Classifications

• • 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
  • C12P7/6436—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 Cyclotella, in particular of the species Cyclotella cryptica.
• 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 docosahexaenoic acid (DHA).
• EPA eicosapentaenoic acid
• DHA docosahexaenoic acid
• the method thus makes it possible to select Cyclotella cryptica 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 Cyclotella cryptica, particularly suitable for the production of fatty acids.
• This new strain of Cyclotella cryptica is useful for producing ⁇ (eicosapentaenoic acid) and docosahexaenoic acid (DHA) in mixotrophic mode.
• ⁇ eicosapentaenoic acid
• DHA docosahexaenoic 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 medium of organic carbon substrates or organic matter is not favorable to them and does not improve their growth.
• a certain Many species of microalgae, of families and of very diverse origins prove not to be 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.
• AGH I highly unsaturated
• PUFA-CJU3 eicosapentaenoic acid
• DHA or C22: 6 ⁇ 3 docosahexaenoic acid
• PUFA-CJU6 arachidonic acid
• ARA or AA or eicosatetraenoic acid C20: 4 ⁇ 6 have a recognized nutritional importance and have high potential in terms of therapeutic applications.
• DHA is necessary for the normal and functional development of cells, and plays a crucial role in various biochemical processes and functions. Its polyunsaturated nature gives it a crucial importance vis-à-vis the properties of the cell membrane, in plants as in animals: fluidity, flexibility and selective permeability allowing, for example, effective adaptation, and even survival, at low temperatures. especially in fish.
• DHA is a major structural constituent of the human brain and is its main AG. DHA represents 15-20% of the cerebral cortex (the brain of an adult contains at least 20 g of DHA) and 30-60% of the retina. It is essential for the development of the central nervous system and retinal function, by incorporation into cell membranes, and plays a vital role in the satisfactory acquisition and maintenance of the mechanisms of vision and memory.
• New sources of these fatty acids such as ⁇ , DHA and TARA must therefore be sought in order to meet, in the future, the growing market demand for this type of polyunsaturated 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.
• 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.
• 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, DHA and ARA in the lipid profiles vary according to the species and the culture conditions.
• the main microalgae of interest, producing EPA and DHA, are marine species. However, of 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) derived from marine phytoplankton. They are generally characterized by active production of EPA, but often produce rather low levels of DHA.
• 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.
• the microalgae be grown under optimal conditions to increase the yield of (s) fatty acid (s) to produce.
• the highest possible yield for example biomass above 30 g / l of material dry, and more than 50% of fatty acids by weight relative to the total weight of the dry matter.
• Cyclotella cryptica is a diatom (a bacillariophyte originally isolated from brackish water in Massachusetts, USA) known to produce ⁇ as well as DHA in autotrophy.
• German Patent Application DE4018820 discloses the cultivation of the Cryptomonas ovata, Chlorella fusca, Scenedesmus accuminatus and Cyclotella cryptica strains under autotrophic conditions to produce ⁇ .
• Table 1 of this application shows comparative results between strains: the yield of the strain Cyclotella cryptica, is 45.6 g of lipids per 100 grams of dry matter, with 22.0% by weight of PUFA and 17.4 % by weight of EPA on the total weight of dry matter.
• an alternative to the autotrophic culture would be to practice the cultures in heterotrophic mode, that is to say in the absence of light with a supply of energy in the form of carbon substrates organic, or in mixotrophic mode, that is to say with a light input of less intensity and in the presence of a supply of organic substrate.
• 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 Cyclotella cryptica 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 DHA, in fermentors benefiting from a reduced light input, and therefore should to save energy compared to autotrophic farming methods.
• the subject of the present invention is therefore a process for cultivating microalgae of the genus Cyclotella, in particular of the Cyclotella cryptica species, in mixotrophic mode.
• the process according to the invention makes it possible to enrich the microalgae of the Cyclotella genus with polyunsaturated fatty acids and more particularly with EPA and DHA.
• the illumination has intensity variations whose amplitude is generally between 5 ⁇ . 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 docosahexaenoic acid (DHA).
• This method can also be used to select strains of the genus Cyclotella, in particular of the Cyclotella cryptica species, of a mixotrophic nature, and having a high yield of polyunsaturated fatty acids, especially ⁇ (eicosapentaenoic acid) and / or DHA. (docosahexaenoic 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 even more preferably from 100 mM to 150 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, substrates organic carbon 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 Cyclotella (Division: Bacillariophyta, Order: Thalassiosirales, Family: Stephanodiscaceae) [ITIS Catalog of Life, 2010] selected for their mixotrophic nature, especially for their ability to be cultivated with a light input greater than 10 ⁇ l, in a mineral medium, for example F / 2 + medium if [Guillard, RRL (1975); Culture of phytoplankton for feeding marine invertebrates, pg. 26-60. in Smith WL and Chanley M. H (eds.) Culture of Marine Invertebrate Animais. Plenum Press, New York, USA], in which is added an organic carbon substrate.
• the organic carbon substrate comprises glucose, lactate, in a concentration equivalent to or greater than 5 mM.
• These new strains of Cyclotella, more particularly Cyclotella cryptica can be isolated and selected according to the method of selection and culture according to the invention described below.
• a representative strain of the Cyclotella cryptica strains according to the invention is the strain FCC 971 isolated by the applicant and deposited at the CCAP, under the number CCAP 1070/7.
• Such strains are capable of producing significant amounts of biomass as well as lipids and more particularly of EPA and DHA when they are cultivated in mixotrophic mode with a variable or discontinuous light supply, according to the invention.
• the CCAP 1070/7 strain belongs to the species Cyclotella cryptica.
• the invention relates to any strain of the species Cyclotella cryptica, capable of growing in mixotrophic culture conditions as described in the present application, and capable of producing fatty acids, such as DHA and ⁇ .
• the invention also relates to any microalgae species of the genus Cyclotella, capable of growing under mixotrophic culture conditions as described in the present application, and capable of producing fatty acids, such as DHA and ⁇ .
• the strains of Cyclotella cryptica 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 DHA, said EPA being able to represent more than 40%, or more than 50% , and said DHA may represent more than 10%, or more than 20%, or more than 30% of the total lipids contained in microalgae.
• FCC 971 isolated by the applicant, from a culture under 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 culture conditions with an identical culture medium, but without the addition of light.
• the subject of the invention is thus a process for the cultivation of microalgae of the genus Cyclotella, in particular of the Cyclotella cryptica species in mixotrophic mode, in the presence of a variable or discontinuous illumination over time, for example in the form of flashes, in particular to produce polyunsaturated fatty acids, such as ⁇ and DHA.
• the subject of the invention is thus a process for the selection of microalgae of the genus Cyclotella, in particular of the Cyclotella cryptica species with a mixotrophic nature, and having a high yield of polyunsaturated fatty acids such as ⁇ and DHA, in the presence of an illumination. variable and / or discontinuous over time.
• 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 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 one 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 one second, or 9/10 of a second.
• the illuminance, or flash is usually longer than 15 seconds. It 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 ⁇ . m "2 , s " 1 , preferably between 5 and 500 ⁇ . m "2 , s " 1 , or 50 and 400 ⁇ .
• 1 ⁇ . 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 ⁇ .
• m “2 , s " 1 , the time period of the frequency flashes are between 10 seconds and 60 minutes for a flash time 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 light intensity variation is generally between 5 and 1000, or between 50 and 800, or between 100 and 600 ⁇ . m “2 , s " 1 .
• the intensity of the light can also vary between 5 and 400 ⁇ . m “2 , s " 1
• the amplitude of the light intensity variation is between 70 and 300 ⁇ . m “2 , s " 1 and more preferably between 100 and 200 ⁇ . m “2 , s “ 1 .
• Said luminous intensity can successively achieve, under variable lighting conditions, for example, the values 50 ⁇ . m “2 , s “ 1 and 100 ⁇ . m “2 s “ 1 , or 5 and 400 ⁇ . m “2 s “ 1 , or 50 and 800 ⁇ . m “2 , s “ 1 several times each hour.
• Said luminous intensity can successively reach, preferably the values 50 and 200 ⁇ . m “2 , s " 1 .
• said luminous intensity can successively, several times in the hour, for example, the values 0 and 50 ⁇ . m “2 , s “ 1 , the values 0 and 100 ⁇ .
• the intensity of the light brought to the culture varies according to the cell density.
• 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 can be between 5 and 15 ⁇ . m “2 , s “ 1 , preferably between 5 and 10 ⁇ . m “2 , s “ 1 .
• the light intensity can be increased to between 15 and 200 ⁇ . m “2 , s “ 1 , for example, preferably between 20 and 50 ⁇ . m “2 , s " 1 .
• the luminous intensity can be increased to between 50 and 400 ⁇ . m “2 , s “ 1 , for example, preferably between 50 and 150 ⁇ . m “2 , s “ 1 .
• the intensity of the light may be greater compared to the values mentioned above.
• the light intensity can be between 5 and 200 ⁇ . m “2 , s “ 1 , preferably between 5 and 100 ⁇ . m “2 , s " 1 .
• the light intensity can be increased to between 30 and 500 ⁇ . m “2 , s “ 1 , for example, preferably between 50 and 400 ⁇ . m “2 , s “ 1 .
• the luminous intensity can be increased to between 100 and 1000 ⁇ .
• m “2 , s " 1 for example, preferably between 200 and 500 ⁇ .
• 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 ⁇ . m "2. It can be between about 4000 and 200,000 ⁇ . 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.
• culture is illuminated with 20 flashes per hour, each flash having a duration of 30 seconds and an intensity of 20 ⁇ . m “2 , s " 1 . The latter gives a total light input per hour of 12,000 ⁇ . m “2.
• the culture is irradiated with 45 flashes per hour, each flash having a duration of 15 seconds and an intensity of 5 ⁇ .
• the culture is illuminated with 120 flashes per hour, each flash having a duration of 10 seconds and an intensity of 200 ⁇ . m “2 , s " 1 , which gives a total light input 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 input in the hour is generally between about 1500 and 8000, preferably 1500 and 6000 ⁇ . m “2 , more preferably between 2000 and 5000 ⁇ . m " 2 .
• the total light supply in the hour can be increased to between 6000 and 67 000 ⁇ . m "2 , preferably between 6000 and 50 000 and more preferably between 12 000 and 45 000 ⁇ . m " 2 , for example.
• the total light supply in the hour can be increased to between 45,000 and 300,000, for example preferably between 45 to 300,000. 000 and 200 000 ⁇ . 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 a intensity between 5 and 10 ⁇ . m “2 , s " 1 , which gives a total light input per hour of 2250 ⁇ . m “2 to 4500 ⁇ . 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 ⁇ .
• m "2 , s " 1 which gives a total light input per hour of 13,500 to 45,000 ⁇ . 2. Then, at final stage of the culture (at a cell density between 10 7 and 10 8 cells per ml), the culture is illuminated 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 input per hour of 45,000 to 135,000 ⁇ . m "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 ⁇ . m “2 , s " 1 , which gives a total light input per hour of 15,000 ⁇ . m “2 to 30,000 ⁇ . 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 output per hour of 100,000 to 150,000 ⁇ . m "2.
• the culture is illuminated with 120 flashes per hour, each flash having a duration of 10 seconds and an intensity between 350 and 450 ⁇ m "2 , s " 1 , which gives a total light output per hour of 420,000 to 540,000 ⁇ . 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 Cyclotella, in particular of the Cyclotella cryptica species with a mixotrophic nature, similar to that isolated by the applicant and filed at the CCAP under the number CCAP 1070/7, and having a high yield of polyunsaturated fatty acids, especially DHA and EPA.
• This process for producing EPA and / or DHA 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.
• the cultivation in mixotrophic mode is carried out under discontinuous and / or variable illumination conditions over time, the illumination having intensity variations whose amplitude is between 5 ⁇ . m “2 , s “ 1 and 400 ⁇ . m “2 , s “ 1 , these variations taking place between 2 and 200 times per hour
• various strains of the genus Cyclotella in particular of the species Cyclotella cryptica, can be cultured, in parallel, on microplates in the same enclosure, with precise monitoring of the conditions and the evolution of the different cultures. It is thus easy to know the response of the different strains to the discontinuous and / or variable illumination and, where appropriate, the addition of one or more organic carbon substrates in the culture medium. Strains that respond favorably to discontinuous and / or variable illumination and to organic 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 a higher proportion of EPA and / or DHA).
• the microalgae can be selected in a fermentor from a heterogeneous population and we seek to select variants favored by the selection mode according to the invention combining discontinuous and / or variable light having a range of light intensity and a specific frequency, with mixotrophic culture 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:
• the culture method according to the invention can also be applied to any species of the genus Cyclotella, capable of growing under the mixotrophic conditions according to the invention, and capable of producing ⁇ and / or DHA.
• the culture method according to the invention makes it possible to optimize the production of the biomass obtained from the culture. It also makes it possible to enrich the microalgae thus cultivated with polyunsaturated fatty acids, more particularly eicosapentaenoic acid (EPA) and / or docosahexaenoic acid (DHA).
• polyunsaturated fatty acids more particularly eicosapentaenoic acid (EPA) and / or docosahexaenoic acid (DHA).
• the invention therefore also aims at optimizing the production of biomass, as well as the production of lipids, in particular fatty acids, via the cultivation of microalgae of the genus Cyclotella of a mixotrophic nature, preferably cultivated or selected according to the methods previously referred to, then the recovery of microalgae cultivated to extract the lipid content, especially ⁇ and / or DHA. Strains of the species Cyclotella cryptica are especially concerned.
• the invention also relates to microalgae of the genus Cyclotella, 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 30%, often more than 40% EPA and / or more than 10% DHA relative to the total percentage of lipids.
• the cultures of Cyclotella cryptica FCC 971 were carried out in fermenters (bioreactors) of 1, 5L 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 23 ° 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 reactors are inoculated with a pre-culture carried out on a stirring table (140 rpm) in thermo-steady enclosure (23 ° C.) and illuminated between 80 and 100 ⁇ .
• Pre-cultures and bioreactor cultures are performed in modified SK medium (Stephen L. Pahl, David M. Lewis, Feng Chen, Keith D. King, J Appl Phycol 2010 April, 22 (2): 165-171)
• 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 GFB filter, Whatman, then drying in an oven at 100 ° C for a minimum of 24 hours before weighing).
• the crop is illuminated with 30 flashes per hour, each flash having a duration of 30 seconds and an intensity of 80 ⁇ . m “2 , s " 1 .
• LEDs Electro Luminescent Diodes
• Computer control triggers the power supply of the LEDs for lighting times or flashes.

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