Polyhydroxyalkanoates (PHA) are promising biodegradable biopolymers, yet their industrial production is strongly limited by downstream processing requirements. Efficient recovery of intracellular PHA typically requires cell disruption followed by selective removal of non-PHA cell material (NPCM). This study investigated a combined mechanical–enzymatic strategy for PHA purification using Cupriavidus necator biomass. Mechanical disruption was performed in a cyclic stirred bead mill to evaluate the effects of bead size and circulation velocity on disruption behaviour. Smaller beads consistently achieved higher disruption rates, indicating more efficient shear transfer and collision frequency, while the kinetic model remained applicable across all tested conditions. Enzymatic post-treatment further improved polymer purity, with protease-based formulations outperforming glycoside hydrolases. Enzyme combinations showed clear synergistic effects, supporting the need for complementary catalytic activities to remove structurally diverse NPCM. Integrating both steps demonstrated that bead milling provides the necessary accessibility for enzymatic action, while enzymatic digestion supplies the selectivity required for high-purity PHA under solvent-minimised conditions. The findings outline key parameters governing mechanical and enzymatic purification and provide a basis for developing more sustainable downstream workflows for intracellular biopolymers.
