## 4 Utilizing the Marx Generator in Biomass Processing
### Application of the Marx Generator for PEF Treatment of Microalgal Biomass
#### 4.1.1 Materials and Methods of Algal Treatment  
The cells of _C. vulgaris_ (UTEX 395, Arizona State University) were cultured in a BG-11 medium. The stationary-stage algal culture was used to inoculate the glass bubble column photobioreactor (PBR) (final optical density 0.1 at 750 nm), which was a 1000 mL glass vessel (61 cm in length and 5 cm in diameter) with a downstream aeration tube. The algal suspension was cultured at 22 \(\pm\) 1 \({}^{\circ}\)C with fluorescent lamps at a photosynthetic photon flux density of 90 \(\mu\)mol/m\({}^{2}\)/s, a 16:8 h light/dark cycle with a supply of CO\({}_{2}\) and an air-gas mixture until it was harvested at the stationary growth phase (7 days).  
Before PEF treatment, the algal suspension was concentrated by centrifugation (3000\(\times\)\(g\), 15 min) at a final concentration of 5 g/L, which was determined using optical density (OD) at 750 nm versus a dry biomass calibration curve (R\({}^{2}\) = 0.962). The final electrical conductivity and pH of the concentrated suspension were determined using a conductivity meter with InLab 738-ISM probe (Mettler Toledo, Columbus, OH, USA) and Orion 720 Aplus (Thermo Scientific(tm), JAV, Waltham, MA, USA), which were in the range of 2 mS/cm, pH \(\sim\)7.3.  
The algae suspension obtained was processed at room temperature using both modules of the generator described above. The capacitors of the modules were charged from 250 V to 2500 V. A two-electrode electroporation cuvette filled with algae suspension with spacing at \(d\) = 2 mm (VWR International, Taiwan) was used as the load of the generator. The applied voltage and pulse duration were estimated with the oscilloscope. After PEF treatment, cell permeability, and quantitative and qualitative protein characteristics were evaluated. As a positive control, representing a fully disrupted cell population, the cell suspension was disrupted using the Vibra Cell ultrasonicator (Sonics & Materials Inc., USA) [35].  
The permeability of the algal cell membrane after PEF treatment was determined using the dye SYTOX Green (Thermo Fisher Scientific, Newtown, CT, USA). Normally, it is a dye that is impermeable to intact cells. Only after the loss of membrane integrity can SYTOX Green enter the cell, bind to the nucleic acid, and emit fluorescence. In brief, after incubation at room temperature for 2 and 24 h, the treated and untreated samples were diluted to OD 1 (at 750 nm wavelength) with a sterile BG-11 medium. Immediately after dilution, each sample was mixed with a fluorescent dye (final concentration 2 \(\mu\)M) and incubated for 5 min in the dark. The fluorescence intensity was then determined using the LS-50B luminescence spectrometer (PerkinElmer Inc., Buckinghamshire, UK) (excitation at 490 nm, emission at 500-550 nm, peak intensity at 525 nm, scan speed 200 nm/min). To detect the leakage of nucleic acid into the supernatant, SYTOX green fluorescence was also measured in the supernatant after an additional centrifugation step (6000\(\times\)\(g\), 1 min).  
The concentration of soluble protein in the sample supernatant after 24 h of incubation was determined using the ROTI Quant Universal Assay (Roth, Germany) according to the manufacturer's protocol. The supernatant obtained after centrifugation was also analyzed by SDS-PAGE on a TV200Y electrophoresis instrument (SciE-Plas Ltd., Cambridge, UK) following the modified protocol described in [36; 37].