AbstractIn this work, stale bread and waste apple pulp were used as feedstocks for the production of bacterial cellulose (BC). A glucose-rich solution was prepared from stale bread by dilute acid hydrolysis, while an extract comprising fructose and glucose was obtained from the waste apple pulp, which was used for cultivating Komagataeibacter xylinus DSM 2004, either as sole feedstocks or supplemented with Hestrin-Schramm medium. Supplementation significantly improved BC production: 3.38 ± 0.09 g/L for waste apple pulp extract and 2.07 ± 0.22 g/L for stale bread hydrolysate. There was no significant impact on BC chemical structure or fiber diameter, but the biopolymer produced from waste apple pulp extract had slightly higher crystallinity (CI = 59–69%) and lower thermal degradation temperature (Tdeg = 341–350 ℃) than that of the stale bread hydrolysate (CI = 55%; Tdeg = 316–320 ℃). Moreover, supplementation of the waste apple pulp extract led to the preparation of thicker membranes, with higher Young’s modulus, tension, and deformation at break but lower water uptake capacity and lower permeability to O2 and CO2. These results show that waste apple pulp and stale bread are suitable feedstocks for BC production and the cultivation conditions can be adjusted for tailoring the biopolymer’s mechanical and barrier properties to suit different applications.