R. Straessner

Plant actin controls membrane permeability

The biological effects of electric pulses with low rise time, high field strength, and durations in the nanosecond range (nsPEFs) have attracted considerable biotechnological and medical interest. However, the cellular mechanisms causing membrane permeabilization by nanosecond pulsed electric fields are still far from being understood. We investigated the role of actin filaments for membrane permeability in plant cells using cell lines where different degrees of actin bundling had been introduced by genetic engineering. We demonstrate that stabilization of actin increases the stability of the plasma membrane against electric permeabilization recorded by penetration of Trypan Blue into the cytoplasm. By use of a cell line expressing the actin bundling WLIM domain under control of an inducible promotor we can activate membrane stabilization by the glucocorticoid analog dexamethasone. By total internal reflection fluorescence microscopy we can visualize a subset of the cytoskeleton that is directly adjacent to the plasma membrane. We conclude that this submembrane cytoskeleton stabilizes the plasma membrane against permeabilization through electric pulses.

Pulsed Electric Field Treatment of Microalgae—Benefits for Microalgae Biomass Processing

In this paper, we demonstrate how pulsed electric field (PEF) treatment of microalgae biomass opens promising downstream processing options for an energetic use of algae. The fact that lipid droplets remain intracellular after treatment facilitates selective processing. After separation of the water-soluble cell contents, the algae lipids can be extracted with adequate solvents. The use of the environmental-friendly solvent ethanol results in an extraction yield from wet and dry biomass that, on average, is four times higher compared with untreated samples. Especially, the use of wet biomass opens a promising processing route for an energetic use of microalgae, because the energy consumed conventionally for drying of the biomass is considerably higher than the energy required for PEF treatment, i.e., ~ 1.5 MJ/kg of dry biomass.

Monitoring of Pulsed Electric Field-Induced Abiotic Stress on Microalgae by Chlorophyll Fluorescence Diagnostic

Application of a pulsed electric field (PEF) on biological cells, in general, results in stress reactions of the affected organisms. Depending on pulse parameters, reactions such as growth stimulation or apoptosis can be observed for short pulses on the nanosecond (ns) time scale. Cell inactivation usually occurs at longer pulse duration and appropriate high treatment energy values. In this paper, the impact of short PEFs on chloroplasts of green microalgae Auxenochlorella protothecoides was investigated, with closer inspection of the photosystem II (PS II), located in the thylakoid membrane. For this purpose, a pulse amplitude modulated (PAM) fluorescence diagnostic was employed, which is a common method for monitoring changes in the photosynthesis apparatus. In particular, alterations of the PS II can be identified by fluorescence quenching analysis, sensitively. For PEF treatment of microalgae suspensions, the high-voltage pulse duration was adjusted to 100 and 1000 ns. The treatment energy was varied between 2 and 78 kJ/kg. The electric field amplitude was constant throughout the experiments (ECuv=40 kV/cm). After PEF treatment, the samples were periodically analyzed by chlorophyll fluorescence analysis for 1 h, using the saturation pulse method. For the evaluation of the physiological status of the microalgae, the maximum photochemical quantum yield of PS II, Fv/Fm, was chosen. The obtained results showed that the influence of PEFs on PS II is significant. Contrary to commonly accepted explanations that intracellular organelles are predominantly affected by short ns-pulses, a large influence of PEF exposure on chloroplasts, particularly on PS II, could be identified for longer pulses. In this paper, the diagnostic method, applied pulse protocols, and the results of the PAM fluorescence measurements will be discussed.

Effect of Pef Treatment on Extraction of Valuable Compounds from Microalgae C. Vulgaris

Effect of PEF Treatment on Extraction of Valuable Compounds from Microalgae C. vulgaris Gianpiero Pataro, Martina Goettel, Ralf Straessner, Christian Gusbeth, Giovanna Ferrari, Wolfgang Frey a Department of Industrial Engineering, University of Salerno, via Giovanni Paolo II 132, 84084 Fisciano (SA), Italy b Karlsruhe Institute of Technology (KIT), Institute for Pulsed Power and Microwave Technology (IHM), 76344 EggensteinLeopoldshafen, Germany c ProdAlScarl – University of Salerno, via Ponte don Melillo, 84084 Fisciano (SA), Italy gpataro@unisa.it

Study of Transmembrane Voltage Kinetics during 100 μ s Pulse Using Voltage Sensitive Dyes

When a cell is subjected to an external electric field, a voltage establishes on the membrane and adds to the resting transmembrane voltage (TMV). If the intensity and duration of the external field are sufficient, the membrane becomes conductive, which is the first step in the electroporation process. The increase of conductivity results in a collapse of the previously established TMV. Using the fluorescent voltage sensitive dye Annine-6, we investigated the kinetics of TMV on mammalian cells DC3F submitted to pulses of 100 μs with field magnitudes ranging from 60 kV.m-1 to 200 kV.m-1. The results contradict the common opinion which suggests that the TMV decreases very quickly as soon as it exceeds a socalled ‘threshold’ of electroporation. On the contrary, at all field strengths tested, experiments show that the TMV remains stable for several microseconds. The duration of the stable phase decreases exponentially as the electric field increases. These results strongly challenge the current models on electro-permeabilisation and provide quantitative data to develop new approaches.