## 4 Utilizing the Marx Generator in Biomass Processing
### Application of the Marx Generator for PEF Treatment of Microalgal Biomass
#### 4.1.2 Microalgae Cell Responses to PEF Treatment
vulgaris_ protein compounds are involved in the hydrolysis of structures associated with the algal cell wall [39]. In addition to PEF-induced cell damage and cell death, one of the most valuable compounds--protein release--was also determined in this study (see Figure 8). The results showed that protein release only occurred when the integrity of the algal cell membrane was compromised, which is consistent with the threshold values observed in membrane permeabilization experiments. For shorter pulses (range 1), the threshold for protein release was 20 kV/cm. For longer pulses (range 2), however, this threshold was already triggered at an electric field strength of 16 kV/cm. It should be also noted that even at the maximum applied voltage, the temperature of the suspension did not rise by more than 7 degrees (see Table 1). However, further increases in electric field strength did not affect the increase in protein extraction efficiency, which reached a saturation level for water-soluble proteins. Figure 8: Extracted protein concentration of _C. vulgaris_ after treatment with different electric field strengths and operation ranges. Figure 7: SYTOX green fluorescence in microalgae suspension and supernatant after PEF treatment using Range 1 (**a**) and Range 2 (**b**) of the electroporator under different electric field strengths 2 or 24 h post-treatment. Similar results of PEF-extracted protein yield and its saturation phenomena have been observed in previous studies, suggesting that PEFs induce soluble protein release, which is closely linked to active biological processes, such as the release of factors that trigger programmed cell death. This link proceeds beyond the solely physical phenomena of pore formation [25; 38]. However, it is important to note that the parameters of the PEFs used were different in the various studies, although they produced the same effect. Likely reasons for these differences are variations in cultivation and extraction conditions. These include not only the chemical composition of the medium (TAP instead of BG-11) and physical differences (electrical conductivity, pH) but also the scale of cultivation (Erlenmeyer flask instead of a bubble bioreactor). These factors influence not only the cell size and wall composition of the _C. vulgaris_ cells but also the efficiency of protein synthesis [40]. The validity of the PEF-induced protein extraction and its quantitative analysis was confirmed using the SDS-PAGE gel images (Figure 9). The gels clearly showed that protein extraction only occurred when the applied electric field (_E_) exceeded the selectively determined threshold. In particular, the most noticeable protein fractions were obtained in the 1 st range at an \(E\) above 30 kV/cm, while the threshold value for the 2 nd range was 20 kV/cm. However, no distinct protein fractions were observed at \(E\) = 20 kV/cm and 26 kV/cm for the 1 st range and 16 kV/cm for the 2 nd range despite the effect on the algae shown in the permeabilization results (Figures 8 and 9). The observed inconsistency was probably due to the much smaller scale of biological processes triggered when PEF was applied in the threshold region. Therefore, the release of water-soluble proteins, which is an enzymatically driven process, requires a longer period to produce a comparable amount of protein, as observed at a higher _E_[25]. Moreover, a further increase in E did not lead to the release of additional proteins. The profiles of these fractionated proteins remained consistent across all lanes, with prominent bands at 55, 35, 25, and 15 kDa. These profiles are consistent with known protein fractions obtained after the PEF treatment of _C. vulgaris_, with specific bands associated with proteins, such as ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo, found in the chloroform-plast), cytochrome c oxidase subunit II (COXII, found in mitochondria), histone H3 (found in the nucleus), and actin (filaments in the cytosol) [25].