## 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  
To assess the effect of electrical pulses on microalgae, we first determined the duration and voltage of the pulses generated for an algae suspension with a conductivity of \(\sigma\) = 2 mS/cm and a resistance of \(R\) = 50 \(\Omega\). Single pulses with a duration of \(\Delta t\) at about 4 \(\upmu\)s and 11 \(\upmu\)s were generated using the 1 st and 2 nd range of the generator, respectively. The pulse duration was calculated at the level of 37% of the maximum amplitude. The maximum charging voltages of the capacitor reached 2500 V and 2000 V for ranges 1 and 2, respectively. The characteristic voltage pulses at the cuvette and the current through the cuvette for two ranges of the generator are shown in Figure 6.  
The electric field in the cuvette was calculated according to the equation \(E=\mathrm{V_{out(max)}}/d\), using the peak value of \(\mathrm{V_{out(max)}}\). The specific energy \(W\) was determined by integrating the voltage and current pulses, taking into account the mass of the suspension. The rise in temperature was also calculated using the adiabatic model. These data are listed in Table 1. Voltages above these thresholds led to sparking in the cuvette. The effect of the generated electric field on microalgae was further investigated. To minimize the effects of reversible electroporation, cell permeability measurements were performed at 2 and 24 h after PEF treatment (Figure 7). In addition, indirect effects, such as the leakage of DNA and soluble proteins from the cells, were investigated in PEF-treated and untreated samples. The data obtained showed that the application of a 4 us pulse (Range 1) induced an increase in the SYOX green fluorescence signal only when the applied \(E\) exceeded 20 kV/cm or \(W\) reached 2.39 J/mL (Figure 7a). In contrast, a lower \(E\) of 16 kV/cm and \(W\) of 3.43 J/mL were required to induce cell permeability in the second range (with a pulse duration of 11 us) (Figure 7b). During the following day, the SYOX signal increased even more in the samples exposed to electric field strengths above the threshold. In addition to the increased permeability of the algal membrane, the leakage of nucleic acids into the supernatant was also observed, with the intensity being highest after prolonged incubation (24 h after PEF). The observed leakage of nucleic acids is a hallmark of programmed cell death, which is a process known to be triggered in \(C\). _vulgaris_ by the PEF-induced release of a cell death factor that was recently described here [38]. Their studies showed that \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Range 1 (Pulse Length -4 μs)** & \multicolumn{4}{c}{**Range 2 (Pulse Length -11 μs)**} \\ \hline \(E\) (kV/cm) & \(W\) (J/mL) & \(\Delta T\) (K) & \(E\) (kV/cm) & \(W\) (J/mL) & \(\Delta T\) (K) \\ \hline
2.8 & 0.05 & 0.01 & 4 & 0.21 & 0.05 \\
6.35 & 0.25 & 0.06 & 10 & 1.34 & 0.32 \\
10 & 0.61 & 0.14 & 16 & 3.43 & 0.82 \\
14.8 & 1.33 & 0.32 & 23 & 7.08 & 1.69 \\
19.8 & 2.39 & 0.57 & 29 & 11.26 & 2.68 \\
25.6 & 3.99 & 0.95 & 34 & 15.48 & 3.69 \\
30.8 & 5.77 & 1.37 & 41 & 22.51 & 5.36 \\
34.8 & 7.37 & 1.76 & 47 & 29.58 & 7.04 \\
46 & 12.88 & 3.07 & & & \\
48.8 & 14.5 & 3.45 & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 1: PEF parameters applied to \(C\). _vulgaris_ processing. Figure 6: Typical waveforms of the output voltage across the cuvette with microalgae solution (cuvette resistance about 50 \(\Omega\)). (**a**)—Output voltage when the cuvette is connected to the first module (Range 1); (**b**)—when connected to the second module (Range 2). programmed cell death occurs independently of PEF treatment with low or high energy (when 1 \(\upmu\)s, 40 kV/cm pulses are used). Furthermore, they identified cell debris rich in cell death-inducing factors that could lead to death in intact/untreated algae [38]. Although the origin of this compound remains unclear, [PERSON] et al. identified several _C.