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
  • C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
• • 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 mixotrophic culture method of a microalga of the genus Botryococcus, in particular species of Botryococcus braunii and Botryococcus sudeticus. It allows the enrichment of microalgae grown and capric acid (also known as decanoic acid, C10: 0 and C10) compared to the conditions in classical autotrophic where capric acid is almost absent. In the presence of a discontinuous and / or variable light illumination, the method makes it possible to obtain a very high biomass yield. The method thus makes it possible to select Botryococcus braunii and Botryococcus sudeticus strains of a mixotrophic nature, and having a high yield of capric 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 of microalgae species, from families and from very different origins do not appear 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.
• 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.
• Microalgae are currently the subject of many industrial projects because some species are able to accumulate or secrete significant amounts of lipids, including hydrocarbons and fatty acids. Such fatty acids, including polyunsaturated fatty acids and saturated fatty acids, have many industrial applications.
• Capric acid (also known as decanoic acid, C10: 0 or C10) is a saturated fatty acid with the chemical formula C10H20O. It has no double bond.
• Capric acid is used in several industrial sectors, for example as a solvent for inks, a degreasing agent, and as an emollient in cosmetic formulations and in soaps. It is also used in the manufacture of perfumes, lubricants, plastics and dyes. Glycerol esters of capric acid are used in foods and dietary products.
• capric acid is mainly obtained by the plant production route (in particular coconut oil and palm kernel oil) or by chemical means.
• Capric acid accounts for 6 to 10% of total fatty acids in coconut oil, and 3 to 14% of total fatty acids in palm kernel oil. This low yield of capric acid has the consequence of making this production route expensive.
• Capric acid also known as decanoic acid, C10: 0 or C10
• C10 0 or C10
• CrOa chromium trioxide
• capric acid by microalgae is not known.
• the production of capric acid by microalgae represents an advantageous alternative compared to the production by the chemical way as well as by the vegetable route, for the reasons mentioned above.
• 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 microalgae in lipids including hydrocarbons and polyunsaturated fatty acids and saturated fatty acids.
• the relative proportions of lipids, in particular in the lipid profiles vary according to the species and the culture conditions.
• 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.
• it is preferable to have the highest possible yield for example, above 50 g / l of dry matter and more than 60% of fatty acids relative to the dry matter).
• Botryococcus braunii is the species that has so far been the most studied because of the quality of its hydrocarbons and its ability to be grown in autotrophic mode.
• the cultivation of this green algae, ponderedly highly pigmented, is generally carried out under conditions of high brightness, between 500 and 1500 ⁇ . m "2 , s " 1 .
• B. braunii var. Showa is known to accumulate up to 30% of its dry weight in botryococcenes.
• the fatty chains of these botryococcenes comprise between 30 and 37 carbon atoms [Plant Patent US 6, 169]. The genome of this strain is currently being sequenced.
• Botryococcus braunii var. Ninsei has been reported to secrete botryococcal to extracellular matrix [US 2006/0265800].
• This secretion has the effect of making the Botryococcus colonies floating relative to their liquid culture medium, which advantageously makes it possible to harvest the algae loaded with botryococcenes on the surface of the culture medium.
• Botryococcus braunii strain B70 isolated by Tanoi and Kawaachi, [Tanoi and Kawaachi (2011) Effects of carbon source on growth and morphology of Botryococcus braunii, J. Appl Phycol. 23: 25-33], is able to grow under autotrophic, heterotrophic and mixotrophic conditions. In the presence of glucose in dark or bright conditions, the cells and granules containing the oil had a size significantly greater than that obtained in the absence of glucose.
• the strains concerned are respectively named FCC 828 (CCAP 807/6) and FCC 841 (CCAP 807/4).
• the strain FCC 828 belongs to the species of Botryococcus braunii and the strain FCC 841 belongs to the species Botryococcus sudeticus. These strains were deposited on 20 October 2010 with the CCAP (Culture Collection of Algae and Protozoa, Scottish Association for Marine Science, Dunstaffnage Marine Laboratory, Oban, Argyll PA371QA, Scotland, United Kingdom) in accordance with the provisions of the Treaty of Budapest.
• the cultivation and selection process consisted more particularly of cultivating the microalgae under conditions of mixotrophy with an organic carbon substrate.
• the light input can be continuous, that is to say in classic mixotrophy mode.
• Culture can also be carried out in the presence of a variable and / or discontinuous illumination, especially in the form of flashes, with a range of light intensity variations and a specific frequency.
• the yield of capric acid by the microalgae cultivated in mixotrophic mode is 30 to 80% of the total fatty acids.
• the yield of biomass, and thus of capric acid is improved by using the process in the presence of a discontinuous variable illumination, in particular in the form of flashes. Under such growing conditions, the yield of microalgae to capric acid may be greater than 55% of total fatty acids.
• Percentage of fatty acids from the FCC 828 strain (CCAP 807/6) and the FCC 841 strain (CCAP 807/4) in autotrophic and mixotrophic culture.
• CCAP 807/6 Percentage of fatty acids from the FCC 828 strain
• CCAP 841 strain CCAP 807/4
• the percentage of the respective fatty acids relative to the percentage of total lipids appears in white for the autotrophic conditions and in black for the mixotrophic conditions.
• the subject of the present invention is therefore a process for the cultivation of microalgae of the genus Botryococcus, in particular of the Botryococcus braunii and Botryococcus sudeticus species in the mixotrophic mode, for the production of capric acid.
• the culture can be carried out in "classical" mixotrophy mode, that is to say with a contribution of natural light (outdoor culture) or a continuous and constant light supply.
• the culture is carried out under conditions of continuous or discontinuous illumination and / or variable over time.
• the illumination has intensity variations whose amplitude is generally between 5 ⁇ . m “2 , s “ 1 and 1000 pmol. 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 illumination discontinuous and / or variable, with intensity variations that may have the same 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 capric acid.
• This method can also be used to select strains of the genus Botryococcus, particularly Botryococcus braunii and Botryococcus sudeticus species of mixotrophic nature, and having a high yield of capric acid.
• the mixotrophic culture of this microalgae 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 more preferably from 100 mM to 500 mM of 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 preferred substrate is sodium acetate.
• 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 carbonates adapted to the mixotrophic culture of microalgae according to the invention.
• This process is more particularly intended for the implementation of microalgae strains of the genus Botryococcus (Phylum: Chlorophyta, Order: Chlorococcales, Family: Dictyosphaeriaceae) [ITIS Catalog of Life, 2010] selected for their mixotrophic nature, especially for their ability to to be cultivated with a light input greater than 10 ⁇ l, in the medium 3NBBM + V (CCAP - Culture Collection of Algae and Protozoa) in which is added an organic carbon substrate.
• the organic carbon substrate comprises acetate in a concentration equivalent to or greater than 5 mM.
• Botryococcus more particularly Botryococcus braunii and Botryococcus sudeticus, can be isolated and selected according to the selection and culture method according to the invention.
• a representative strain of the strains of Botryococcus braunii according to the invention is the strain FCC 828, isolated by the applicant and filed with the CCAP on October 20, 2010, under the numbers CCAP 807/6.
• Another representative strain of the Botryococcus sudeticus strains according to the invention is the strain FCC 841, isolated by the applicant and deposited at the CCAP on October 20, 2010, under the number CCAP 807/4.
• Such strains are capable of producing significant amounts of capric acid when grown in a mixotrophic mode.
• the biomass yield of these strains is even higher when the light input is variable or discontinuous according to the invention.
• CCAP 807/6 and CCAP 807/4 belong to the species Botryococcus braunii, race A and Botryococcus sudeticus respectively.
• the invention relates to any strain of the species Botryococcus braunii or Botryococcus sudeticus, capable of growing in mixotrophic culture conditions, as described in the present application, and capable of producing capric acid.
• the invention also relates to any species of microalgae of the genus Botryococcus, capable of growing under conditions of mixotrophic culture, as described in the present application, and capable of producing capric acid.
• the culture of the Botryococcus strains identified in the present application under mixotrophic culture conditions according to the invention makes it possible to produce, under conventional mixotrophic conditions, significant amounts of biomass as well as lipids very rich in capric acid which can represent more than 30%, more than 45%, more than 60%, or more than 75% of the total lipids contained in microalgae (see Figure 1).
• Figure 1 shows the lipid profiles of FCC 828 and FCC 841 strains grown in mixotrophic mode (with 5mM sodium acetate as organic carbon substrate) or in autotrophic mode.
• FCC strain 828 ( Figure 1A) grown in autotrophic mode does not produce capric acid.
• the majority of the fatty acids obtained from the autotrophic culture consist of C18: 1 n9, C18: 3n3, C16: 0, C18: 2n6t, and C20: 5n3 (listed in order of importance).
• the physiological nomenclature is used in the figure to identify fatty acids.
• C18: 1n9 is a fatty acid having a carbon chain length of 18 carbons, and a double bond which is located at the level of the ninth carbon from the terminal methyl group.
• Capric acid now accounts for the majority of the fatty acids obtained, with small percentages of C18: 1 n9, C18: 3n3, C16: 0, C18: 3n3, C18: 2n6t and C20: 5n3.
• FCC strain 841 (FIG. 1B) cultured in autotrophic mode does not produce capric acid either.
• the majority of the fatty acids obtained from the autotrophic culture consist of C16: 0, C18: 1 n9, C18: 3n3 and C18: 2n6t (listed in order of importance), and of C20 and C22 chain acids in small quantities .
• Capric acid is by far the predominant fatty acid obtained, with small percentages of C18: 1 n9, C18: 3n3, C16: 0, C18: 3n3, C18: 2n6t and C20: 5n3.
• capric acid accounts for more than 70% of the total fatty acids obtained.
• the culture is carried out in mixotrophic conditions in the presence of a variable and / or discontinuous illumination, in particular in the form of flashes.
• the biomass obtained with the FCC 828 and FCC 841 strains is 10 to 20%, more generally 20 to 50%, higher than that of a culture with the same strain carried out in conventional mixotrophic mode.
• conventional mixotrophic mode is meant culture conditions with an identical culture medium, but with a contribution of natural light (outdoor culture) or a continuous and constant light supply.
• the subject of the invention is thus a process for the cultivation of microalgae of the genus Botryococcus, in particular of the Botryococcus braunii and Botryococcus sudeticus species in the mixotrophic mode, in order to produce capric acid.
• the invention also relates to such a method in the presence of a variable and / or discontinuous illumination over time, for example in the form of flashes.
• the subject of the invention is thus a process for the selection of microalgae of the genus Botryococcus, in particular of the Botryococcus braunii and Botryococcus sudeticus species with a mixotrophic nature, and having a high yield of capric acid, in particular in the presence of variable illumination 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 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. 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. seconds and 6 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 ⁇ . m "2 , s " 1 , or 50 and 400 ⁇ .
• 1 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 time period of 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 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 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 “ ⁇ or 5 and 400 ⁇ . 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 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 pmol.
• 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 can be between 5 and 15 ⁇ . 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 , for example, preferably between 50 and 400 pmol.
• the light intensity can be increased to between 100 and 1000 pmol. m “2 , s “ 1 for example, preferably between 200 and 500 pmol. 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 pmol. 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 pmol. m “2 , s " 1 , which gives a total light input per hour of 9000 pmol. 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, to give a 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 pmol. 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 input in the hour is generally between about 1500 and 8000, preferably 1500 and 6000 ⁇ . 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. m "2 , preferably between 6000 and 50,000 and more preferably between 12,000 and 45,000 pmol m- 2 , for example.
• the total light contribution 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 more preferably between 50,000 and 150,000 pmol 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 pmol. m 2 to 4500 pmol m -2 .
• the intermediate stage at 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 pmol.
• 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 pmol 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 pmol. 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 pmol. 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 pmol m -2 , s -1 , giving 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 Botryococcus, in particular species of Botryococcus braunii and Botryococcus sudeticus, of a mixotrophic nature, similar to those isolated by the applicant and deposited with the CCAP under the numbers CCAP 807. / 6 and CCAP 807/4, and having a high yield of capric acid.
• This cultivation process is characterized in that it comprises the following steps: a) Mixotrophic culture of one or more strains of the genus Botryococcus under discontinuous or variable illumination conditions over time, the illumination exhibiting intensity variations whose amplitude is between 5 pmol. m “2 , s " 1 and 1000, preferably between 5 and 400 ⁇ . m “2" s "1 , these variations occurring between 2 and 3600, preferably 5-400 times per hour,
• 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 Botryococcus in particular of the species Botryococcus braunii or Botryococcus sudeticus, can be cultured, in parallel, on microplates in the same enclosure, with precise monitoring of the conditions and evolution of the strains. different cultures. It is thus easy to know the response of the different strains to discontinuous and / or variable illumination and, where appropriate, to 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 the lipid production in terms of quality (more generous capriic acid in the lipid profile) and quantitative (the lipids contain a proportion higher capric acid).
• 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 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:
• lipid recovery step of microalgae, and possibly e) extraction of capric acid recovered lipids.
• the culture method according to the invention can also be applied to any species of the genus Botryococcus, capable of growing under the mixotrophic conditions according to the invention, and capable of producing capric acid.
• 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 fatty acids, more particularly with capric acid.
• 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 Botryococcus genus of a mixotrophic nature, preferably cultivated or selected according to the methods referred to above, then the recovery of the microalgae thus cultivated to extract the contents lipid, in particular capric acid.
• the strains of the species Botryococcus braunii and Botryococcus sudeticus are especially concerned.
• the invention also relates to the microalgae of the genus Botryococcus braunii and of the genus Botryococcus sudeticus, which can be obtained according to the process of the invention as previously described. These microalgae are enriched in capric acid.
• the total lipids of such microalgae generally comprise more than 30%, more than 45%, more than 60% or more than 75% of the total lipids contained in microalgae.
• Botryococcus braunii and Botryococcus sudeticus strains in classical and autotrophic mixotrophic mode The cultures of Botryococcus braunii were carried out in 2L fermentors (bioreactors) useful with dedicated automata and computer station supervision.
• 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 3 stirring wheels placed on the shaft according to the Rushton configuration (three-blade propellers with downward pumping).
• the bioreactor is equipped with an external lighting system surrounding the transparent tank.
• 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.
• the pre-cultures and cultures in bioreactors are carried out in the 3NBBM + V medium (CCAP-Culture Collection of Algae and Protozoa), In the case of mixotrophy, organic carbon in the form of sodium acetate (100 and 150 mM) was added to the culture medium.
• the total biomass concentration is monitored by measuring the dry mass (GFC filter filtration, Whatman, then drying in a vacuum oven, 65 ° C and -0.8 bar, for 24 hours minimum before weighing).
• Lipid extraction methods are known to those skilled in the art and are, for example, described by Bligh, EG and Dyer, WJ (1959).
• the analysis of the fatty acids is carried out by gas chromatography after trans-esterification of the lipids, according to methods known to those skilled in the art.

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