The selection and optimization of carbon and nitrogen sources are essential for enhancing pullulan production in Aureobasidium pullulans. In this study, combinations of carbon (sucrose, fructose, glucose) and nitrogen sources ((NH4)2SO4, urea, NaNO3) were screened, where sucrose and NaNO3 offered the highest pullulan yield (9.33 g L-1). Plackett-Burman design of experiment identified KH2PO4, NaCl, and sucrose as significant factors, which were further optimized using a central composite design. A hyperparameter-optimized artificial neural network (ANN) model with a 3-6-2-1 architecture demonstrated superior predictive accuracy (R2: 0.96) and generalizability (R2CV: 0.74) over a reduced quadratic model (R2: 0.82). The predicted pullulan yield (31.9 g L-1) under ANN model optimized conditions (sucrose: 79.9 g L-1, KH2PO4: 0.25 g L-1, NaCl: 4.3 g L-1) closely matched with the observed yield (30.17 g L-1), while quadratic model showed a significant deviation (39.7 g L-1 vs. 21.0 g L-1), highlighting the reliability of the ANN model.
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