Energy-equivalent cyclic pulsed electric fields enable reversible membrane permeabilization and sustainable protein...

The rigid cell wall of microalgae limits efficient intracellular protein extraction. Here, three pulsed electric field (PEF) energy distribution strategies-high-voltage few-pulse (HP), low-voltage multi-pulse (LP), and a medium-voltage cyclic protocol (MP)-were systematically compared at approximately equivalent specific energy inputs using Nannochloropsis sp. HP treatment caused extensive and largely irreversible membrane disruption, resulting in rapid but early-saturating protein release, whereas LP treatment induced only marginal permeabilization and limited extraction. In contrast, the MP protocol achieved controlled membrane permeabilization (51.77%) while preserving overall cellular morphology, enabling sustained protein release without early saturation under the tested isoenergetic conditions. Under doubled energy input, MP yielded the highest protein concentration (=0.259 mg mL^-1). These results suggest that cyclic redistribution of electrical energy can improve the balance between membrane permeabilization and structural preservation compared with the tested HP and LP regimes.