Microalgae are high in protein, lipids, carbohydrates, minerals and vitamins, in addition to containing chlorophyll a and b as the main pigments and antioxidants (Ahmad, Shariff, Md. Yusoff, Goh, & Banerjee, 2020; Giannoglou, Andreou, Thanou, Markou, & Katsaros, 2023), such as lutein, α-carotene, β-carotene, ascorbic acid and α-tocopherol (Bhuvana, Sangeetha, Anuradha, & Ali, 2019; Spolaore, Joannis-Cassan, Duran, & Isambert, 2006). It is also a very interesting source of polyunsaturated fatty acids (PUFAs) (Lupette & Benning, 2020; Tokuşoglu & Üunal, 2003). Meanwhile, microalgae also have unique physiological adaptations, thus it has attracted increasing attention in the food industry. However, the extraction of valuable bio-molecules from the microalgae is not an easy task (R. Zhang, Lebovka, Marchal, Vorobiev, & Grimi, 2020). These bio-molecules are commonly located in the intracellular compartments, protected by the cell walls and cell membrane, and membranes surrounding the cytoplasm and the internal organelles (Postma et al., 2017). Traditional extraction methods generally use solvents such as methanol and chloroform. However, these solvents have negative impacts on both human health and the environment which are inconsistent with the requirements for sustainable development (Zhou et al., 2022). Therefore, some green solvents have been developed in the hope of replacing traditional toxic solvents, but their extraction efficiency is currently not comparable to that of conventional solvents (Parniakov et al., 2015; Zbinden et al., 2013). In order to increase the efficiency of green solvents, pretreatments can be used to disrupt cell walls or membranes (Parniakov et al., 2015).

There are many techniques that have been applied to cell disruption, such as bead milling, high-pressure homogenization and ultrasonication. These techniques, while improving the extraction efficiency significantly, also have the disadvantages of high energy consumption, thermal generation and non-selective extraction (Gunerken et al., 2015). To overcome these disadvantages, an advanced non-thermal technique, pulsed electric field (PEF), can be used (Corrêa, Morais Júnior, Martins, Caetano, & Mata, 2021; Naliyadhara et al., 2022). In recent years, there are many examples that have obtained higher efficiency using PEF-assisted solvent extraction. A more than 130% increase in initial lipid yield compared to control samples was obtained when using PEF-assisted ethyl acetate extraction of lipids from microalgae (Zbinden et al., 2013). Nordic microalgae Haematococcus pluvialis pretreated by PEF followed by incubation in its own growth medium increased the subsequent astaxanthin extraction yields using ethanol to 96% of the total content (Martínez et al., 2019). Using PEF-assisted binary mixtures of organic solvents and water to extract nutritional compounds from microalgae Nannochloropsis spp. can improve the extraction of proteins and pigments (Parniakov et al., 2015).

PEF is a technology of very short duration (from several nanoseconds to several milliseconds) with electric pulse amplitude from 100 V/cm to 40 kV/cm. The principle of PEF improving extraction is to induce electroporation in the cell membrane, promoting the target to be released from the cell or the extraction solvent to enter the cell. Among the several theories aiming to explain electroporation, the most accepted one proposes that the formation of pores is due to electro-mechanical stress (Freire, Lattanzio, Orera, Mañas, & Cebrián, 2021). The cell is isolated from the outer environment through its membrane. This cell membrane contains dielectric compounds (Zimmermann, Pilwat, & Riemann, 1974). When the electric field is applied to the cell, the dielectric compound gets converted to charged entities and move towards the opposite electrode (Arshad et al., 2020). The deposition of oppositely charged ions at both sides of the membrane leads to the generation of transmembrane potential. The movement of ions in the cell membrane creates electro-mechanical stress inside the cell membrane. For this purpose, an external electric field (Ee) above the critical electric field (Eth), i.e., the threshold, should be applied to ensure the poration in the cell membrane. Depending on the process parameters applied (Ee > Eth or Ee > > Eth), the poration in the cell membrane can be categorized as reversible or irreversible (Kotnik et al., 2015). The improvement effect of PEF-assisted extraction depends largely on the efficacy of electroporation.

The parameters influencing the efficacy of electroporation are process parameters (e.g., treatment time, electric field strength, pulse width, and specific energy), products characteristics (e.g., conductivity and bio-molecules composition, extraction yield and purity, size, etc), in which the process parameters have the greatest influence (Arshad et al., 2020; Coustets et al., 2015; Khan et al., 2021; Li, Yang, & Zhao, 2021; Luengo, Martínez, Bordetas, Álvarez, & Raso, 2015). The influence of basic electrical parameters such as electric field strength and pulse width on electroporation has been extensively studied. The higher the electric field strength, the wider the pulse width and the higher the specific energy, the more effective the electroporation (Fesmire, Petrella, Kaufman, Topasna, & Sano, 2020; Khan et al., 2021; S. Qin et al., 2014; Sano, Arena, DeWitt, Saur, & Davalos, 2014). However, the effect of different pulse waveforms on electroporation varies due to the different response properties of biological cells to electric field frequencies. For example, when a composite pulse consisting of a wide square wave pulse and a narrow square wave pulse was used to inactivate Aspergillus spp. in rice, an 80% increase in effectiveness was achieved compared to the conventional square wave pulse (Wu, Tseng, & Hung, 2004). When inactivating Escherichia coli (ATCC11229), Saccharomyces cerevisiae (ATCC16664), and Bacillus subtilis (ATCC9372) with pulses of different waveforms, it was found that the unipolar rectangular pulses were 60% more efficient than the exponentially decaying pulses (B.-L. Qin, Zhang, Barbosa-Canovas, Swanson, & Pedrow, 1994). When a electropermeabilization experiments were conducted on Chinese hamster fibroblasts, bipolar rectangular pulses were found to be significantly more effective than unipolar rectangular pulses (Kotnik, Mir, Flisar, Puc, & Miklavčič, 2001). These researches proved that better electroporation can be achieved by using suitable pulse waveforms. However, on the one hand, few studies have examined the influence of the pulse waveform on electroporation or extraction of microalgal cells; on the other hand, the waveforms used in these studies were still limited to conventional waveforms due to the limitations of the treatment system, which may not allow for optimal treatment results. These two aspects may severely limit the flourishing of microalgae in the food industry.

Therefore, the goal of this study was to investigate the influence of pulse waveforms on electroporation in Chlorella vulgaris. To achieve this objective, a pulsed power generator capable of outputting high-voltage pulses with arbitrary waveforms was developed. This device utilized to generate pulsed electric fields of nine different waveforms with the same amplitude and energy to conduct electroporation experiments on Chlorella vulgaris. Besides, a numerical modeling of electroporation was established to analyze the experimental results.