Abstract
<jats:p>Bacterial cellulose (BC) is a highly pure, crystalline, and nanofibrillar polysaccharide with broad potential in biomaterials, packaging, and biomedical applications, but its static-culture production remains limited by low yield and inconsistent process reporting. This study used a two-stage design-of experiments framework to optimize BC biosynthesis from a kombucha-derived mixed culture and to evaluate how yield normalization affects the interpretation of static fermentation performance. A definitive screening design first assessed sucrose concentration, tea concentration, initial pH, culture volume, air-liquid interfacial area, and fermentation duration. Interfacial area, tea concentration, and fermentation duration were identified as the dominant factors controlling BC dry mass, whereas culture volume, sucrose concentration, and initial pH were not significant within the investigated ranges. These factors were then optimized using a CCD-inspired response surface design. The fitted quadratic model showed strong explanatory and predictive performance and identified the highest BC dry mass within the experimental domain at high interfacial area, elevated tea concentration, and extended fermentation time. Independent validation experiments confirmed the robustness of this high-yield region. FTIR, XRD, and SEM analyses verified that optimized production increased BC mass without detectable changes in cellulose I chemical structure, crystallinity, or nanofibrillar morphology. Because BC formation in static culture is governed by the aerobic air-liquid interface, volumetric yield alone can be misleading when used without consideration of surface area, fermentation time, and reactor geometry. We therefore 2 recommend reporting dry mass together with surface-area-normalized yield and areal productivity, while retaining volumetric yield for comparison among studies employing similar vessel geometries and for continuity with the existing literature.</jats:p>