Integrating fed‐batch pulse feeding and flux balance analysis for <scp>PHB</scp> production by <i>Cupriavidus necator</i> from cassava‐based dextrose

Abstract BACKGROUND The growing demand for biodegradable polymers has positioned polyhydroxybutyrate (PHB) as a sustainable alternative to petrochemical plastics. Microbial synthesis from renewable, low‐cost substrates such as cassava starch‐derived dextrose offers a promising route, but feeding strategies and metabolic understanding remain critical. This study aimed to evaluate the effect of fed‐batch pulse feeding on PHB production by Cupriavidus necator DSM 428 and to integrate experimental results with flux balance analysis (FBA). RESULTS Three pulse‐feeding strategies were assessed. Two pulses containing carbon and nitrogen at 24 and 48 h (C/N ratio 73) led to 33.55% (w/w) PHB accumulation. A single dextrose‐only pulse at 48 h led to 25.12% (w/w), while delaying this pulse to 60 h increased PHB accumulation to 50.40% (w/w). Feeding at the onset of stationary phase enhanced biomass growth but reduced PHB storage, whereas late feeding favored polymer accumulation, likely due to nitrogen limitation. PHB was characterized by X‐ray diffraction, Fourier transform infrared spectroscopy, scanning electron microscopy, energy‐dispersive X‐ray spectroscopy, thermogravimetric analysis, and differential scanning calorimetry, confirming its expected structural and thermal properties. FBA with i CNH2025A showed strong correlation with experimental data, accurately predicting growth and carbon flux redirection toward PHB synthesis. CONCLUSION The integration of fed‐batch pulse feeding with FBA provides new insights into the metabolic and process factors driving PHB accumulation from cassava‐based substrates. The results highlight the importance of feeding timing for optimizing yields and demonstrate the value of metabolic modeling in guiding sustainable bioprocess design. © 2026 Society of Chemical Industry (SCI).