WO2019121630A1 - Essentially non-destructive method for extracting intracellular water-soluble molecules from unicellular microalgae - Google Patents

Abstract

An essentially non-destructive method for extracting intracellular water-soluble molecules from unicellular microalgae, the method comprising: - a step of providing unicellular microalgae lacking a secondary cell wall or having a secondary cell wall with a thickness less than or equal to 0.5 μm; - a step of placing the microalgae in a medium with low electrolytic conductivity in order to form a suspension of microalgae, the total electrolytic conductivity of the suspension of microalgae having a value ranging from 15 to 40 μS.cm-1 at 25°C; - a step of applying a pulsed electric field to the suspension of microalgae in order to obtain a suspension of temporarily permeabilised microalgae, the pulsed electric field having an electric force ranging from 0.5 to 1 kV.cm-1; and, - a step of incubating the suspension of temporarily permeabilised microalgae, in order to obtain microalgae depleted in intracellular water-soluble molecules in suspension in a medium enriched in extracted water-soluble molecules, the incubation step allowing the diffusion of intracellular water-soluble molecules from the at least temporarily permeabilised microalgae into the medium with low electrolytic conductivity.

Description

 Method for extracting essentially non-destructive intracellular water-soluble molecules from unicellular microalgae

1. Field of the invention

The field of the invention is that of processes for extracting intracellular water soluble molecules from unicellular microalgae.

 More specifically, the invention relates to extraction methods comprising the application of a pulsed electric field.

 2. Prior Art

The cells of the microalgae are endowed with chloroplast (s). The chloroplast is mainly known for its ability to perform photosynthesis. Another characteristic of the chloroplast is to possess customizable deoxyribonucleic acid (DNA) using molecular biology methods to produce proteins of interest. It has therefore been envisaged that chloroplasts of terrestrial plants and microalgae will produce proteins of economic interest in order to replace current methods of production. To date, the green microalgae Chlamydomonas reinhardtii has in particular been used to produce proteins of therapeutic interest. Microalgae thus have a significant biotechnological potential because they produce interesting molecules, including proteins of interest, for different industrial sectors: food, health, parapharmacy, energy, cosmetics, etc.

 The current biotechnological processes for extracting molecules of interest from microalgae generally have the following successive stages:

 (1) Obtain microalgae biomass;

 (2) Induce the production and accumulation of molecules of interest in microalgae;

(3) Harvest biomass; and

 (4) Extract and purify molecules of interest.

One of the brakes slowing the development of these processes on an industrial scale is related to the high cost of the post-culture process and the re-cultivation of microalgae. Innovative methods are therefore sought to reduce the costs of production / extraction of molecules of interest, including proteins of interest, from microalgae so that they are competitive with current methods of producing animal and / or vegetable proteins. The phenomenon of electroporation, or electro-permeabilization, was observed for the first time in 1754 by Nollet: it corresponds to an increase in the membrane permeability of cells when they are subjected to an electric field. But it was not until the 1980s that the first applications of pulsed electric field (PEC) processes were developed. The CEP methods were first used in the field of medicine to transfer DNA into cells. They then took off in the field of biotechnology through many other applications. Among these can be mentioned genetic transformation and inactivation of microorganisms as well as the extraction of biomolecules.

 Several recent studies have focused on the extraction of water-soluble microalgae compounds through a process involving treatment involving pulsed electric fields (CEP treatment). These studies are based either on the application of CEP treatment alone or on a succession of different extraction processes including the application of a CEP treatment. Among the methods consisting of a succession of processes, there is in particular a combination of a CEP treatment with a heating of the suspensions of microalgae or processes including a CEP treatment followed by extraction by solvent. None of the extraction methods used in the studies mentioned above were found to be completely satisfactory. They all have the disadvantage of being destructive of the biomass to be extracted. They therefore produce organic or organic waste in the case of using extraction solvents. In addition, growing new biomass is extremely expensive and time consuming.

 Some methods also accumulate other disadvantages such as non-selectivity of the extracted molecules and therefore the need for post-extraction purification steps, a low yield of protein extraction (only exceeding in very rare cases 10% total cellular protein content), poor energy efficiency (over-consumption of electricity), or poor production efficiency (time required for abnormally long extraction).

 3. Objectives of the invention

The invention aims to overcome at least some disadvantages of the prior art. The invention particularly aims to provide a method for extracting intracellular water-soluble molecules of unicellular microalgae to preserve the viability of most of the biomass extracted.

 Another object of the invention is, at least according to certain advantageous embodiments, to provide a method for the extraction of intracellular water-soluble molecules of microalgae, especially proteins, mainly from chloroplasts.

 Another object of the invention is, at least according to certain advantageous embodiments, to provide a method for the extraction of intracellular water-soluble molecules of microalgae, especially proteins, with good yields.

 Another object of the invention is, at least according to certain advantageous embodiments, to provide a simple, fast, energy efficient and low cost process.

 Another object of the invention is, at least according to some embodiments, to provide a method for obtaining proteins at a comparable cost or less than the animal and vegetable proteins currently available on the market.

 4. Presentation of the invention

The present invention relates to a method of extracting substantially non-destructive intracellular water soluble molecules from unicellular microalgae. The method comprises:

 a step of supplying unicellular microalgae, the microalgae being devoid of secondary cell wall or having a secondary cell wall of thickness less than or equal to 0.5 miti;

a step of placing the microalgae in a medium of low electrolytic conductivity to form a suspension of microalgae, the total electrolytic conductivity of the microalgae suspension having a value ranging from 15 to 40 pS.cm ¹ at 25 ° C .;

a step of applying a pulsed electric field to the suspension of microalgae in order to obtain a suspension of microalgae temporarily permeabilized, the pulsed electric field having an electric force ranging from 0.5 to 1 kV.cm ¹ ; and,

a step of incubation of the suspension of microalgae temporarily permeabilized, in order to obtain microalgae impoverished by intracellular water-soluble molecules in suspension in a medium enriched in water-soluble molecules extracted, the incubation step for the diffusion of intracellular water-soluble molecules of microalgae at least temporarily permeabilized in the medium of low electrolytic conductivity.

 For the purposes of the present invention, the term "essentially non-destructive" means that the process makes it possible to keep microalgal cells subjected to the pulsed electric field as viable as possible. The process according to the present invention makes it possible in particular to keep at least 40%, preferably at least 60%, and very preferably at least 80% of the microalgae cells viable.

 The term "water-soluble intracellular molecules", including proteins, carbohydrates or ions inside the cells before extraction.

The inventors of the present invention have been able to demonstrate experimentally that it is possible to effectively extract water-soluble intracellular molecules from microalgae while at the same time optimally preserving the viability of the latter, thus making it possible to reuse them later for a new extraction according to the invention. extraction process of the invention. In particular, the inventors have demonstrated that a pulsed electric force electric field ranging from 0.5 kV.cm ¹ to 1 kV.cm ¹ , it is possible to reversibly and sufficiently permeabilize (for a non-electrical extraction). negligible water-soluble molecules) the plasma membrane and, where appropriate, the different walls overhanging it, in particular the primary cell wall and / or the secondary cell wall, of unicellular microalgae lacking a secondary cell wall or having a secondary cell wall of lesser thickness or equal to 0.5 pm. Conversely, a pulsed electric field of electric force strictly less than 0.5 kV.cm ¹ is too weak to sufficiently permeabilize the plasma membrane and, where appropriate, the different walls overlooking the microalga and does not allow the extraction in quantity. non-negligible intracellular water-soluble molecules. Furthermore, a pulsed electrical force field strictly greater than 1 kV.cm ¹ results in irreversible permeabilization of the plasma membrane and, where appropriate, overlying cell walls for some or all of the microalgae, without any additional gain in efficiency. in extraction of intracellular water-soluble molecules. An irreversible permeabilization of the plasma membrane and, where appropriate, the overlying cell walls ultimately implies the destruction of the cells subjected to the pulsed electric field, which is not desired in the present invention. The placement of microalgae in a medium of low electrolytic conductivity, the total electrolytic conductivity of the medium of low electrolytic conductivity including microalgae having a value ranging from 15 to 40 pS.cm ¹ at 25 ° C, ensures that the microalgae of the suspension of microalgae heat up little or very little during the application of the pulsed electric field. Indeed, the higher the conductivity of the microalgae suspension medium, the more it is able to store heat during the application of the pulsed electric field. However, too high temperatures lead to the denaturation of the extracted molecules and the destruction of the microalgae cells.

 The first step of the process according to the invention is a step of supplying unicellular microalgae, that is to say microscopic algae formed by a single cell.

 The plasma membrane of unicellular microalgae separates the intracellular medium from the extracellular medium. Depending on the different stages of development of the unicellular microalga, a primary wall may cover the plasma membrane and a secondary wall may cover the primary wall. The secondary cell wall is less flexible than the primary cell wall.

 The microalgae used in the process according to the invention do not have a secondary wall or alternatively have a secondary cell wall with a thickness of less than or equal to 0.5 micrometers (μm).

 Preferably, the microalgae used in the process according to the invention have a mean diameter of between 15 and 30 μm.

 In particular, unicellular microalgae may belong to the order Chlamydomonadales, class Chlorophyceae, phylum Chlorophyta. Microalgae of this order are green microalgae with two flagella at a vegetative stage. Their cytoplasm comprises at least one chloroplast. As indicated above, thanks to genetic engineering, chloroplasts can be advantageously used to produce water-soluble molecules of interest.

 Specifically, unicellular microalgae may belong to the Haematococcaceae family.

Even more specifically, unicellular microalgae may belong to the genus Haematococcus. In particular, unicellular microalgae may belong to the species Haematococcus pluvialis, such as the strain of microorganisms deposited in the Culture Collection of Algae and Protozoa (CCAP), Scottish Marine Institute, United Kingdom having as order number: CCAP 34 / 19 Haematococcus pluvialis.

 The microalgae of the species Haematococcus pluvialis can be found in puddles, gutters and other shallow water points. Its ability to survive in these environments that can be considered hostile is linked to its life cycle, which is both very complex and flexible. This microalgae has three main stages of development: macrozoid, palmella and cyst. The macrozoid and palmella stages are observable during the vegetative growth phase (growth by mitosis) whereas the cyst stage only appears after the cell divisions have stopped.

 Microalgae Haematococcus pluvialis macrozoid stage are mobile ovoid cells whose diameter can vary between 8 and 50 pm, the average diameter is generally between 20 and 25 pm. They have two flagella of equal length and are surrounded by a multilayer glycoprotein matrix traversed by cytoplasmic prolongations. At this stage the cells multiply by simple mitosis: a mother cell gives two daughter cells which will then separate, surround themselves with their own gangue and thus become independent.

 The second stage observed during the vegetative phase is the palmella stage. Palmellas also generally have a mean diameter of between 20 and 25 μm. In contrast to macrozoids, palmellas do not have flagella and are spherical in shape. The glycoprotein matrix surrounding the cell is thinner than in macrozoids and a primary wall is set up between this matrix and the plasma membrane. The division of cells at this stage of development can lead to the formation of either new palmellas or new macrozoids. The palmella stage is observable when environmental conditions are favorable for microalgae. However, it can also occur when these conditions begin to degrade and cells must be prepared to enter a rest phase during which they will be at the cyst stage.

The appearance of cells in the cyst stage is preceded by an increase in the size of the microalgae, whose diameter may exceed 50 pm. Due to a large accumulation of astaxanthin (up to 99% of total cell carotenoids and 5% of dry weight of biomass), the pigment composition of the cells also undergoes important variations. The encystment phase is also accompanied by both structural and compositional modifications of the extracellular wall, leading to progressive thickening of the latter. Indeed, the different layers of the glycoprotein matrix characterizing the macrozoic stage disintegrate one after the other during the encystment and several new structures are gradually put in place to replace them. The first structure put in place is the primary wall, composed largely of cellulose (the primary wall, present in the palmella stage, is transient since it disappears during the maturation of cysts). The second structure, also set up is a trilaminar sheath. It is composed of algaenan, a non-hydrolysable biopolymer. The third and last structure put in place corresponds to the secondary wall. Its major constituent is mannan (a term comprising polysaccharides essentially composed of mannose), and more particularly granular, non-fibrillar mannan. The maximum thickness reached by the secondary wall of the cysts is about 2.5 μm.

 Advantageously, the microalgae of the species Haematococcus pluvialis used in the process according to the present invention is at a vegetative stage, macrozoid and / or palmella. Indeed, it is known that the microalgae of the species Haematococcus pluvialis at a vegetative stage comprises from 25% to 30% by weight of protein solids. In addition, recent studies have shown that the water-soluble proteins produced at this stage of the Haematococcus pluvialis cell cycle have good emulsifying properties, having a real interest in the fields of cosmetology, and diet (Ba. et al., 2016).

 The microalgae of the species Haematococcus pluvialis used for the method of the present invention can also be at the beginning of a phase of encystment, transition between a macrozoid stage and / or palmella and a cyst stage, when the secondary cell wall has a thickness less than or equal to 0.5 μm.

The second step of the process according to the invention is the microalgae placement step in a medium of low electrolytic conductivity. The medium of low electrolytic conductivity must be biocompatible so as not to damage the microalgae. The total electrolytic conductivity of the medium of low electrolytic conductivity including microalgae has a value ranging from 15 to 40 micro-siemens per centimeter (pS.cm ^_1 ) at 25 ° C (reference temperature of the conductivity measurement). The medium of low electrolytic conductivity makes it possible to guarantee that the microalgae of the microalgae suspension heat up little or very little during the application step of the pulsed electric field. Advantageously, the microalgae suspension is heated by less than 1 ° C during the step of applying the pulsed electric field.

 According to a particular embodiment, the medium of low electrolytic conductivity is water, especially distilled water.

Preferably, the density of the microalgae in the microalgae suspension is less than or equal to 10 ⁶ cells / ml of medium of low electrolytic conductivity. This has the advantage of ensuring that the electric field felt by each cell is substantially uniform during the application step of the pulsed electric field. On the contrary, for microalgae densities strictly greater than 10 ⁶ cells / mL, a screen effect, all the more important as the density of microalgae is important, implies that some microalgae feel a greater electric field than others when of the application step of the pulsed electric field. More preferably, the density of microalgae in the microalgae suspension is of the order of 10 ⁵ cells / ml medium of low electrolytic conductivity.

 Preferably, the suspension of microalgae is maintained at a pH ranging from 6.5 to 8.

 The third step of the method according to the invention is the step of applying a pulsed electric field on the suspension of microalgae to obtain a suspension of microalgae temporarily permeabilized. The application of a pulsed electric field allows the spontaneous formation of pores in the plasma membrane and, where appropriate, in the primary cell wall and the secondary cell wall. This is the cause of permeabilization. In addition, during the application of the electric field, rapid electrophoretic transport takes place, resulting in charged molecules and intracellular ions.

A pulsed electric field is an electric field formed by means of electrical pulses. Depending on the amplitude of the electrical pulses, different pulsed electric field electric forces can be obtained. The electric force is expressed here in kilovolt per centimeter (kV.cm ^-1 ) The electric force of the pulsed electric field ranges from 0.5 to 1 kV.cm ¹ . Since the electric force is less than or equal to 1 kV.cm ¹ , the permeabilization is made essentially temporary, or in other words, essentially reversible. This is the best way to preserve the viability of microalgae. Conversely, for higher field electric forces, a larger portion of the microalgae or even all the microalgae are irreversibly permeabilized, without a significant gain in the extraction yield of extracted water-soluble molecules is observed. In addition, it appears that field electric forces greater than or equal to 0.5 kV.cm ¹ are required to ensure sufficient pore opening.

 It is not necessary to apply a large number of pulses to obtain temporary permeabilization of microalgae. The number of pulses is greater than or equal to one. The number of pulses may in particular be greater than or equal to 2. In this case, the pulsed electric field is preferably periodic. The pulsed electric field may for example have a frequency having a value ranging from 0.1 to 19 Hz, in particular a value of 9.62 Hz. In the case where the number of pulses is greater than or equal to 2, the pulses may alternatively a positive sign and a negative sign. The pulsed electric field is then called bipolar. A bipolar electric field prevents the oxidation of the electrodes and increases the life of the latter. In addition, it reduces the formation of reactive oxygen species.

 The number of pulses may also be greater than or equal to 5. According to one embodiment, the number of pulses is equal to 5.

The pulsed electric field pulses can have a duration of between 1 microsecond (ps) and 10 milliseconds (ms). This range of values has the advantage of allowing the permeabilization of the plasma membrane and, where appropriate, the different walls overlooking the microalgae, while having a limited impact on the cytoskeleton, nucleus and DNA of the microalgae necessary for its viability. It should be noted that the longer the pulse duration, the larger the pores formed. Preferably, the pulses have a duration of between 0.1 ms and 5 ms. This also makes it possible to target the permeabilization of chloroplasts. In this same perspective, the duration of the pulses is advantageously chosen between 1 ms and 3 ms. The fourth step of the process according to the invention is a step of incubating said suspension of microalgae temporarily permeabilized, in order to obtain microalgae depleted of intracellular water-soluble molecules in suspension in a medium enriched in extracted water-soluble molecules. The pores formed during the step of applying the pulsed electric field being temporarily open, a diffusion of intracellular water-soluble molecules of the temporarily permeabilized microalgae takes place in the medium of low electrolytic conductivity. Even if the electrophoretic transport, taking place during the application of a pulsed electric field, is more effective than the diffusion in quantity of molecules extracted per unit of time, it lasts much less. Indeed, the period during which the plasma membrane is permeabilized following the application of the CEP may extend over several minutes, or even a few tens of minutes, while the duration of a pulse does not exceed 10 ms. The diffusion time is thus about 10,000 times higher than that of the pulses. The majority of the water-soluble molecules extracted thus pass from the intracellular medium to the extracellular medium via the diffusion phenomenon during the incubation step of the suspension of microalgae temporarily permeabilized. The incubation step may have a duration ranging from 3 to 45 minutes. Preferably, it has a duration ranging from 5 to 10 minutes.

 Among the intracellular water-soluble molecules, the method according to the invention makes it possible in particular to extract proteins.

These proteins may in particular have a molecular weight of up to 120 kilo-Daltons (kDa-1 Dalton being defined as 1/12 of the mass of a nuclide atom ¹² C). According to advantageous embodiments of the invention, where a high permeabilization (essentially reversible) is obtained, the molecular weight of the extracted proteins can be up to 150 kDa on electrophoresis gel (in particular polyacrylamide gel) and go up to at 265 kDa (approximately) by mass spectrometry.

Proteins can be derived from cytoplasm, chloroplasts, Golgi apparatus, endoplasmic reticulum or mitochondria. In particular, the proteins may be derived, at least in part, from chloroplasts of microalgae. In some embodiments, at least 50% of the extracted proteins are derived from microalgae chloroplasts. This may especially be the case when the pulse duration of the pulsed electric field during the step of applying a pulsed electric field is between 1 ms and 3 ms. The extraction process according to the invention may further comprise a separation step after the step of incubating the suspension of microalgae temporarily permeabilized. The separation step makes it possible to separate the depleted microalgae into extracted water-soluble molecules and the medium enriched in extracted water-soluble molecules. It is implemented by usual methods, such as centrifugation. The water-soluble molecules extracted, in particular the proteins, can then be isolated from their medium by usual methods, in particular by chromatography and / or electrophoresis.

 The extraction process according to the invention may further comprise a step of recovery / regeneration of microalgae depleted in water-soluble molecules extracted after the separation step. The recovery / regeneration step consists in incubating the microalgae depleted in water soluble molecules extracted, in a nutrient medium adapted to the culture of microalgae. This step makes it possible in particular to restore the correct functioning of the photosynthetic machinery (recovery) of cells depleted in water-soluble molecules extracted after the latter have begun to synthesize de novo the electro-extracted molecules. This also allows the cells to multiply again (regeneration).

A nutrient medium adapted to the culture of microalgae can in particular be a growth medium for microorganisms comprising nitrogen, phosphorus and a source of organic carbon. In particular, the nutrient medium adapted to the culture of microalgae may be tri-acetate-phosphate medium (TAP). The so-called TAP medium is well known to those skilled in the art for the cultivation of microalgae, in particular for the highly studied microalgae Chlamydomonas reinhardtii. The TAP medium consists of Trishydroxymethylaminomethane (buffer, known as TRIS), acetate ions, a phosphate solution, Beijerinck salts (NH ₄ Cl + MgSO ₄ + CaCl ₂ ) and trace elements. form of traces.

The recovery / regeneration step has, if necessary, a duration greater than or equal to one day, preferably a duration greater than or equal to two days and, very preferably, a duration greater than three days. In particular, it is estimated that after three days of recovery / regeneration, the microalgae cells recovered a physiological state similar to that of untreated cells. The process comprising a final recovery / regeneration step makes it possible in particular to keep at least 85%, preferably at least 95%, and most preferably at least 99% of the microalgae cells viable.

 5. List of figures

 Figure 1 shows a typical bipolar pulse pattern (abscissa: Time in milliseconds (ms) - ordinate: Millivolt voltage (mV)).

 Figure 2 represents the concentration of water-soluble proteins extracted (in the extracellular medium) as a function of the electric force of the pulsed electric field applied (number of pulses 5 or 9).

 Figure 3 represents heating of the suspensions as a function of the electric force of the applied pulsed electric field (reference temperature: 21.1 ° C).

 Figure 4 shows the percentage of moving cells as a function of the electric force of the applied pulsed electric field.

 Figure 5 shows the percentage of cells permeabilized as a function of the electric force of the applied pulsed electric field.

 Figure 6 shows the crude photosynthetic activity ("APb") / respiration rate ("R") as a function of the electric force of the pulsed electric field applied.

Figure 7 shows the maximum quantum efficiency of the PS II (F _Ro ) as a function of the electric force of the applied pulsed electric field.

 Figure 8 shows the percentage of mobile cells as a function of recovery / regeneration time.

 Figure 9 shows the percentage of permeabilized cells as a function of recovery / regeneration time.

 Figure 10 shows cell density versus recovery / regeneration time.

 Figure 11 represents crude photosynthetic activity as a function of recovery / regeneration time.

Figure 12 shows the maximum quantum efficiency of PS II (F _Ro ) as a function of recovery / regeneration time.

Figure 13 shows the respiration rate as a function of recovery / regeneration time. 6. Detailed description of an embodiment

 6.1 - Obtaining an Axenized Strain of Haematococcus pluvialis

 A strain was collected in a natural environment in a rainwater retention pond. It was first isolated and then axenized by treatment with sodium hypochlorite.

 The new isolated and axenized strain was identified on the basis of morphological criteria as belonging to the species Haematococcus pluvialis. In order to complete the morphological identification of the new strain, a phylogenetic analysis was performed. The taxonomic positioning of the new strain was clarified following the creation of a phylogenetic tree based on a dataset containing the sequence of the 18S rRNA gene from several species belonging to the taxonomic order of Chlamydomonadales, including that of the new isolated strain. The results obtained show that our new strain belongs to the same clade as the species Haematococcus pluvialis and Haematococcus lacustris (both denominations being taxonomic synonyms).

 The isolated and axenized strain was deposited on 11 December 2017 at the Culture Collection of Algae and Protozoa (CCAP), Scottish Marine Institute, in the United Kingdom and has as order number: CCAP 34/19 Haematococcus pluvialis

 6.2 - Obtaining green vegetative cells (macrozoid and palmella stages)

Microalgae of the isolated and axenized strain, hereinafter called "microalgae", were cultured under sterile conditions in 250 or 500 mL Erlenmeyer flasks containing a quarter of their volume capacity in culture medium. The initial cell density of each culture was fixed at 10 ⁴ cells. mL ¹ , the inoculum being composed of cells from a parent culture aged about 3 to 4 weeks. The temperature of the culture chamber was maintained at 19 ° C.

The best culture parameters were obtained in TAP medium in mixotrophic Conditions (exposed to illumination 35 pmol photons. M ^{2. 1} s with a photoperiod of 14 h).

With these culture parameters, microalgae are not observed at the cyst stage. The encystment phase could nevertheless be caused by nitrogen deficiency (stress conditions). The cultivation parameters for other species of the order Chlamydomonadales can be reasonably arranged without representing an excessive burden for the skilled person. The parameters to be adapted include the culture temperature and / or the autotrophic, mixotrophic or heterotrophic conditions.

 6.3 - Preparation of a suspension of microalgae

The microalgae grown (see section 6.2) were collected by centrifugation (5 min at 400 g, 19 ° C) before being transferred under sterile conditions into distilled water to obtain a suspension of microalgae with a density of 10 ^5. cells / mL. The suspension has a conductivity of about 20 pS.cm ¹ .

 6.4 - Application of pulsed electric fields and incubation of the suspension

temporarily permeabilized - experimental study

 An electro-permeabilization bench was used for the application of pulsed electric fields. The main equipment composing the electro-permeabilization bench used in this study were the following:

 two interconnected pulse generators (DEEX-Bio, Tech, France) allowing the application of bipolar pulses to cell suspensions,

- two electroporation chambers (CNRS). Depending on the desired field strength, either of the two available chambers has been selected. Indeed, the distance between the two electrodes located inside each chamber being 6 or 3 mm, the maximum electric force of the pulses that can be delivered between these two electrodes is respectively 3 or 6 kV.cm ¹ .

A bipolar pulse train was delivered to each cell suspension passing through the electroporation chamber through the action of a peristaltic pump (Fisher Scientific Variable-Flow Peristaltic Pump, Fisher Scientific, Pittsburgh, USA). The flow rate to be applied by this pump (Q. in mL.s ¹ ) was calculated using the following equation:

 Q. = (F x V) / N

where N is the average number of pulses delivered to each microalga as it passes through the chamber, F is the repetition frequency of the pulses (F = 9.62 Hz) and V is the volume capacity of the electroporation chamber (0.54 or 0.27 mL for the chamber of 6 or 3 mm, respectively).

A typical profile of the bipolar pulses delivered by the two generators is shown in Figure 1. For each selected number of pulses (N = 5 or 9), five different field strengths were tested (0.5, 1, 2, 3 and 6 kV.cm ¹ ). The field strengths equal to 0.5 and 1 kV.cm ^{1 respectively} correspond to the field electric forces according to the invention. The field strengths equal to 2, 3 and 6 kV.cm ¹ do not correspond to the field electric forces according to the invention.

The duration of each pulse was set at 2 ms. The condition "0 kV.cm ^1,1 was used as a control condition: the treatment protocol was identical to that used in the case of the other forces except that no pulse was delivered during the passage of the suspension in In addition, in order to evaluate the Joule warming of microalgae suspensions due to the treatment with PEL, the temperature of each of them was measured just before and immediately after the application of the electrical impulses with the aid of The same procedure was used to evaluate the impact of the CEP on the conductivity of the suspensions.

 Following these measurements, cell suspensions were incubated for 45 min at room temperature to allow sufficient time for intracellular compounds to diffuse out of the cells. After a centrifugation step (5 min at 400 g, 19 ° C), the supernatants were collected in order to assay the extracted water-soluble proteins which diffused through the plasma membrane of the treated microalgae.

 6.4-A. Influence of force and number of pulses on protein extraction efficiency

 6.4-A.i) Impact of incubation of cells in distilled water on the diffusion of water-soluble proteins

 Three different controls were prepared:

- a control condition "0 kV.cm ¹ " for which the same treatment protocol as that used in the case of the other forces was applied, except that no electrical pulse was delivered during the passage of the suspension in the electroporation chamber.

a negative control for which the cells were incubated for 45 min (same duration as that of the post-treatment incubation period) in distilled water, just like the treated cells, without any other treatment being given to them; be applied. This control was performed to determine the amount of protein diffusing out of the cells placed in a hypotonic medium for a specified duration (45 min). a positive control for which the microalgae were ground using glass microbeads in order to extract all the water-soluble proteins.

According to Figure 2, no significant difference in terms of extracted protein concentration could be detected between the negative control and the control condition "0 kV.cm ¹ ". This observation therefore suggests that the mechanical forces applied to the cells due to the use of the peristaltic pump do not lead to additional diffusion (compared to the negative control) of the proteins towards the extracellular medium.

6.4-A.ii) Influence of field strength on the diffusion of water-soluble proteins

The different CEP treatments lead to a significant increase in the concentration of extracted proteins compared to the negative control and the control condition (Figure 2). However, the efficiency of protein extraction is not proportional to the strength of the field: it increases steadily and significantly (up to a factor of 5, compared to the control condition) with lower field forces or equal to 1 kV.cm ¹ , then it tends to stabilize when higher field strengths are used. The membrane permeabilization induced by the CEP treatment (1 kV.cm ¹ ) is such that after 45 minutes of post-treatment incubation, approximately 50% of the water-soluble proteins extracted by grinding with glass microbeads (positive control) can be collected. (Figure 2).

6.4-A.iii) Influence of the number of pulses on the diffusion of water-soluble proteins

The evolution of the concentration of proteins in the extracellular medium as a function of the force of the field always follows the same trend, whether with one or the other of the two numbers of pulses (N = 5 or 9; 2). In addition, an increase in the value of this parameter (from 5 to 9) does not significantly influence the concentration of electro-extracted proteins assayed in the supernatant (FIG. 2). These results therefore make it possible to conclude that the number of pulses has no synergistic effect with the strength of the electric field on the extraction efficiency of the water-soluble proteins, and therefore potentially on the degree of permeabilization of the plasma membrane.

 Therefore, the number of pulses is set at a value of 5 for subsequent experiments.

 6.4-B. Influence of the force of the pulses on the physiological state of the treated cells

The impact of electric field intensity on the physiological state of microalgae was evaluated by measuring the following five parameters: cell mobility, permeabilization of the plasma membrane, photosynthetic activity, yield maximum quantum of PS II photochemistry (F _Ro ) and respiration rate. These measurements were carried out after a 30 min recovery period during which the microalgae were returned to the previously defined mixotrophic culture conditions (TAP medium, illumination of 35 pmoles of photons, m ^{~ 2,} s ¹ ).

 The advantage of this recovery period is to allow time for the cells transferred into TAP medium to recover (at least in part) from the stress induced by their stay in distilled water, and thus be able to mainly study the impact of CEP on their viability.

6.4-B.i) Joule heating of suspensions

According to Figure 3, heating for pulsed electric fields whose electric force is equal to 0.5 or 1 kV.cm ¹ is less than 1 ° C. The maximum heating observed is 7.1 ° C for the pulsed electric field whose electric force is equal to 6 kV.cm ¹ .

6.4-B.ii) The mobility of microalgae

Cell mobility was analyzed to evaluate the ability of PECs to affect the locomotor system (consisting of two flagella) of the microalga H. pluvialis at the macrozoid stage. The results obtained indicate that only 40% of the total cells of the control condition are mobile (see Figure 4). The higher proportion of microalgae at the palmella stage than at the macrozoid stage within the parent culture explains this observation, which is not particularly singular since it coincides with the results obtained on cells cultured for 8 days in conditions mixotrophs. In fact, the proportion of mobile cells calculated under these conditions is 52 ± 1.8%, which is not biologically different from the value measured for the control condition (40 ± 8%). Whatever the strength of the electric field, the CEP treatment causes a decrease in the percentage of moving cells (compared to the control condition). The loss of mobility between the cells of the control condition and those of the condition "0.5 kV.cm ^1.1 is 50% .The subsequent increase in the force of the pulses (0.5 to 2 kV.cm ¹ ) causes a proportional decrease in the percentage of mobile cells.For the highest intensities of CEP (3 and 6 kV.cm ¹ ), the value of this percentage remains close to 4 %.The absence of mobility does not necessarily imply cell death without mobility: the musculoskeletal system can be affected by the CEP treatment without necessarily involving the death of the cells, whereas the presence of cell mobility indicates the viability of the cells.

6.4-B.iii) Permeabilization of the plasma membrane

The impact of different CEP treatments on the plasma membrane has been studied using the blue Evans. It is a permeability marker capable of crossing the plasma membrane when the latter is permeabilized. The cells having incorporated the marker are stained blue whereas the microalgae whose plasma membrane is intact remain green. The main limitation of this marker is that it does not allow the differentiation of permeabilized cells transiently (and therefore always viable) irreversibly permeabilized cells that are already dead or who will become so. Nevertheless, according to the staining protocol used, the permeability measurements were carried out 75 min after the end of the treatment (45 min of incubation post-treatment + 30 min of recovery in TAP medium (similar to the recovery step regeneration presented in section 8), which is sufficient for a majority of transiently permeabilized cells to return to their original conformation (of the order of a few minutes to ten minutes when the duration of the pulses is the order of the ms).

 The results related to the use of Evans blue which will be discussed in the following give an indication, but do not allow to conclude in a quantitative way, the ability of CEPs to kill microalgae via irreversible permeabilization of their plasma membrane. Only the qualitative aspect of the results (see section 8 for more details on the relevance of this method at the quantitative level), incorporation, respectively non-incorporation, of the dye is to be considered and indicates that the cells are permeabilized (irreversibly but also in some measure reversibly), respectively the cells are non-permeabilized.

The results obtained for the control condition (0 kV.cm ¹ ) indicate that 24.2% of the cells not exposed to the CEP are permeabilized (see Figure 5). This means that 24.2% of the cells would not be viable from the start in the control culture. The concentration of proteins assayed in the supernatant for the control condition and the condition "0.5 kV.cm ^1.1 (Figure 2) is in favor of overestimating the proportion of permeabilized cells for the control condition. the concentration of proteins assayed after the application of 0.5 kV.cm ¹ CEP is on average three times greater than that recorded for the control condition, and if the proportion of permeabilized cells for the control condition is in fact between 8 and 10%, while it is three times lower than the proportion calculated for the condition "0.5 kV.cm ¹ ", which eliminates the inconsistency between the extraction data and those of permeabilization. shown in Figure 5 will therefore be described and discussed on the basis of a proportion of permeabilized cells for the 9% control condition.

Regardless of the strength of the pulses delivered, the proportion of permeabilized cells increases relative to the control condition (Figure 5). However, this increase is not constant: with PECs of 0.5 and 1 kV.cm ¹ , the proportion of cells stained by Evans blue is multiplied by a factor of 3 (27% of permeabilized cells) and 6 ( 54% permeabilized cells) compared to the control condition, whereas with the strongest electric field strengths (2 to 6 kV.cm ¹ ), the proportion of permeabilized cells stabilizes at a value of on average 67 %.

 6.4-B.iv) Photosynthetic parameters

In order to study the impact of the CEP on the functioning of the photosynthetic microalgae device, two parameters were measured: the raw photosynthetic activity (APb) and the maximum quantum yield of the photochemistry of the PS II (FRo). The raw photosynthetic activity provides information on the actual capacity of the cells to perform photosynthesis since it corresponds to the amount of 0 ₂ produced in the light during the oxidation reaction of water. The F _o parameter translates the maximum photochemical efficiency of PS II. This parameter is based on the principle that the light picked up by PS II collector antennas can be used to perform photochemistry, or be re-emitted into the environment in the form of heat and / or fluorescence. The parameter F _o is proportional to the maximum quantum yield of photochemistry. The optimum value of F _o varies between 0.78 and 0.85. The impact of PEL on the operation of the photosynthetic apparatus depends on the strength of the pulses. As can be seen in FIGS. 6 and 7, the values of the photosynthetic activity (FIG. 6) and of the parameter F _o (R 1 ut 7) are not significantly different from those of the control condition only with the forces of fields greater than or equal to 1 kV.cm ¹ .

 The fact that PECs cause a decrease in the value of these two parameters implies that long pulses (> 1 ps), such as those used in this study, associated with sufficiently high field strength can impact chloroplasts as well as thylakoïdes, and thus the smooth running of the processes that take place there.

 6.4-B.v) Cellular respiration

The impact of the strength of the electric field on the rate of cellular respiration is similar to that observed for photosynthetic activity (see Figure 6). In conclusion of sections 6.4-A and 6.4-B, the results indicate that the best compromise between protein extraction efficiency and microalgae survival is achieved for a pulsed electric field with an electrical force of 0.5 to 1 kV .cm ¹ .

 7) Identification of electro-extracted proteins

Protein extracts obtained with 0.5 and 1 kV.cm ¹ were separated by 1D electrophoresis. The strips were collected to identify all the proteins present on them. Thanks to Nano-LC ESI MS-MS analyzes, 52 proteins have been characterized and identified (at least in part). Of these, 36 proteins were electroextracted only with CEPs of lkV.cm ¹ . The remaining 16 proteins were electro extracted with both CEP conditions. The increase in the number of "match peptides" for the condition "1 kV.cm ¹ " (compared to the condition "0.5 kV.cm ¹ ") suggests that, in addition to the variety of proteins, the extracted from the same protein increases with the strength of the pulses.

Electro-extracted proteins with both field strengths were also separated by 2D electrophoresis. The electrophoretic profiles made it possible to study the impact of the force of the pulses on the number and the intensity of the spots. The increase in the number (new spots) and intensity of the spots (for those already present with 0.5 kV.cm ¹ ) visible for the condition "1 kV.cm ^-1 " confirms that membrane electro-permeabilization and the electro-extraction of proteins depends on the strength of the pulses. Nevertheless, the silver staining having been used to visualize the spots, only the proteins present for the condition "1 kV.cm ¹ " but absent for the condition "0.5 kV.cm ¹ " were collected in order to be analyzed by mass spectrometry (microsequencing). Only 3 could be characterized (numbers "88", "186", "205"). In addition, four spots (A, B, C and D) more intense for 1 kV.cm ¹ than for 0.5 kV.cm ¹ were randomly selected for mass spectrometry analysis.

The results obtained are summarized in Table 1 below. The first three proteins (n ° 88, 186 and 205) correspond to the spots, taken from 2D electrophoresis gels, which are visible only for the condition "1 kV.cm ¹ ". The following four proteins (Nos. A, B, C and D) correspond to spots, taken from the same gels, which are more intense for the condition "1 kV.cm ¹ " than for the condition "0.5 kV. cm ¹ ". The following 52 proteins (# 1 to 52) come from collected strips

after electrophoresis 1D SDS-PAGE. Proteins 1 to 16 were electro-extracted with CEP values of 0.5 and 1 kV.cm ¹ while 36 others (nos. 17 to 52) were electro-extracted only with CEP of lkV.cm ¹ .

 Table 1

The characterization of the electro-extracted proteins and the analysis of the visible spots on the electrophoretic profiles corresponding to the conditions "0.5 kV.cm ^_1 " and "1 kV.cm ^_1 " have showed that the efficiency of the CEP in terms of protein variety and, for the same protein, amount of electro-extracted material depends on the strength of the pulses. The identification of the electro-extracted proteins with CEP of 1 kV.cm ¹ allowed us to determine the initial intracellular localization of these and therefore the compartments affected by the CEP. According to the results obtained, the cytoplasm, the chloroplast, the Golgi apparatus, the endoplasmic reticulum and, potentially, the mitochondria belong to it. CEPs of 1 kV.cm ¹ are therefore strong enough to permeabilize the membrane (s) of the organelles and affect the processes that take place there. In contrast, no cell wall protein was identified among the extracted proteins. In addition, nearly half of the identified proteins are located (before extraction) in the chloroplast.

 8) Recovery / Regeneration of microalgae

The cell pellets obtained after the centrifugation step are placed in TAP culture medium. The cell density of the new suspensions thus obtained was fixed at 5 × 10 ⁴ cells. mL ¹ .

In order to study the recovery / regeneration capacity of microalgae, various parameters reflecting their physiological state were measured during the three days following their exposure to PECs of 1 kV.cm ¹ (N = 5): the mobility, the permeabilization of plasma membrane, cell multiplication, photosynthetic activity, maximal quantum yield of PS II photochemistry and respiration. The values obtained were compared to that of a control condition (0 kV.cm ¹ ) for which the same treatment protocol was used except that no pulse was delivered during the passage of the cells in the chamber. electroporation.

Under both conditions (0 and 1 kV.cm ¹ ), the percentage of mobile cells increases from the first day of the recovery period and continues to increase over the next two days to finally reach 85% of the total number of microalgae in both populations (see Figure 8). To explain this result, the following hypothesis can be formulated: the increase in mobility is the result of the appearance of new flagellated cells (macrozoid stage) thanks to the resumption of cellular divisions. The proportional increase in cell density (see Figure 10) and the percentage of mobile cells in both conditions (0 and 1 kV.cm ¹ ) reinforce the validity of this hypothesis. In the case of the condition "1 kV.cm ¹ ", the proportion of moving cells increases more strongly than for the control condition. A second hypothesis (valid only for cells with the condition "1 kV.cm ¹ ") can therefore be considered: during the recovery period, the flagellar machinery of the cells treated with CEP resumes normal functioning (damage repair , increase of ATP synthesis).

The percentage of permeabilized cells (FIG. 9) within the suspensions treated with 1 kV.cm ¹ CEP is significantly higher (55% on average of the total number of cells) than that calculated for the control condition during the first 24 hours. the payback period. It returns to a basal level within the next 24 hours. In the case of the control condition (0 kV.cm ¹ ), the proportion of permeabilized cells remained stable and less than 10% (on average) of the total population throughout the follow-up period. Now a plasma membrane transiently permeabilized following the application of electrical pulses of the order of the MS regains its initial conformation in just a few minutes (at most). The first permeabilization measurement being carried out 75 min after the end of the treatment, the percentage of permeabilized cells at day 0 (57.5%) should therefore correspond to the proportion of irreversibly permeabilized cells. These microalgae should therefore no longer be viable. The decrease in the proportion of permeabilized cells observed between days 1 and 2 would therefore be a priori due to the multiplication of the cells that remained viable following the treatment (that is to say the 42.5% of non-permeabilized cells). Nevertheless, the comparison of the evolution of the proportion of (irreversibly) permeabilized cells relative to that of the cell density reveals an inconsistency at day 1. Indeed, the cell density of cultures treated with 1 kV.cm CEP ¹ increases continuously during the follow-up period and (almost) at the same rate as the control condition cultures (Figure 10). As the number of cells doubled between day 0 and day 1, the proportion of cells that were exposed to the CEP in the total population is thus halved. However, the proportion of permeabilized cells does not vary significantly during the first 24 hours, which would indicate that some of the cells from divisions that occurred after treatment would be permeabilized whereas these "daughter" cells were not exposed to electrical impulses. Because of their ability to divide, the cells incorporating Evans blue are therefore obviously still viable. This result calls into question the relevance of the choice of Evans blue as a quantitative marker of reversible / irreversible permeabilization, and therefore of viability / mortality.

 The cell density of CEP-treated cultures increases early in the recovery period. The first post-treatment divisions therefore take place during the 24 hours following the transfer of the cells into fresh TAP medium. The microalgae, however, divide a little more slowly (0.5 versus 0.63 d-1) than untreated cells (Figure 10) even if the difference in cell density between the two conditions is not significant (p value> 0.05) during the three days of follow-up.

 The rapid increase in cell density of the treated cultures indicates that the microalgae very quickly produce enough energy in the form of ATP to multiply. Two processes are responsible for ATP synthesis: respiration and photophosphorylation (photosynthesis). During the first two days of the recovery period, the respiration rate of the treated cells increases (Figure 13), which could indicate an increase in ATP production. Nevertheless, the parallel increase in cell respiration rate of the control condition suggests bacterial contamination of the treated (treated and untreated) cultures early in the follow-up period. Therefore, it is likely that bacteria are responsible for increasing the measured respiration rate for both conditions.

At the same time, the values of the photosynthetic activity (Figure 11) and the maximal quantum yield of the photochemistry of the PS IIs (Figure 12) of the treated cells remain significantly lower than those of the cells of the control condition (0 kV.cm ¹ ). The energy provided by photophosphorylation is therefore reduced during this period. Since ATP (missing) is not produced by more intense respiration, the capacity of the treated cells to achieve photochemistry therefore seems to be large enough to provide the energy necessary for the multiplication of microalgae.

From the 2nd day of recovery, the values of the photosynthetic activity and the parameter F _Ro of the cells treated with CEP increase to reach values which are no longer significantly lower than those of the untreated cells (FIGS. 11 and 12). As the cell density of the treated cultures increases from the beginning of the monitoring period, the proportion of cells that have been exposed to the CEP in these cultures decreases over time. However, since the values of the photosynthetic parameters increase only later, the damage caused by the CEP to the photosynthetic machinery seems to be "transmitted" to cells from post-treatment divisions. The results presented above strongly suggest that the CEP treatment leads to the permeabilization of chloroplast envelopes and thylakoid membranes, thus allowing the diffusion of proteins and possibly chlorophyll and carotenoid molecules. The hypothesis that the slow recovery (72h) of photosynthetic machinery following CEP treatment is related to the time required (1) to de novo synthesis of electro-extracted molecules and (2) to the photosynthetic electron transport chain to function again normally can be issued.

The results presented in this section to conclude that the cells treated with CEP kV.cm 1 ¹ (N = 5) are able to regain a physiological condition similar to that of untreated cells within 3 days of application of the treatment.

 9) Optimization of the incubation step

The objective has been to improve the physiological state of the treated cells without reducing the extraction yield of the water-soluble proteins. Distilled water is not an optimal medium for the culture of H. pluvialis, the impact of the duration of the post-treatment incubation period of cells in distilled water was studied. The CEP parameters previously defined (1 kV.cm ¹ and N = 5) have not been modified.

The results show that a reduced incubation period of 5 min constitutes a good compromise between the extraction efficiency of the proteins and the cell viability.

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Claims

 A method of extracting substantially non-destructive intracellular water-soluble molecules from unicellular microalgae, said method comprising:

 a step of supplying unicellular microalgae, said microalgae being devoid of secondary cell wall or having a secondary cell wall of thickness less than or equal to 0.5 miti;

a step of placing said microalgae in a medium of low electrolytic conductivity to form a microalgae suspension, the total electrolytic conductivity of said microalgae suspension having a value ranging from 15 and 40 μS cm ^-1 at 25 ° C .;

a step of applying a pulsed electric field on said suspension of microalgae in order to obtain a suspension of microalgae temporarily permeabilized, said pulsed electric field having an electric force ranging from 0.5 to 1 kV.cm ¹ ; and,

 a step of incubating said suspension of microalgae temporarily permeabilized, in order to obtain microalgae depleted of intracellular water-soluble molecules in suspension in a medium enriched in extracted water-soluble molecules, said incubation step allowing the diffusion of intracellular water-soluble molecules of said microalgae at least temporarily permeabilized in the medium of low electrolytic conductivity.

2. Extraction process according to claim 1, wherein said microalgae are unicellular microalgae of the order of Chlamydomonadales, preferentially of the family Haematococcaceae and more preferably of the genus Haematococcus.

3. Extraction process according to claim 2, wherein said microalgae being unicellular microalgae of the species Haematococcus pluvialis, such as the strain deposited in the Culture Collection ofAlgae and Protozoa, Scottish Marine Institute, in the United Kingdom and having as number Order: CCAP 34/19 Haematococcus pluvialis.

4. Extraction process according to any one of the preceding claims, wherein the density of said microalgae in said microalgae suspension is lower or equal to 10 ⁶ cells / mL medium of low electrolytic conductivity; and preferably of the order of 10 ⁵ cells / ml medium of low electrolytic conductivity.

5. Extraction method according to any one of the preceding claims, wherein said pulsed electric field has pulses of duration between lps and 10 ms; preferably pulses of duration between 0.1 ms and 5 ms; and, very preferably, pulses of duration between 1 ms and 3 ms.

6. Extraction process according to any one of the preceding claims, wherein said incubation step has a duration ranging from 3 to 45 minutes; preferably has a duration ranging from 5 to 10 minutes.

The extraction method according to any one of the preceding claims, wherein said water-soluble molecules are proteins.

8. The method of claim 7, wherein said proteins have a molecular weight of up to 120 kDa and preferably a molecular weight of up to 150 kDa on electrophoresis gel and up to 165 kDa by mass spectrometry.

9. Extraction process according to one of 7 or 8, wherein at least 50% of said extracted proteins is derived from the chloroplasts of said microalgae.

10. Extraction process according to any one of the preceding claims, said method comprising a step of separating the microalgae depleted in water-soluble molecules extracted and the medium enriched in said water-soluble molecules extracted.

11. Extraction method according to claim 10, said method comprising a step of recovery / regeneration of said microalgae depleted in water-soluble molecules extracted after said separation step, said recovery / regeneration step consisting of incubation of said microalgae depleted in water-soluble molecules in a nutrient medium adapted to the culture of said microalgae.