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 (E-Cuv = 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, F-v/F-m, 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.