Flow Process for Electroextraction of Total Proteins from Microalgae

Abstract

Classical methods for protein extraction from microorganisms, used for large-scale treatments such as mechanical or chemical processes, affect the integrity of extracted cytosolic protein by releasing proteases contained in vacuoles. Our previous experiments on flow-process yeast electroextraction proved that pulsed electric field technology allows us to preserve the integrity of released cytosolic proteins by keeping intact vacuole membranes. Furthermore, large volumes are easily treated by the flow technology. Based on this previous knowledge, we developed a new protocol in order to electroextract total cytoplasmic proteins from microalgae (Nannochloropsis salina and Chlorella vulgaris). Given that induction of electropermeabilization is under the control of the target cell size, as the mean diameter for N. salina is only 2.5 μm, we used repetitive 2-ms-long pulses of alternating polarities with stronger field strengths than previously described for yeasts. The electric treatment was followed by a 24-h incubation period in a salty buffer. The amount of total protein released was evaluated by a classical Bradford assay. A more accurate evaluation of protein release was obtained by SDS-PAGE. Similar results were obtained with C. vulgaris under milder electrical conditions, as expected from their larger size. This innovative technology designed in our group should become familiar in the field of microalgae biotechnology.

A biorefinery for Nannochloropsis: Induction, harvesting, and extraction of EPA-rich oil and high-value protein

“…These authors were able to obtain 25% protein yield from N. gaditana CCFM-01 with the aid of enzymes to break the cell wall. Testing of other methods were used to extract protein from crops like soybean (Sari et al, 2013) and rapeseed (Sari et al, 2013), and from other microalgal species like Chlorella fusca (Sari et al, 2013), C. vulgaris (Coustets et al, 2013;Ursu et al, 2014), Scenedesmus sp. (Garcia-Moscoso et al, 2013).…”

“…(Garcia-Moscoso et al, 2013). These methods include enzyme digestion (Samarakoon et al, 2013;Sari et al, 2013), pressurized extraction (Garcia-Moscoso et al, 2013Ursu et al, 2014) and electroextraction (Coustets et al, 2013).…”

The Use of Pulsed Electric Fields for Protein Extraction from Nanochloropsis and Chlorella

“…The PEF delivery system was built with 2 pulse generators (DEEX-Bio, Betatech, France) and flow through applicators (CNRS) designed to apply trains of pulses with alternating polarities on the flow [12]. In the pulsing chamber for C. Vulgaris, the distance between electrodes was 6 mm.…”

A Microfluidic Device for the Real-Time Characterization of Lipid Producing Algae Cell Population Submitted to a Pulsed Electric Field

“…Studies concerning the use of pulsed electric fields (PEF) to permeabilize algae cell wall for their content extraction are in constant increase. The targets are numerous: extraction of lipids [12][13][14][15], pigments [16], proteins [17] lysis of toxic algae [18] or even to increase biomethane production [19]. However, the efficiency of electropermeabilization applied to algae cells for the extraction of large compounds remains unsatisfactory.…”

“…However, the efficiency of electropermeabilization applied to algae cells for the extraction of large compounds remains unsatisfactory. In addition, this process is mostly employed using millimeter or centimeter scale electroporation cuvettes [13,[15][16][17]20]. This paper investigates real time observation of algae cells being electroporated within a devoted microfluidic system.…”