C. Gusbeth

Pulsed electric field treatment for bacteria reduction and its impact on hospital wastewater.

During the last years the pulsed electric field (PEF) method entered several fields of application. A promising application is the decontamination of hospital wastewater effluents, which are loaded with pathogenic and increasingly with antibiotic-resistant bacteria. For this study, Pseudomonas putida suspended in buffer solution or wastewater from university hospital was used as reference strain. To prove whether the descendent of the survival bacteria develop an adaptation to electric field, surviving PEF treated bacteria were recultivated and pulsed in serial experiments with 10 pulses (100kVcm(-1) and 600ns pulse duration). This procedure was repeated for 30 generations. The inactivation rate was calculated with 3.5+/-0.8 log of colony forming units and remained constant over 30 cycles. Investigations of the variable intergenic spacer region of the ribosomal operon demonstrated no visible changes in this highly variable part of the genome structure during the serial PEF treatment experiments. The mutagenicity of PEF treated hospital wastewater, buffer solutions and tap water was analyzed by the umu-test. Most hospital wastewater samples exhibit a considerable genotoxicity already before PEF treatment, but this was not increased by the PEF treatment, not even for higher treatments energies over 250JmL(-1). No genotoxicity was induced in buffer solutions and tap water by PEF treatment. This study supports, that PEF treatment is a sustainable non-chemical method for bacterial decontamination without any adverse effects.

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.

Influence of Pulsed Electric Field (PEF) treatment on the extraction of lipids from the microalgae Auxenochlorella protothecoides

Microalgae are a promising source of lipids and biopolymers. In most cases algal lipids are stored intracellular. During the extraction of intracellular products from cells the cell wall and the cell membrane are natural barriers, preserving the cell content. Pulsed Electric Field (PEF) treatment results in the electroporation (EP) or the electropermeabilization of the cell membrane. The formation of pores across the membrane often leads to an enhanced extraction process since solvents can penetrate the biomass matrix more efficiently.

Fluorescence Diagnostics for Lipid Status Monitoring of Microalgae during Cultivation

Microalgae are known to accumulate considerable amounts of triglycerides when starving for nitrogen [1-2] or when interacting with bacteria [3]. Due to depleting mineral oil resources and the accumulation of greenhouse gases in the atmosphere, microalgae biomass is a promising sustainable option for the production of biofuels [4] and a possible future feedstock for biochemical industry. Since costs for microalgae biomass production nowadays still exceed revenues from the biofuel market, current challenges for an energetic use of microalgae biomass Abstract

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

Environmental Applications, Food and Biomass Processing by Pulsed Electric Fields

Pulsed electric field (PEF) treatment is a physical method which exhibits specific advantages over conventional processing in various applications and was proven for feasibility on pilot and industrial scale. For bacterial inactivation in wastewater and liquid food and for eradication of Cyanobacteria in surface waters, PEF-based techniques are demonstrated to be energy saving and persistent in efficacy without adding harmful chemicals and in particular do not cause adverse effects to food matrices or to the aquatic environment. For component extraction, the specific advantages of PEF treatment, i.e., low heat influx, low-energy demand, and selectivity of compound release, promote PEF processing in winemaking, extraction of sugar from sugar beets and valuable components from fruits and vegetables, and PEF-downstream processing of microalgae. These promising applications of PEF processing will be introduced in more detail in the following sections.

Nanosecond pulsed electric fields trigger cell differentiation in Chlamydomonas reinhardtii

Nanosecond pulsed electric fields (nsPEFs) have great potential for biotechnological and medical applications. However, the biological mechanisms causing the cellular responses are still far from understood. We used the unicellular green algae Chlamydomonas reinhardtii as experimental model to dissect the immediate consequences of electroporation from the developmental cellular responses evoked by nsPEFs. We observe that nsPEFs induce a short-term permeabilization of the membrane, accompanied by swelling and oxidative burst. These response are transient, but are followed, several days later, by a second wave of oxidative burst, arrested cell division, stimulated cell expansion, and the formation of an immobile palmella stage. This persistent oxidative burst can be suppressed by specific inhibitor diphenyl iodonium (DPI), but not by the unspecific antioxidant ascorbic acid (Asc). Treated with natural and artificial auxins allow to modulating the cell cycle and cell expansion, and natural auxin can suppress the spontaneous formation of palmella stages. However, when administered prior to the nsPEFs treatment, auxin cannot mitigate the elevated formation of palmella stages induced by nsPEFs. We interpret our findings in terms of a model, where nsPEFs generate a developmental signal that persists, although the other immediate responses remain transient. This signal will initiate, several days later, a developmental programme comprising halted cell cycle, stimulation of cell expansion, a persistent activation of NADPH oxidase activity causing a second wave of oxidative burst, and the irreversible initiation of palmella stages. Thus, a short transient nsPEFs treatment can initiate a stable response of cellular differentiation in Chlamydomonas reinhardtii.

Lifetime and design considerations for anodes for pulsed underwater corona discharges

Efficient decontamination of wastewater by pulsed underwater corona discharges was demonstrated in many studies. Recently, the streamer density and length, the major factors determining decontamination performance, could be increased by using Titan anodes which previously were coated with an Almandine ceramic. However, the expensive and sophisticated ceramic coating and the short lifetime, of about 105 pulses make this technique inefficient for industrial application. Metal wires, with a diameter smaller than 300 μm, provide a cheaper anode alternative. These anodes could be continuously replaced to avoid ruptures during operation by using a withdrawable unit. Based on these constructive ideas, the length and density of streamers, generated by various metal wires with different diameters and configurations, were investigated by means of image processing. The analyses of the anode surface by electron microscopy has shown, that the high temperature generated in the streamer leads to local melting of the anode surface. Also because of the high melting point of tungsten (3422°C), tungsten wires with diameters between 100 and 300 μm turned out to be suitable for anode use. Anodes metals with melting points lower than the streamer temperature (>3400°C) would erode faster. The analysis of images of the underwater corona discharges proved that for all types of anodes the length of streamers lineary increased with anode voltage and was almost independent of the type of anode. The water volume interspersed by streamers, normalized to the total treatment volume was significantly higher when applying Ti-Almandine anodes. However, the efficiency of corona discharges to decontaminate Enterococcus f. bacteria in wastewater was similar for all anode types.

Effect of nanosecond pulsed electric field treatment on cell proliferation of microalgae

Photoautotrophic microalgae based biorefinery concepts are currently not competitive compared to other established production systems. Therefore, innovative upstream processes need to be developed to increase the competitiveness of photoautotrophic microalgae biorefinery concepts. Abiotic sub-lethal stress induction via nanosecond pulsed electric field (nsPEF) treatment might be a viable process to increase the efficiency of photoautotrophic microalgae cultivation. In this work, an increased cell growth after nsPEF treatment was observable. Application of nsPEF to highly proliferating cells in a repetitive process resulted in a statistical significant increase in cell growth (p = 0.009). The effect was most pronounced after five days wherefore cellular structures and processes were analyzed to reveal a possible mechanism. Within this work, a protocol for increased cell proliferation with a possible mechanism was derived, which improves competitiveness of photoautotrophic microalgae biorefineries in the future. However, based on the derived mechanism, the results are also relevant for other microorganisms.