.25%COS was added in three batches after 2 days fermentation.The main roles of COS are as follows:1)Increasing film yield by attachment and inducing the self-assembly of BCF well;2)Inhibiting G6PD activity and glucose oxidation;3)Inhibiting the formation of gluconic acid through antioxidation;4)Changing the microstructure of BCF,reducing the relaxation of BC chain,improving the mechanical properties of BCF and reducing its swelling properties.（3）Construction of BC/COS/γ-PGA composite film based on double network interpenetrating covalent crosslinking:The activatedγ-PGA was combined with the COS embedded in the BCF by covalent crosslinking to form a composite film of BC/COS/γ-PGA.The swelling and mechanical properties of the BCF were balanced by regulating the hydrogen bonds,electrostatic interactions,and amide bonds in the dual network system,the Young’s modulus was increased by more than 8 times based on maintaining 91%the swelling behavior of native BCF.Meanwhile,the loading capacity and sustained release time of BAC were significantly improved.This study attempted to remove the drawbacks of native BCF in the yield and the disequilibrium between the swelling and mechanical properties,and the findings laid a theoretical foundation for the fundamental study of BC,and would provide a referable strategy for its further development and application.