EP2825628A1

EP2825628A1 - Production of eicosapentaenoic acid and/or arachidonic acid in mixotrophic mode using euglena

Info

Publication number: EP2825628A1
Application number: EP13715323.5A
Authority: EP
Inventors: Khadidja Romari; Pierre Calleja
Original assignee: Fermentalg SA
Current assignee: Fermentalg SA
Priority date: 2012-03-16
Filing date: 2013-03-15
Publication date: 2015-01-21
Also published as: JP2015510762A; WO2013136023A1; FR2988096B1; FR2988096A1; US20150044738A1

Abstract

The invention relates to new strains of microalgae belonging to the Euglena genus, allowing production of lipids, in particular of EPA and/or ARA, in mixotrophic mode, and to a method for selecting and culturing such strains, using a variable and/or discontinuous light source, in particular a flashing light.

Description

Production of eicosapentaenoic acid and / or arachidonic acid in mixotrophic mode by Eualena

The invention relates to a method of cultivation in mixotrophic mode, especially in the presence of a discontinuous illumination and / or variable light, of a microalga of the genus Euglena, in particular of the species of Euglena gracilis. The method makes it possible to obtain a high yield of biomass and an enrichment of the microalgae thus cultivated with lipids and more particularly with eicosapentaenoic acid (EPA) and / or arachidonic acid (ARA). The method thus makes it possible to select Euglena gracilis strains of a mixotrophic nature, and having a high yield of lipids and more particularly of polyunsaturated fatty acids. The invention also relates to a new microalgae strain belonging to the species Euglena gracilis, particularly suitable for the production of fatty acids. This new strain of Euglena gracilis is useful for producing ΙΈΡΑ (eicosapentaenoic acid) and arachidonic acid (ARA) in mixotrophic mode. Preamble

Microalgae are photosynthetic microorganisms of autotrophic nature, that is to say having the ability to grow autonomously by photosynthesis.

Microalgae grow in marine as well as in fresh and brackish waters, as well as in various terrestrial habitats.

Most microalgae species found in freshwater or oceans are usually autotrophic, that is, they can only grow by photosynthesis. For these, the presence in their environment of carbon substrates or organic material is not favorable to them and does not improve their growth. However, a number microalgae species, families and very diverse origins, prove to be not strictly autotrophic. Thus some of them, called heterotrophic, are able to develop in the total absence of light, by fermentation, that is to say by exploiting the organic matter.

Other microalgae species, for which photosynthesis remains essential for their development, are able to take advantage of both photosynthesis and organic matter present in their environment. These intermediate species, called mixotrophs, can be grown both in the presence of light and organic matter.

This particularity of so-called "mixotrophic" algae seems to be linked to their metabolism, which allows them to operate simultaneously photosynthesis and fermentation. Both types of metabolism coexist with a positive overall effect on algal growth [Yang, C. et al. (2000); Biochemical Engineering Journal, 6: 87-102].

Microalgae are currently the subject of many industrial projects because some species are able to accumulate or secrete significant amounts of lipids, including polyunsaturated fatty acids.

Among these polyunsaturated fatty acids, certain highly unsaturated omega-3 (PUFA-3) series, in particular eicosapentaenoic acid (EPA or C20: 5 ω3) and docosahexaenoic acid (DHA or C22: 6 ω3), and of the omega-6 series (PUFA-ωθ), in particular arachidonic acid (AA or ARA or eicosatetraenoic acid C20: 4 ω6), have a recognized nutritional importance and have high potential in terms of therapeutic applications. .

Fish oils from the fishing industry are currently the main commercial source of this type of fatty acid. However, while these oils find new applications (food supplement in aquaculture, incorporation in margarines), marine fisheries resources are becoming scarce due to intensive fishing activity. New sources of these fatty acids such as ΙΈΡΑ and TARA must therefore be sought in order to meet, in the future, the growing market demand for this type of polyunsaturated fatty acids.

In addition to their capacity to synthesize de novo fatty acids, microalgae offer several advantages over fish oils: they are cultivable in vitro under controlled conditions, which allows the production of a biomass of relatively constant biochemical composition and, d. On the other hand, unlike fish oils, they do not have an unpleasant smell and their lipids contain little or no cholesterol.

Finally, the lipids produced by microalgae have a simpler fatty acid profile than that of fish oils, which limits the separation steps of the fatty acids of interest.

At present, the classification of algae is still largely based on morphological criteria and the nature of photosynthetic pigments that contain their cells. As a result, it is not very indicative of the autotrophic, heterotrophic or mixotrophic nature of algal species, whereas the latter cover a very large diversity of species and forms [Dubinsky et al. (2010); Hydrobiologia, 639: 153-171].

The taxonomic classification of eukaryotic algae contains 14 phyla. Among the species of the different classes composing these phyla, which produce fatty acids, there are significant variations in the content of polyunsaturated fatty acids in microalgae. Moreover, the relative proportions of lipids, in particular EPA and ARA in the lipid profiles, vary according to the species and the culture conditions.

The main microalgae of interest, producing EPA and ARA, are marine species. However, among the hundreds of thousands of marine microalgae species, only a small number have a high content of both of these fatty acids at the same time and sufficient capacity to be cultured in vitro. The species of interest are mainly Bacillariophytes (or diatoms) from marine phytoplankton. They are generally characterized by an active production of EPA.

Although rich in α-linolenic acid (C18: 3 u) 3), freshwater microalgae generally do not contain EPA [Pencreac'h et al. (2004) Marine microalgae: alternative source of EPA and DHA, Lipids, 11 (2): 118-222].

On the other hand, the production of ARA is quite rare for marine algae. Such microalgae include, but are not limited to Euglenophytes (eg, Euglena), Rhodophytes (eg, Porphyridium) and Chlorophytes (eg, Parietochloris).

Several strains of freshwater microalgae are able to synthesize and accumulate arachidonic acid. Among others, the triglycerides of Chlorophyte Parietochloris incisa, an isolated algae from Mount Tateyama in Japan, are naturally rich in arachidonic acid. ARA is predominantly present as triglycerides which constitute the dominant lipid class [Bigogno C. et al. (2002); Lipid and fatty acid composition of the green oleaginous alga Parietochloris incisa, the richest plant source of arachidonic acid, Phytochemistry, 60 (5): 497-503].

To implement the production of fatty acids by microalgae on an industrial scale, several factors must be taken into account. For example, the cultures can be carried out in autotrophic, mixotrophic or heterotrophic conditions depending on the strain, the temperature, the light conditions and the size of the fermenters. For example, crops can also be grown in one-liter containers, in a laboratory, in photobioreactors, and in 100,000-liter containers or in open ponds (several hectares). However, energy expenditure and other resources such as labor and the ease of continuing cultivation must be taken into account by developing ideal growing conditions.

In any case, it is desirable that the microalgae be grown under optimal conditions to increase the yield of (s) fatty acid (s) to produce. So, it's best to have the most return high potential (eg biomass above 30 g / 1 dry matter, and more than 15% fatty acids relative to dry matter).

International Patent Application WO 03/079810 discloses cultures of Parietoch loris incisa for the production of ARA for animal food products. These cultures were performed under autotrophic conditions with an ARA yield of at least 10% ARA by weight based on the weight of these cells.

Japanese patent applications JP9252764 and JP60087798 thus describe strains of Monodus subterraneus grown in autotrophic mode that can accumulate an amount of EPA of up to 3.8% of their dry weight. These strains were cultured under laboratory conditions, ie in inorganic culture media, in flasks or bioreactors of low volumetric capacity with a continuous light supply.

In the perspective of industrial exploitation, such a mode of cultivation proves to be unsuitable. Indeed, to be profitable, the production of biomass must be able to be carried out in closed large photo-bioreactors. However, such a culture method is difficult to achieve in autotrophic mode, because when the cell density increases in the culture medium, the cells have more and more difficulty in capturing light from outside the reactor. It is then necessary to actively stir the culture medium, which requires a significant energy expenditure.

It would be desirable to be able to obtain higher EPA and ARA yields for more efficient and profitable industrial exploitation.

Euglena is a common genus of Flagellate Protista and belongs to the Euglenoids family, which does not have a rigid wall, which gives them a characteristic shape mobility during their displacement, which is called the euglenoid movement. Euglenes can lose their chloroplasts and produce depigmented heterotrophic individuals that are slightly different from flagellated protozoa. The Euglenes have a stigma, orange spot that fulfills the role of photoreceptor and allows the microorganism to move. The flagella are permanently present in the Euglenes. The metabolism of Euglenes is versatile. In the presence of light, they are autotrophic, and in the absence of light or after the loss of their chloroplast, they become heterotrophic and are particularly able to metabolize lactate [Lechevalier, Hubert A. (1977); Fungi, Algae, Protozoa, and Viruses, CRC Handbook of Microbiology, 2nd Edition, Vol. He, p. 874, Florida, Laskin, Allen I.].

In the scientific article "Biomass production in mixotrophic culture of

It has been shown that the biomass yield obtained with Euglena gracilis grown in mixotrophic conditions (with continuous light) is 15%, 1522, 1523-1228. at 19% higher than the yield obtained with culture under heterotrophic conditions, high chlorophyll levels of 39.4 mg per liter and carotenoids of 13.8 mg per liter were also obtained in mixotrophic cultures.

In the scientific article by Hayashi M et al: "Enriching Euglena with unsaturated fatty acids", BIOSCIENCE BIOTECHNOLOGY BIOCHEMISTRY, vol 57, No. 2, January 1, 1993, p 352-353, the enrichment of microalgae of the genus Euglena gracilis EPA is described The microalgae are cultivated under heterotrophic conditions with an addition of EPA or DHA of 0 to 1% to the culture medium in order to obtain microalgae with a 75.6% EPA relative to to their total lipids, 0.5% EPA had to be added to the culture medium, which represents a costly and unprofitable step for industrial exploitation.

Thus, it was at the end of many experiments under unusual light conditions and by the addition of different substrates that the applicant managed to isolate microalgae strains of the Euglena gracilis species, cultivable in mixotrophic mode, allowing, in the conditions of the present invention, high production yield of polyunsaturated fatty acids, in particular EPA and ARA.

A strain (FCC 540) representative of the new strains of Euglena gracilis thus isolated and selected, was deposited with the CCAP (Culture Collection of Algae and Protozoa, Scottish Association for Marine Science, Dunstaffnage Marine Laboratory, Oban, Argyll PA371QA, Scotland , United Kingdom) under the provisions of the Budapest Treaty, accession number CCAP 1224/49.

The cultivation and selection process consisted more particularly in cultivating microalgae under mixotrophic conditions, in the presence of a variable and / or discontinuous illumination, in particular in the form of flashes, with a range of variations in light intensity and a frequency specific.

The close alternation of illuminated phases and dark or less luminous phases, generally perceived as stressful by microalgae, has allowed, surprisingly, to obtain strains of Euglena gracilis a high production of biomass, lipids and more. especially polyunsaturated fatty acids. This implementation of the strains according to the invention opens the prospect of an industrial production of polyunsaturated fatty acids, in particular EPA and ARA, in fermenters benefiting from a reduced light input, and should therefore make it possible to carry out energy savings compared to autotrophic farming methods.

The various aspects and advantages of the invention are detailed below.

detailed description

The subject of the present invention is therefore a process for culturing microalgae of the genus Euglena, in particular of the Euglena gracilis species, in mixotrophic mode, under discontinuous and / or variable illumination conditions over time. The illumination has intensity variations whose amplitude is generally between 5 pmol. m ^"2 , s ^{" 1} and 1000 μιηοΙ. m ^"2 , s ^{" 1} , preferably between 30 and 400 μητιοΙ. m ^"2 , s ^{" 1} . These variations can generally take place between 2 and 3600 times per hour, preferably between 2 and 200 times per hour. These cultivation conditions make it possible to provide a defined quantity of light. This luminous contribution may comprise phases of discontinuous and / or variable illumination, with variations in intensity that may have identical or different amplitudes. The illumination can be in particular in the form of flashes.

This process has the advantage of increasing the yield of biomass obtained from the culture. It also has the advantage of enriching the microalgae thus cultured in polyunsaturated fatty acids, more particularly in eicosapentaenoic acid (EPA) and / or arachidonic acid (ARA). This method can also be used to select strains of the genus Euglena, in particular Euglena gracilis, of a mixotrophic nature, and having a high yield of polyunsaturated fatty acids, in particular ΙΈΡΑ (eicosapentaenoic acid) and / or TARA ( arachidonic acid).

The mixotrophic culture of this microalga is preferably carried out in the presence of 5 mM to 1 M, preferably from 50 mM to 800 mM, more preferably from 70 mM to 600 mM, and still more preferably from 100 mM to 500 mM. an organic carbon substrate. The supply of the substrate is ensured continuously during the culture, to allow the cells to accumulate a high concentration of lipids. Additional substrate is added to the culture medium during the culture process to maintain a constant concentration. This organic carbon substrate preferably comprises, in pure form or as a mixture: glucose, cellulose derivatives, lactate, starch, lactose, sucrose, acetate and / or glycerol.

The organic carbon substrate contained in the culture medium may consist of complex molecules or a mixture of substrates. Products resulting from the biotransformation of starch, for example from corn, wheat or potato, in particular starch hydrolysates, which consist of small molecules, constitute, for example, carbon substrates adapted to the mixotrophic culture of microalgae according to the invention.

This process is more particularly intended for the implementation of new microalgae strains of the genus Euglena (Phylum: Euglenoza, Order: Euglenales, Family: Euglenaceae) [ITIS Catalog of Life, 2010] selected for their mixotrophic nature, particularly for their ability to be cultivated with a light input greater than 10 μl, in a mineral medium, for example Euglena medium [Andersen, RA; Jacobson, D.M. & Sexton, J.P. (1991) - Provasoli-Guillard Center for Culture of Marine Phytoplankton, Catalog of Strains. 98pp. West Boothbay Harbor, Maine, USA] in which an organic carbon substrate is added. Preferably, the organic carbon substrate comprises glucose and / or lactate, in a concentration equivalent to or greater than 5 mM.

These new Euglena strains, more particularly Euglena gracilis, can be isolated and selected according to the method of selection and culture according to the invention described below.

A representative strain of the Euglena gracilis strains according to the invention is the strain FCC 540 isolated by the applicant and deposited at the CCAP, under the number CCAP 1224/49. Such strains are capable of producing significant quantities of biomass as well as lipids, and more particularly of ΙΈΡΑ and ARRA when they are cultivated in mixotrophic mode with a variable and / or discontinuous light supply, according to the invention.

According to ongoing taxonomic analyzes, strain CCAP 1224/49 belongs to the species Euglena gracilis. The invention relates to any strain of the species Euglena gracilis, capable of growing under mixotrophic culture conditions as described in the present application, and capable of producing fatty acids, such as TARA and ΙΈΡΑ. The invention also relates to any species of microalgae of the genus Euglena, capable of growing under mixotrophic culture conditions as described in the present application, and capable of producing fatty acids, such as TARA and ΙΈΡΑ. The euglena gracilis strains isolated according to the invention make it possible to produce, under mixotrophic conditions, significant amounts of biomass as well as lipids rich in EPA and / or ARA, said EPA and / or ARA being able to represent more than 10%, more than 25%, or more than 40% of the total lipids contained in microalgae.

In the present invention, the biomass obtained with the FCC 540 strain, isolated by the applicant, from a culture in mixotrophic conditions in the presence of a variable and / or discontinuous illumination, in particular in the form of flashes, is from 10 to 60% , more generally from 20 to 50%, greater than that of a culture with the same strain carried out in heterotrophic mode. Heterotrophic mode means identical culture conditions, except for the absence of illumination.

The subject of the invention is thus a process for culturing microalgae of the genus Euglena, in particular of Euglena gracilis species in mixotrophic mode, in the presence of a variable and / or discontinuous illumination with the passage of time, for example in the form of flashes, especially for producing polyunsaturated fatty acids, such as ΙΈΡΑ and TARA.

The subject of the invention is therefore a process for the selection of microalgae of the genus Euglena, in particular of Euglena gracilis species with a mixotrophic nature, and having a high yield of polyunsaturated fatty acids such as ΙΈΡΑ and TARA, in the presence of variable illumination. and / or discontinuous over time.

It appeared that a variable and / or discontinuous illumination of crops, particularly in the implementation of a mixotrophic culture, had a favorable impact on the development of algae and made it possible to increase the productivity of these crops. , particularly with regard to their lipid production. Without being bound by theory, the inventor believes that a discontinuous and / or variable light supply to microalgae has the effect of causing a "stress" favorable to the growth and synthesis of lipids. This may be explained, in part, by the fact that in nature, microalgae tend to accumulate lipid reserves to withstand the stresses of their environment. By discontinuous illumination, it is necessary to hear an illumination punctuated by periods of darkness. The periods of darkness may occupy more than a quarter of the time, preferably half or more of the time, during which the algae are grown.

According to a preferred aspect of the invention, the illumination is discontinuous and more preferably in the form of flashes. A flash, within the meaning of the invention, is a short period of illumination, that is to say less than 30 minutes. The duration of the flash may be less than 15 minutes, preferably less than 5 minutes or more preferably less than 1 minute. According to some embodiments of the invention, the flash duration may be less than one second. For example, the flash duration can be 1/10 of a second, or 2/10 of a second, or 3/10 of a second, or 4/10 of a second or 5 / 10 of a second, or 6/10 of a second, or 7/10 of a second, or 8/10 of a second, or 9/10 of a second. The illuminance, or flash, is usually longer than 15 seconds. The duration of the flash is generally between 5 seconds and 10 minutes, preferably between 10 seconds and 2 minutes, more preferably between 20 seconds and 1 minute.

In general, the number of flashes is between about 2 and 3600 per hour. It can be, for example, between 100 and 3600 flashes per hour. It can also be between 120 and 3000, or between 400 and 2500, or between 600 and 2000, or between 800 and 1500 flashes per hour. It may also be between 2 and 200, preferably between 10 and 150, more preferably between 15 and 100, and even more preferably between 20 and 50 per hour. Flashes can be emitted at regular intervals or not in time. In case of transmission at regular intervals, the number of flashes per hour then corresponds to a frequency (F) having a time period (T), being considered that F = 1 / T. This time period can be between 1 second and 30 minutes, or between 1 second and 36 seconds, or between 1, 2 seconds and 30 seconds, or between 1.44 seconds and 9 seconds, or between 1.8 seconds and 6 seconds. seconds, or between 2.4 seconds and 4.5 seconds. This frequency can also be between 18 seconds and 30 minutes, preferably between 24 seconds and 6 minutes, more preferably between 36 seconds and 4 minutes, and even more preferably between 72 seconds and 3 minutes. The number of flashes per hour is chosen according to the intensity and duration of the flashes (see below). In general, the intensity of the light provided in the form of flashes is between 5 and 1000 pmol. m ^"2 , s ^{" 1} , preferably between 5 and 500 pmol. m ^"2 , s ^{" 1} , or 50 and 400 pmol. m ^"2 , s ^{" 1} , and more preferably between 150 and 300 pmol. m ^"2 , s ^{" 1} . By definition, 1 pmol. m ^"2 , s ^{" 1} corresponds to 1 μΕ m ^"2 , s ^{" 1} (Einstein), a unit often used in the literature.

According to a particular embodiment of the invention, the intensity of the light is between 50 and 200 pmol. m ^"2 , s ^{" 1} , the flash frequency is between 10 seconds and 60 minutes for a flash duration of between 1 second and 1 minute.

According to another embodiment of the invention, the illumination may be variable, which means that the illumination is not interrupted by dark phases, but that the light intensity varies over time. This variation in light intensity is regular and can be periodic or cyclic. According to the invention, it is also possible to carry out a light supply combining continuous and discontinuous illumination phases.

According to the invention, whatever the lighting conditions, the light intensity provided to the algae in culture, expressed in micromoles of photons per second per square meter (pmol.m ^-2 , s ^-1 ), varies at least one times in one hour. The amplitude of this variation in light intensity is generally between 5 and 1000, or between 50 and 800, or between 100 and 600 pmol. m ^"2 , s ^{" 1} . The intensity of the light can also vary between 5 and 400 pmol. m ^"2 , s ^{" 1} . Preferably, the magnitude of the light intensity variation is between 70 and 300 pmol. m ^"2 , s ^{" 1} and more preferably between 100 and 200 pmol. m ^"2 , s ^{" 1} .

Said luminous intensity can successively reach, under conditions of variable illumination, for example, the values 50 pmol. m ^"2 , s ^{" 1} and 100 pmol. m ^"2 s ^{" 1} , or 5 and 400 pmol. m ^"2 , s ^{" 1} , or 50 and 800 pmol. m ^"2 , s ^{" 1} several times each hour. Said luminous intensity can successively reach, preferably, the values 50 and 200 pmol. m ^"2 ^{s -1.} Alternatively, batch lighting conditions, said light intensity can reach successively several times within one hour, for example, the values 0 and 50 pmol. ^{m" 2} s ^"1, the values 0 and 100 pmol.m.sup.- ² , s.sup.- ¹ or, more preferably, the values 0 and 200 pmol.m.sup.- ² , s.sup.- ^1, It may also successively, several times in the hour, for example, the values 0 and 300 pmol m ^-2 , s ^-1 , the values 0 and 600 pmol m ^-2 , s ^-1 , the values 0 and 800 pmol m ^-2 , s ^-1, or the values 0 and 1000 pmol. m ^"2 , s ^{" 1} .

According to a mode of the invention and whatever the conditions of illumination, the intensity of the light brought to the culture varies according to the cell density. The denser the culture, the more intense the light. The cell density is the number of cells per ml and is measured according to the techniques known to those skilled in the art.

In the initial stage of the culture when the cell density is between about 10 ⁵ and 5 x 10 ⁵ cells per ml, the light intensity may be between 5 and 15 pmol. m ^"2 , s ^{" 1} , preferably between 5 and 10 pmol. m ^"2 , s ^{" 1} . When the culture reaches a density between 10 ⁶ and 10 ⁷ cells per ml, the light intensity can be increased to between 15 and 200 pmol. m ^"2 , s ^{" 1} , for example, preferably between 20 and 50 pmol. m ^"2 , s ^{" 1} . When the culture, at the final stage, reaches a density between 10 ⁷ and 10 ⁸ cells per ml, the light intensity can be increased to between 50 and 400 pmol. m ^"2 , s ^{" 1} for example, preferably between 50 and 150 pmol. m ^"2 , s ^{" 1} .

According to some embodiments, for example when the duration of the flashes is for example less than one minute, or less than one second, the intensity of the light may be greater compared to the values mentioned above. In the initial stage of the culture, when the cell density is between about 10 ⁵ and 5 x 10 ⁵ cells per ml, the light intensity may be between 5 and 200 pmol. m ^"2 , s ^{" 1} , preferably between 5 and 100 pmol. m ^"2 , s ^{" 1} . When the culture reaches a density between 10 ⁶ and 10 ⁷ cells per ml, the light intensity can be increased to between 30 and 500 pmol. m ^"2 , s ^{" 1} , by for example, preferably between 50 and 400 μιτιοΙ. m ^"2 , s ^{" 1} . When the culture, at the final stage, reaches a density between 10 ⁷ and 10 ⁸ cells per ml, the light intensity can be increased to between 100 and 1000 pmol. m ^"2 , s ^{" 1} for example, preferably between 200 and 500 μιτιοΙ. m ^"2 , s ^{" 1} .

According to a mode of the invention, the quantity of light brought to the culture in the hour remains between certain values. It is between about 2000 and 600 000, preferably between 2000 and 300 000 mol. m ^"2. It can be between about 4000 and 200 000 pmol. ^{m" 2} per hour.

According to a mode of the invention, the culture is illuminated with 30 flashes per hour, each flash having a duration of 30 seconds and an intensity of 10 μητιοΙ. m ^"2 , s ^{" 1} . The latter gives a total light input per hour of 9000 μιτιοΙ. m ^"2. In another embodiment of the invention, the culture is irradiated with 20 flashes per hour, each flash having a duration of 30 seconds and an intensity of 20 pmol. ^{m" 2} s ^"1. This gives total light output per hour of 12,000 pmol.m ^-2 . According to another embodiment of the invention, the culture is illuminated with 45 flashes per hour, each flash having a duration of 15 seconds and an intensity of 5 pmol. m ^"2 , s ^{" 1} , which gives a total light input per hour of 3375 pmol. m ^"2. In another embodiment of the invention, culture is illuminated with 120 flashes per hour, each having a flash duration of 10 seconds and an intensity of 200 μιτιοΙ. ^{m" 2} s ^"1, to give a total light output per hour of 240,000 μιηοΙ m- ² .

As described for the light intensity above, and according to a mode of the invention, the amount of light provided to the culture per hour may vary depending on the cell density. In the initial stage of the culture, when the cell density is 10 ⁵ and 5 x 10 ⁵ cells per ml, the total light supply in the hour is generally between about 1500 and 8000, preferably 1500 and 6000 pmol. m ^"2 , more preferably between 2000 and 5000 pmol. m ^{" 2} . When the culture reaches a density of between 10 ⁶ and 10 ⁷ cells per ml, the total light supply in the hour can be increased to between 6000 and 67000 pmol. m ^"2 , preferably between 6000 and 50,000, and more preferably between 12,000 and 45,000 pmol ^" m ^"2 , for example. At final stage of the culture, at a cell density between 10 ⁷ and 10 ⁸ cells per ml, the total light supply in the hour can be increased to between 45 000 and 300 000, for example preferably between 45 000 and 200,000 pmol. m ^"2 , and for example, more preferably between 50,000 and 150,000 μιτιοΙ. m ^" ² .

According to a mode of the invention, at the initial stage of the culture (at a cell density between 10 ⁵ and 5 × 10 ⁵ cells per ml), the culture is illuminated with 30 flashes per hour, each flash having a duration of 30 seconds and an intensity between 5 and 10 pmol. m ^"2 , s ^{" 1} , which gives a total light input per hour of 2250 μητιοΙ. m ² to 4500 pmol m ^-2 . Then, in the intermediate stage (at a cell density between 10 ⁶ and 10 ⁷ cells per ml), the culture is illuminated with 30 flashes per hour, each flash having a duration of 30 seconds and an intensity between 15 and 50 μιτιοΙ. m ^"2 , s ^{" 1} , which gives a total light input per hour of 13,500 to 45,000 pmol. m ^"2. Then, at the final stage of culture (to a cell density of 10 ⁷ and 10 ⁸ cell per ml), the culture is irradiated with 30 flashes per hour, each flash having a duration of 30 seconds and an intensity between 50 and 150 μιηοΙ m ^"2 , s ^{" 1} , which gives a total light output per hour of 45,000 to 135,000 μηποΙ .rrï ² .

According to a mode of the invention, for example, when the duration of the flashes is for example less than one minute, or less than one second, at the initial stage of the culture (at a cell density between 10 ⁵ and 5 x 10 ⁵ cells per ml), the culture is illuminated with 30 flashes per hour, each flash having a duration of 10 seconds and an intensity between 50 and 100 pmol. m ^"2 , s ^{" 1} , which gives a total light input per hour of 15,000 μητιοΙ. m ² to 30,000 pmol m ^-2 . Then at the intermediate stage (at a cell density between 10 ⁶ and 10 ⁷ cells per ml), the culture is illuminated with 50 flashes per hour, each flash having a duration of 10 seconds and an intensity between 200 and 300 μιτιοΙ. m ^"2 , s ^{" 1} , which gives a total light input per hour of 100,000 to 150,000 pmol. m ^"2. Then, at the final stage of culture (at a cell density between 10 ⁷ and 10 ⁸ cell per ml), the culture is illuminated with 120 flashes per hour, each having a flash duration of 10 seconds and intensity between 350 and 450 μιτιοΙ. m ^"2 , s ^{" 1} , which gives a total light output per hour of 420,000 to 540,000 pmol. m ^"2 .

The contribution of light in the cultures can be obtained by lamps distributed around the external wall of the fermenters. A clock triggers these lamps for defined lighting times. Fermentors are preferably located in an enclosure away from daylight, which can control the ambient temperature.

As observed by the applicant, the fact that the strains thus selected have good aptitude to grow in mixotrophic mode, in the presence of discontinuous and / or variable light, predisposes said strains to a higher production of acids. polyunsaturated fats, including EPA and ARA.

The culture method according to the invention thus makes it possible to select strains of the genus Euglena, in particular of the species Euglena gracilis of a mixotrophic nature, similar to that isolated by the applicant and deposited with the CCAP under the number CCAP 1224/49. and having a high yield of polyunsaturated fatty acids. This culture method is characterized in that it comprises the following steps:

a) Mixotrophic culture of one or more strains of the genus Euglena under discontinuous and / or variable illumination conditions over time, the illumination having intensity variations whose amplitude is between 5 μιηοΙ . m ^"2 , s ^{" 1} and 1000, preferably between 5 and 400 pmol. m ^"2 , s ^{" 1} , these variations occurring between 2 and 3600, preferably 5-400 times per hour,

b) a step of maintaining said culture over several generations, in the presence of an organic carbon substrate in the culture medium, and optionally

c) a step of recovery of the microalgae thus cultivated.

By recovery step is meant more particularly the isolation of the strain or strains whose cell number has grown the most during said generations. To carry out the selection of the strains, various strains of the genus Euglena, in particular of the Euglena species gracilis, can be cultured, in parallel, on microplates in the same enclosure, with precise monitoring of the conditions and evolution of the different cultures. It is thus easy to know the response of the various strains to the discontinuous and / or variable illumination and, where appropriate, the addition of one or more carbon substrates in the culture medium. Strains that respond favorably to discontinuous and / or variable illumination and to carbon substrates generally offer a better yield for lipid production in terms of quality (polyunsaturated fatty acids more abundant in the lipid profile) and quantitative (lipids contain higher proportion of EPA and / or ARA).

The microalgae can be selected in a fermentor from a heterogeneous population and the preferred variants of which are to be selected by the selection method according to the invention combining discontinuous and / or variable light having a range of light intensity and a frequency specific, with mixotrophic growing conditions. In this case, the culture is practiced by maintaining the microalgae in cultures over many generations, then an isolation of the components that have become the majority in the culture medium is carried out at the end of the culture.

The culture method according to the invention also makes it possible to produce lipids.

In this case, the method according to the invention also comprises the following steps:

d) a step of recovery of the lipids of the microalgae, and possibly

e) extracting eicosapentaenoic acid (EPA) and / or arachidonic acid (ARA) from the recovered lipids.

The culture method according to the invention can also be applied to any species of the genus Euglena, capable of growing under the mixotrophic conditions according to the invention, and capable of producing ΙΈΡΑ and / or TARA.

The culture method according to the invention makes it possible to optimize the production of the biomass obtained from the culture. It also allows to enrich the microalgae thus cultured in polyunsaturated fatty acids, more particularly in eicosapentaenoic acid (EPA) and / or arachidonic acid (ARA).

The invention therefore also aims at optimizing the production of biomass, as well as the production of lipids, in particular of fatty acids, via the cultivation of microalgae of the Euglena genus of a mixotrophic nature, preferably cultivated or selected according to the methods referred to above, then the recovery of microalgae cultivated to extract the lipids, especially ΙΈΡΑ and / or TARA. Strains of the species Euglena gracilis are especially concerned.

Selective lipid extraction methods of which ΙΈΡΑ and TARA are known to those skilled in the art and are, for example, described by [Bligh, E.G. and Dyer, W.J. (1959); A rapid method of total lipid extraction and purification, Can. J. Biochem. Physiol., 37: 911-917].

The invention also relates to microalgae of the genus Euglena gracilis, which can be obtained according to the process of the invention as previously described. These microalgae are enriched in polyunsaturated fatty acids. The total lipids of such microalgae generally comprise more than 15% or 20%, often more than 30% and sometimes even more than 40% EPA and / or ARA based on the total percentage of lipids.

Example 1 Euglena gracilis FCC 540 cultures are carried out in 2L fermentors (bioreactors) useful with dedicated automata and supervision by computer station. The system is regulated in pH via addition of base (1N sodium hydroxide solution) and / or acid (1N sulfuric acid solution). The culture temperature is set at 22 ° C. Stirring is carried out by means of two stirring shakers placed on the shaft according to the following configuration: Rushton propeller and three-bladed pumping propellers. The stirring speed and the aeration rate are regulated at the minimum = 100 rpm and max = 250 rpm and Qmini = 0.5 wm / Qmaxi = 2 wm respectively. The bioreactor is equipped with an external lighting system surrounding the transparent tank. The intensity as well as the light cycles are controlled by a dedicated automaton and supervised by a computer station.

The reactors are inoculated with a pre-culture performed on a stirring table (140 rpm) in a thermostatically controlled enclosure (22 ° C.) and illuminated between 80 and 100 μE. Pre-cultures and cultures in bioreactors are carried out in medium Euglena medium [Starr, R.C. & Zeikus, J.A. (1993) - UTEX - The Culture Collection of Algae at the University of Texas at Austin. J. Phycol. Suppl. 29]. The organic carbon substrate used for the bioreactor mixotrophic culture is glucose at concentrations between 100 mM and 150 mM.

Crop monitoring:

The total biomass concentration is monitored by measuring the dry mass (filtration on GFC filter, Whatman, then drying in a vacuum oven at 65 ° C and -0.8 bar, for 24 hours minimum before weighing).

Regarding the quantification of total lipids, 10 ⁸ cells / ml were extracted. Lipid extraction methods are known to those skilled in the art.

Illuminance:

The crop is illuminated with 30 flashes per hour, each flash having a duration of 30 seconds and an intensity of 80 pmol. m ^"2 , s ^{" 1} .

The light input into the bioreactor cultures was obtained by LED lamps (Electro Luminescent Diodes) distributed around the outer wall of the fermenter. A clock triggers these LEDs for illumination times or pulses. Results (n = 3):

Dry mass ARA lipids (% of EPA (% of

(g / L) total (% of total lipids) lipids (MS) total)

Mixotrophy 25.6 +/- 1, 2 17.1 +/- 0.7 15.6 +/- 0.3 14 +/- 0.9 Flash

Heterotrophy 17.5 +/- 1 18.7 +/- 0.2 16.1 +/- 0.4 14.3 +/- 1.1

Claims

A method comprising the following step:

a) the mixotrophic culture of one or more strains of the genus Euglena under discontinuous and / or variable illumination conditions over time, the illumination having intensity variations whose amplitude is between 5 and 1000, these variations occurring between 2 and 3600 times per hour.

2. Method according to claim 1, characterized in that the illumination has intensity variations whose amplitude is between 5 pmol. m ^"2 , s ^{" 1} and 400 pmol. m ^"2 , these variations occurring between 2 and 200 times per hour.

3. Method according to claim 1 or 2, characterized in that the microalgae is of the species Euglena gracilis.

4. Method according to claim 1, 2 or 3, characterized in that the culture is carried out in the presence of an organic carbon substrate at a concentration of 5 mM to 1 M, preferably from 50 mM to 800 mM, more preferably from 70 mM to 600 mM, and even more preferably from 100 mM to 500 mM, the organic carbon substrate being chosen from starch, lactate, lactose, sucrose, acetate, glycerol, glucose and derivatives thereof. cellulose and a mixture of these molecules.

5. Method according to claim 4, characterized in that said organic carbon substrate present in the culture medium comprises at least 5 mM glucose.

6. Method according to claim 5, characterized in that the amplitude of the intensity variations is between 30 and 400 μιηοΙ. m ^"2 , s ^{" 1} , preferably between 70 and 300 pmol. m ^"2 , s ^{" 1} , and more preferably between 100 and 200 μιηοΙ. m ^"2 , s ^{" 1} .

7. Method according to any one of claims 1 to 6, characterized in that the light input is in the form of flashes.

8. Method according to claim 7, characterized in that a flash has a duration between 1/10 second and 10 minutes, preferably between 5 seconds and 10 minutes, more preferably between 10 seconds and 2 minutes, more preferably still between 20 seconds and 1 minute.

9. The method of claim 7 or claim 8, characterized in that the number of flashes is between 5 and 3600, preferably between 10 and 150, more preferably between 15 and 100, and more preferably between 20 and 50 times by hour.

10. Process according to any one of claims 1 to 9, characterized in that the total light input per hour in micromoles of the photons is between 2000 to 600 000 pmol. m ^"2 preferably, 2000 to 200,000 pmol. m ^{" 2} .

11. Method according to any one of claims 1 to 10, characterized in that it further comprises the following steps:

b) a step of maintaining said culture over several generations in the presence of an organic carbon substrate in the culture medium, and optionally

c) a step of recovery of the microalgae thus cultivated, and possibly

d) a step of recovery of the lipids of the microalgae, and possibly

12. Microalga of the genus Euglena obtainable by the method of any one of claims 1 to 11.

13. Microalga of the genus Euglena, according to claim 12, characterized in that its total lipids comprise more than 10%, preferably more than 25%, and more preferably more than 40% of EPA and / or ARA relative to to the total percentage of lipids.