Reversible electropermeabilization of Auxenochlorella protothecoides microalgae: tracking mass transfer and membrane resealing dynamics induced by pulsed electric fields

Pulsed Electric Field (PEF) treatment is an emerging technology that enhances mass transfer by inducing cell permeabilization, offering great potential in microalgae bioprocessing. While irreversible permeabilization for extraction has been extensively studied, reversible permeabilization remains largely unexplored for most microalgae species, thus limiting its application in processes requiring viable cells, such as cyclic extraction, gene delivery, and cell fortification. This study investigated how electric field strength and split pulse configuration, either a single long pulse or multiple shorter pulses, affect the reversible electropermeabilization of Auxenochlorella protothecoides. The uptake of the cell membrane-impermeable stain Sytox Green™ was used to assess cell permeabilization, while protein extraction served as a mass transfer indicator. Results showed that a split pulse configuration (2 × 50 μs or 5 × 20 μs pulses at 15 kV cm⁻¹) increased the relative fluorescence intensity fivefold compared with a single long pulse, with only a 12 % increase in energy input. The split pulse approach kept the cells permeable for over 10 min, enabling 38 % more protein extraction with 41 % less energy than a single pulse at 20 kV cm⁻¹, but this higher degree of permeabilization compromised cell viability, inactivating around 70 % of the cells and delaying regrowth during the first 48 h, thereby hindering suitability for live-cell applications. For the first time, the resistance of A. protothecoides to high electric fields has been investigated, e.g., with a single pulse of 15 kV cm⁻¹ permeabilizing the cells for at least 5 min while maintaining good resealing and survival rates comparable to the untreated control. These findings offer valuable insights into maximizing reversible permeabilization, mass transfer, and cell survival in microalgae while minimizing energy consumption in PEF processing.