Ion flux, transmembrane potential, and osmotic stabilization: A new electrophysiological dynamic model for Eukaryotic cells

International audienceSurvival of mammalian cells is achieved by tight control of cell volume while transmembrane potential is known to control many cellular functions since the seminal work of Hodgkin and Huxley. Regulation of cell volume and transmembrane potential have a wide range of implications in physiology, from neurological and cardiac disorders to cancer and muscle fatigue. Therefore understanding the relationship between transmembrane potential, ion fluxes, and cell volume regulation has become of great interest. In this paper we derive a system of differential equations that links transmembrane potential, ionic concentrations, and cell volume. This model demonstrates that volume stabilization occurs within minutes of changes in extracellular osmotic pressure. We infer a straightforward relationship between transmembrane potential and cell volume. Our model is a generalization of previous models in which either cell volume was constant or osmotic regulation instantaneous. When the extracellular osmotic pressure is constant, the cell volume varies as a function of transmembrane potential and ions fluxes thus providing an implicit link between transmembrane potential and cell growth. Numerical simulations of the model provide results that are consistent with experimental data in terms of time-related changes in cell volume and dynamics of the phenomena

Evaluation of pulsed electric fields effect on the microalgae cell mechanical stability through high pressure homogenization

Pulsed Electric Fields, a known technique for permeabilization of cell membranes, can considerably foster intracellular component extraction from microalgae. However, it is currently uncertain in what way, apart from the cell membrane, the cell wall is affected during pulsation. In this study, fresh Auxenochlorella protothecoides and Chlorella vulgaris were subjected to treatment with pulsed electric fields and energy input of 1.5 MJ/kgDryWeight. Subsequently the biomass was fed into a High Pressure Homogenizer for 5 passes at 1500 bar. The percentage of intact cells after each pass was determined through cell counting and compared with Control biomass that underwent the same homogenization. No major difference on the disruption degree of pulsed and control samples was observed, indicating that the resistance to mechanical stress of the cell, a function of the cell wall, is not affected by pulsed electric fields. Scanning Electron Microscopy observation also showed no superficial or structural cell alteration after pulsation

Post-incubation pH Impacts the Lipid Extraction Assisted by Pulsed Electric Fields from Wet Biomass of Auxenochlorella protothecoides

Pulsed electric field (PEF) treatment is a promising technology for efficient lipid extraction from microalgae. This study focusses on under-investigated processing parameters, such as media pH, pulse application, and incubation protocols. The lipid yield and electroporation level of PEF-treated Auxenochlorella protothecoides were determined at a medium pH of 3.0 and 5.0 under variation of the pre- or post-PEF incubation time and for split-dose treatments. Low energetic PEF treatments with 40 kV/cm and 1 μs pulses at 9.6 and 19.2 kJ/L were performed either in batch mode or in continuous flow. Post-PEF incubation significantly increased the shared electroporated cells (>60%) in medium pH 3.0, while no change was observed at pH 5.0. Split-dose PEF treatments at pH 5.0 caused significantly higher electroporation levels and lipid extraction yields than equivalent single-dose treatments. Results have shown that medium pH is critical in the final electroporation and lipid extraction yields of A. protothecoides and therefore should be considered in further studies

Pulsed microwave pretreatment of fresh microalgae for enhanced lipid extraction

Pulsed microwave (PMW) is considered as an energy-saving pretreatment for microalgae. The efficiency of PMW was studied using a generator delivering square-pulsed modulated microwave in continuous flow on fresh Auxenochlorella protothecoides suspension. The efficiency was evaluated by measuring the increase of the suspension\u27s conductivity, the liberation of carbohydrates, the percentage of permeabilized microalgae cells and the lipid yield after solvent extraction. The properties of the pulses i.e. pulse duration, repetition rate and pulse power had little effect on the efficiency and especially on lipid extraction performance. Lipid yield was positively correlated with the energy input and increased from 3.81% to 38.42% with microwave energy input increasing from 1.4 to 2.8 MJ/kgDW (Dry Weight). At a given PMW absorbed energy, the lipid yield decreased with the increase of algal concentration, whereas it increased with the suspension flow rate. Based on comparison with water-bath heating i.e. a pure thermal treatment, results suggest that both the microwave induced heating and non-thermal effects impact the efficiency of PMW treatment. An energy consumption of 2.53 MJ/kgDW achieved 37.29% lipid yield, which confirms that PMW is a potentially competitive, highly efficient and easy to implement method that could benefit downstream processing of microalgae

Post-incubation pH Impacts the Lipid Extraction Assisted by Pulsed Electric Fields from Wet Biomass of "Auxenochlorella protothecoides"

Pulsed electric field (PEF) treatment is a promising technology for efficient lipid extraction from microalgae. This study focusses on under-investigated processing parameters, such as media pH, pulse application, and incubation protocols. The lipid yield and electroporation level of PEF-treated Auxenochlorella protothecoides were determined at a medium pH of 3.0 and 5.0 under variation of the pre- or post-PEF incubation time and for split-dose treatments. Low energetic PEF treatments with 40 kV/cm and 1 μs pulses at 9.6 and 19.2 kJ/L were performed either in batch mode or in continuous flow. Post-PEF incubation significantly increased the shared electroporated cells (>60%) in medium pH 3.0, while no change was observed at pH 5.0. Split-dose PEF treatments at pH 5.0 caused significantly higher electroporation levels and lipid extraction yields than equivalent single-dose treatments. Results have shown that medium pH is critical in the final electroporation and lipid extraction yields of A. protothecoides and therefore should be considered in further studies

Evaluation of pulsed electric fields effect on the microalgae cell mechanical stability through high pressure homogenization

Pulsed Electric Fields, a known technique for permeabilization of cell membranes, can considerably foster intracellular component extraction from microalgae. However, it is currently uncertain in what way, apart from the cell membrane, the cell wall is affected during pulsation. In this study, fresh Auxenochlorella protothecoides and Chlorella vulgaris were subjected to treatment with pulsed electric fields and energy input of 1.5 MJ/kgDryWeight. Subsequently the biomass was fed into a High Pressure Homogenizer for 5 passes at 1500 bar. The percentage of intact cells after each pass was determined through cell counting and compared with Control biomass that underwent the same homogenization. No major difference on the disruption degree of pulsed and control samples was observed, indicating that the resistance to mechanical stress of the cell, a function of the cell wall, is not affected by pulsed electric fields. Scanning Electron Microscopy observation also showed no superficial or structural cell alteration after pulsation

The effect of cell disruption on the extraction of oil and protein from concentrated microalgae slurries

Novel cell-disruption combinations (autolytic incubation and hypotonic osmotic shock combined with HPH or pH12) were used to investigate the fundamental mass transfer of lipids and proteins from Nannochloropsis slurries (140 mg biomass/g slurry). Since neutral lipids exist as cytosolic globules, their mass transfer was directly dependent on disintegration of cell walls. Complete recovery was obtained with complete physical disruption. HPH combinations exerted more physical disruption and led to higher yields than pH12. In contrast, proteins exist as both cytosolic water-soluble fractions and cell-wall/membrane structural fractions and have a complex extraction behaviour. Mass transfer of cytosolic proteins was dependent on cell-wall disintegration, while that of structural proteins was governed by cell-wall disintegration and severance of protein linkage from the wall/membrane. HPH combinations exerted only physical disruption and were limited to releasing soluble proteins. pH12 combinations hydrolysed chemical linkages in addition to exerting physical disruption, releasing both soluble and structural proteins

Evaluation of Downstream Processing, Extraction, and Quantification Strategies for Single Cell Oil Produced by the Oleaginous Yeasts Saitozyma podzolica DSM 27192 and Apiotrichum porosum DSM 27194

Single cell oil (SCO) produced by oleaginous yeasts is considered as a sustainable source for biodiesel and oleochemicals since its production does not compete with food or feed and high yields can be obtained from a wide variety of carbon sources, e.g., acetate or lignocellulose. Downstream processing is still costly preventing the broader application of SCO. Direct transesterification of freeze-dried biomass is widely used for analytical purposes and for biodiesel production but it is energy intensive and, therefore, expensive. Additionally, only fatty acid esters are produced limiting the subsequent applications. The harsh conditions applied during direct esterification might also damage high-value polyunsaturated fatty acids. Unfortunately, universal downstream strategies effective for all yeast species do not exist and methods have to be developed for each yeast species due to differences in cell wall composition. Therefore, the aim of this study was to evaluate three industrially relevant cell disruption methods combined with three extraction systems for the SCO extraction of two novel, unconventional oleaginous yeasts, Saitozyma podzolica DSM 27192 and Apiotrichum porosum DSM 27194, based on cell disruption efficiency, lipid yield, and oil quality. Bead milling and high pressure homogenization (HPH) were effective cell disruption methods in contrast to sonification. By combining HPH (95% cell disruption efficiency) with ethanol-hexane-extraction 46.9 +/- 4.4% lipid/CDW of S. podzolica were obtained which was 2.7 times higher than with the least suitable combination (ultrasound + Folch). A. porosum was less affected by cell disruption attempts. Here, the highest disruption efficiency was 74% after BM and the most efficient lipid recovery method was direct acidic transesterification (27.2 +/- 0.5% fatty acid methyl esters/CDW) after freeze drying. The study clearly indicates cell disruption is the decisive step for SCO extraction. At disruption efficiencies of >90%, lipids can be extracted at high yields, whereas at lower cell disruption efficiencies, considerable amounts of lipids will not be accessible for extraction regardless of the solvents used. Furthermore, it was shown that hexane+ethanol which is commonly used for extraction of algal lipids is also highly efficient for yeasts

Analysis of the lipid extraction performance in a cascade process for Scenedesmus almeriensis biorefinery

Background Microalgae have attracted considerable interest due to their ability to produce a wide range of valuable compounds. Pulsed Electric Fields (PEF) has been demonstrated to effectively disrupt the microalgae cells and facilitate intracellular extraction. To increase the commercial viability of microalgae, the entire biomass should be exploited with different products extracted and valorized according to the biorefinery scheme. However, demonstrations of multiple component extraction in series are very limited in literature. This study aimed to develop an effective lipid extraction protocol from wet Scenedesmus almeriensis after PEF-treatment with 1.5 MJ·kgDW−1. A cascade process, i.e., the valorization of several products in row, was tested with firstly the collection of the released carbohydrates in the water fraction, then protein enzymatic hydrolysis and finally lipid extraction. Biomass processed with high pressure homogenization (HPH) on parallel, served as benchmark. Results Lipid extraction with ethanol:hexane (1:0.41 vol/vol) offered the highest yields from the different protocols tested. PEF-treatment promoted extraction with almost 70% of total lipids extracted against 43% from untreated biomass. An incubation step after PEF-treatment, further improved the yields, up to 83% of total lipids. Increasing the solvent volume by factor 2 offered no improvement. In comparison, extraction with two other systems utilizing only ethanol at room temperature or elevated at 60 °C were ineffective with less than 30% of total lipids extracted. Regarding cascade extraction, carbohydrate release after PEF was detected albeit in low concentrations. PEF-treated samples displayed slightly better kinetics during the enzymatic protein hydrolysis compared to untreated or HPH-treated biomass. The yields from a subsequent lipid extraction were not affected after PEF but were significantly increased for untreated samples (66% of total lipids), while HPH displayed the lowest yields (~ 49% of total lipids). Conclusions PEF-treatment successfully promoted lipid extraction from S. almeriensis but only in combination with a polar:neutral co-solvent (ethanol:hexane). After enzymatic protein hydrolysis in cascade processing; however, untreated biomass displayed equal lipid yields due to the disruptive effect of the proteolytic enzymes. Therefore, the positive impact of PEF in this scheme is limited on the improved reaction kinetics exhibited during the enzymatic hydrolysis step