Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
  • C12P7/6409—Fatty acids
  • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
  • C12P7/6432—Eicosapentaenoic acids [EPA]
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/12—Unicellular algae; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/12—Unicellular algae; Culture media therefor
  • C12N1/125—Unicellular algae isolates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
  • C12P7/6409—Fatty acids
  • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
  • C12P7/6436—Fatty acid esters
  • C12P7/6445—Glycerides
  • C12P7/6472—Glycerides containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/89—Algae ; Processes using algae

Definitions

• 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).
• EPA eicosapentaenoic acid
• ARA arachidonic acid
• 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 ⁇ (eicosapentaenoic acid) and arachidonic acid (ARA) in mixotrophic mode.
• ⁇ eicosapentaenoic acid
• ARA arachidonic acid
• Microalgae are photosynthetic microorganisms of autotrophic nature, that is to say having the ability to grow autonomously by photosynthesis.
• 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.
• 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.
• 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.
• 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.
• PUFA-3 highly unsaturated omega-3
• EPA or C20: 5 ⁇ 3 eicosapentaenoic acid
• DHA or C22: 6 ⁇ 3 docosahexaenoic acid
• PUFA- ⁇ arachidonic acid
• AA or ARA or eicosatetraenoic acid C20: 4 ⁇ 6 arachidonic acid
• 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.
• 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.
• the taxonomic classification of eukaryotic algae contains 14 phyla.
• 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.
• 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.
• freshwater microalgae Although rich in ⁇ -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].
• ARA is quite rare for marine algae.
• microalgae include, but are not limited to Euglenophytes (eg, Euglena), Rhodophytes (eg, Porphyridium) and Chlorophytes (eg, Parietochloris).
• 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.
• 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).
• energy expenditure and other resources such as labor and the ease of continuing cultivation must be taken into account by developing ideal growing conditions.
• 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).
• 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.
• 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.
• 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.
• 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 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 ⁇ . m “2 , s “ 1 , preferably between 30 and 400 ⁇ . 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 ⁇ (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 ⁇ l, 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.
• the organic carbon substrate comprises glucose and / or lactate, in a concentration equivalent to or greater than 5 mM.
• These new Euglena strains 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 ⁇ and ARRA when they are cultivated in mixotrophic mode with a variable and / or discontinuous light supply, according to the invention.
• 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 ⁇ .
• 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 ⁇ .
• 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.
• 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 ⁇ 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 ⁇ and TARA, in the presence of variable illumination. and / or discontinuous over time.
• 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.
• 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.
• 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.
• the flash duration may be less than one second.
• 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.
• 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.
• n pmol. m “2 , s “ 1 corresponds to 1 ⁇ m “2 , s “ 1 (Einstein), a unit often used in the literature.
• 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.
• 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.
• the light intensity provided to the algae in culture 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 .
• 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.
• 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 .
• 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.
• the light intensity may be between 5 and 15 pmol. m “2 , s “ 1 , preferably between 5 and 10 pmol. m “2 , s " 1 .
• 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 .
• 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 .
• the intensity of the light may be greater compared to the values mentioned above.
• the light intensity may be between 5 and 200 pmol. m “2 , s " 1 , preferably between 5 and 100 pmol. m “2 , s " 1 .
• the light intensity can be increased to between 30 and 500 pmol. m “2 , s " 1 , by for example, preferably between 50 and 400 ⁇ .
• 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 ⁇ . m “2 , s “ 1 .
• 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.
• the culture is illuminated with 30 flashes per hour, each flash having a duration of 30 seconds and an intensity of 10 ⁇ . m “2 , s " 1 .
• the latter gives a total light input per hour of 9000 ⁇ . m "2.
• 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 .
• the culture is illuminated with 45 flashes per hour, each flash having a duration of 15 seconds and an intensity of 5 pmol.
• culture is illuminated with 120 flashes per hour, each having a flash duration of 10 seconds and an intensity of 200 ⁇ . m" 2 s "1, to give a total light output per hour of 240,000 ⁇ m- 2 .
• the amount of light provided to the culture per hour may vary depending on the cell density.
• 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 .
• the total light supply in the hour can be increased to between 6000 and 67000 pmol.
• 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 ⁇ . m " 2 .
• 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 ⁇ . m 2 to 4500 pmol m -2 .
• 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 ⁇ .
• the culture is irradiated with 30 flashes per hour, each flash having a duration of 30 seconds and an intensity between 50 and 150 ⁇ m "2 , s " 1 , which gives a total light output per hour of 45,000 to 135,000 ⁇ .rr ⁇ 2 .
• the duration of the flashes is for example less than one minute, or less than one second
• 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 ⁇ . m 2 to 30,000 pmol m -2 .
• the culture is illuminated with 50 flashes per hour, each flash having a duration of 10 seconds and an intensity between 200 and 300 ⁇ .
• m “2 , s " 1 which gives a total light input per hour of 100,000 to 150,000 pmol. m "2.
• the culture is illuminated with 120 flashes per hour, each having a flash duration of 10 seconds and intensity between 350 and 450 ⁇ .
• 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.
• 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:
• recovery step is meant more particularly the isolation of the strain or strains whose cell number has grown the most during said generations.
• 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.
• 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.
• the method according to the invention also comprises the following steps:
• EPA eicosapentaenoic acid
• ARA arachidonic acid
• 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 ⁇ 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).
• EPA eicosapentaenoic acid
• ARA arachidonic acid
• 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 ⁇ and / or TARA. Strains of the species Euglena gracilis are especially concerned.
• 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 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 ⁇ E.
• 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.
• 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).
• 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 .

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