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
  • C12P7/6434—Docosahexenoic acids [DHA]
• • 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 process for the cultivation and selection of microalgae strains belonging to the Isochrysis genus involving a discontinuous light supply in the form of flashes, as well as to selected Isochrysis strains, particularly suitable for the production of polyunsaturated fatty acids, especially EPA (eicosapentaenoic acid) in mixotrophic culture mode.
• EPA eicosapentaenoic 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 strictly autotrophic, that is, they can only grow by photosynthesis. For these, the presence in their medium of carbon substrates or organic material is not favorable to them and even tends to inhibit their growth.
• 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 photosynthesis and the organic matter present. in their midst. 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.
• 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, 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. There are important variations among the different species of the different classes composing these phylums with regard to the content of microalgae in polyunsaturated fatty acids. In addition, the relative proportions of EPA and DHA in lipid profiles vary with species and culture conditions [Yongmanitchai, W. and Ward, O.P. (1989) Omega-3 fatty acids: alternative sources of production. Process. Biochem. 24: 1 17-125].
• microalgae In the perspective of industrial exploitation of microalgae, it is the heterotrophic or mixotrophic species that are currently attracting the most interest from industrialists. Indeed, the reduced dependence of this type of microalgae vis-à-vis the light, can consider their culture in closed containers of large size, as is practiced in fermenters for bacteria or yeasts.
• microalgae of the genus Isochrysis which are flagellated marine microalgae of brown color belonging to the class of Prymnesiophyceae.
• Microalgae of the genus Isochrysis are widely used in fish hatcheries for fish, shrimp, molluscs and crustaceans as a dietary supplement rich in DHA. These microalgae are generally marketed in the form of concentrated long-life microalgae preparations (Instant Algae® , 871 East Hamilton Ave, Campbell, CA 95008, USA). The peculiarity of these microalgae is to store their fatty acids intracellularly in the form of lipid inclusions. As their walls are relatively thin, several studies place Isochrysis among the microalgae best suited to extract their lipids on an industrial scale.
• This process which is the subject of the present invention, consisted more particularly in cultivating microalgae under the conditions of mixotrophy, in the presence of discontinuous illumination, especially in the form of flashes.
• This process has, for the first time, enabled the production of EPA by Isochrysis strains under mixotrophic conditions.
• CCAP Culture Collection of Algae and Protozoa, Scottish Association for Marine Science, Dunstaffnage Marine Laboratory, Oban , Argyll PA371 QA, Scotland, United Kingdom
• strains and the culture method according to the invention thus opens the prospect of an industrial production of polyunsaturated fatty acids, in particular EPA and DHA, using strains of the genus Isochrysis, in fermenters benefiting from reduced light input and energy saving.
• the present invention firstly relates to new microalgae strains of the genus Isochrysis (Isochrysis sp.) Characterized in that they are capable of producing ⁇ under mixotrophic conditions.
• the isolated Isochrysis strains according to the invention are the first ones described as being capable of producing, in a mixotrophic condition, significant amounts of EPA that may represent more than 5% or even more than 10% of lipids. totals contained in microalgae.
• a representative strain of the Isochrysis strains according to the invention is the strain FCC 11 11, deposited at the CCAP on May 27, 2011, under the number CCAP 927/16. This strain is characterized in that it is capable of producing ⁇ in mixotrophic culture mode.
• this strain belongs to the genus Isochrysis [Parke, M. 1949 (1949). Studies on marine flagellates. Journal of the Marine Biological Association of the United Kingdom 28: 255- 288]. Nevertheless, considering that the main species Isochrysis galbana, Isochrysis litoralis or Isochrysis maritima are phylogenetically close, the precise species to which belongs the strain FCC 11 1 1 has not yet been definitively established. Therefore, the invention relates to any species of Isochrysis capable of producing ⁇ in mixotrophic culture mode, as described in the present application.
• the mixotrophic culture of Isochrysis according to the invention is preferentially carried out in a f / 2 type culture medium [Guillard, R.R. and Ryther, J.H. (1962) Studies on marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacaea (Cleve) Gran. Canadian Journal of Microbiology 8: 229-239], in the presence of at least 5 mM, preferably at least 10 mM, more preferably at least 20 mM, and even more preferably more than 50 mM of a carbon substrate.
• This carbon substrate preferably comprises, in pure form or as a mixture, glucose, cellulose (or cellulose derivatives), starch, lactose, sucrose, acetate and / or glycerol.
• the cultivation in the mixotrophic mode of this microalga is preferably carried out in the presence of 10-200 mM and more preferably between 20 and 50 mM of carbon substrate.
• the carbon substrate present in the culture medium comprises at least 5 mM glycerol or lactose.
• 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.
• the culture is thus carried out in the presence of cumulative concentrations of carbon substrate of 5 mM to 1 M, preferably 50 mM to 800 mM, more preferably 70 mM to 600 mM, and still more preferably 100 mM to 500 mM.
• This carbon substrate can consist of mixtures 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.
• the subject of the invention is also a process for cultivating microalgae of the genus Isochrysis in mixotrophic mode in order to produce polyunsaturated fatty acids, in particular ⁇ .
• This process has the effect of enriching microalgae of the genus Isochrysis polyunsaturated fatty acids, which generally results in an increase in the proportion of EPA or DHA present in the total lipids produced by said microalgae.
• the efficiency of microalgae in EPA is higher when microalgae are cultivated in the presence of a variable or discontinuous light input, that is to say when the light flux brought to the microalgae in culture is variable or discontinuous over time. .
• microalgae tend to accumulate lipid reserves to withstand the stresses of their environment.
• 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, that is to say over periods of short duration.
• the successive phases of illumination are then generally between 5 seconds and 10 minutes, preferably between 10 seconds and 2 minutes, more preferably between 20 seconds and 1 minute.
• the illumination may be variable, which means that the illumination is not interrupted by dark phases, and that the light intensity varies over time.
• This light variation can be periodic, cyclic or even random.
• the illumination can vary continuously, that is to say that the light intensity is not constant and varies continuously over time (dpmol (photons) / dt ⁇ 0).
• the invention aims, in particular, a microalgae culture process of the Isochrysis genus, characterized in that said algae are cultivated in the dark with a discontinuous or variable light input during of time, whose intensity in micromoles of photons varies by an amplitude equal to or greater than 10 ⁇ .
• m “2 , s " 1 at several times per hour, preferably equal to or greater than 50 ⁇ .
• m "2 , s " 1 more preferably equal to or greater than 100 ⁇ . m “2 , s “ 1 .
• the common point of these different modes of illumination lies in the fact that, according to the invention, the light intensity provided to the algae in culture, expressed in micromoles of photons per second per square meter ( ⁇ .m “2 , s " 1 ), varies at least once in the same hour.
• the amplitude of this variation of light intensity is generally greater than 10 pmol. m “2 , s “ 1 , preferably greater than or equal to 20 ⁇ . m “2 , s " 1 , more preferably greater than or equal to ⁇ ⁇ . ⁇ 2 . s "1.
• the light intensity reaches every hour, preferably several times in an hour, a high and low value, whose difference is equal to or greater than that indicated above.
• said intensity The luminous value successively reaches the values 50 pmol.m.sup.- 2 , s.sup.- 1 and 100 pmol.m.sup.- 2 , s.sup.- 1 every hour, more preferably values 0 and 50 pmol.m.sup.- 2 . s more preferably still values 0 and 100 ⁇ . m "2 , s 1 .
• the intensity of the light provided to the culture can be increased depending on the cell density.
• the duration of the flashes can be increased to 20 seconds with an intensity of 50-100 ⁇ . m "2 , s " 1 .
• the flashes can have a duration of 30 seconds and an intensity of 100-200 ⁇ . m "2 , s 1 .
• 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 therefore makes it possible to select Isochrysis strains of a mixotrophic nature, having a high yield of polyunsaturated fatty acids and capable of producing ⁇ in mixotrophic mode, such as the FCC strain 1 1 11, deposited at the CCAP under the number CCAP 927/16.
• Such a method generally comprises one or more of the following steps:
• strains of Isochrysis 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 illumination and, where appropriate, the addition of one or more carbon substrates in the culture medium. Strains that respond favorably to discontinuous illumination and 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 EPA).
• the microalgae can be selected in a fermenter from a pool of diversified microalgae, the preferred variants of which are to be selected by the selection method according to the invention, combining discontinuous or variable light with mixotrophic culture conditions.
• the culture is practiced by maintaining the microalgae in culture 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 process according to the invention is characterized more particularly in that the culture of the strains is carried out over several generations, preferably in the mixotrophic mode, and in that the cells loaded with lipids are harvested.
• the object of the invention is therefore also the production of lipids, in particular of fatty acids, via the cultivation of microalgae of the genus Isochrysis of a mixotrophic nature, preferably cultivated or selected according to the processes referred to above, then the recovery of the microalgae thus cultivated for to extract the lipid content, in particular ⁇ .
• the invention also relates to microalgae of the Isochrysis genus enriched in polyunsaturated fatty acids, which can be obtained according to the process of the invention as previously described.
• the total lipids of such microalgae generally comprise more than 20%, often more than 40% and sometimes even more than 50% EPA.
• Such microalgae can be used as a dietary supplement, especially in fish farming.
• the cultures are made in fermentors (bioreactors) of 2L 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 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 intensity as well as the light cycles are controlled by a dedicated automaton supervised by a computer station.
• the reactors are inoculated with a pretreatment performed on a stirring table (140 rpm) in a thermostatically controlled enclosure (22 ° C.) and lit continuously at 100 ⁇ m- 2 sec -1 .
• Pre-cultures and cultures in bioreactors are carried out in f / 2 medium supplemented with 10 ⁇ g / L of Biotin as well as vitamin B12.
• the organic carbon used for the bioreactor mixotrophic culture is glycerol at final concentrations between 20 and 30 g / L.
• the organic carbon substrate is added to the culture medium in "fed-batch" mode.
• 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).
• lipid extraction methods are known to those skilled in the art and are, for example, described by Bligh, EG and Dyer, WJ [A rapid method of total lipid extraction and purification (1959) Can. J. Biochem. Physiol 37: 911-917].
• the contribution of light in bioreactor cultures was obtained by LED lamps distributed around the outer wall of the fermenters.
• a clock triggers these LEDs for illumination times or flashes between 20 and 200 ⁇ m "2 , s " 1 .
• the duration of the illumination times is between 15 and 30 seconds.
• the intensity and duration of illumination time vary depending on the cell density.
• the culture has a total duration of about 10 days.
• the flashes For the first three days of culture, the flashes have a duration of 15 seconds and an intensity of 30 ⁇ m "2 , s " 1 . From the fourth day, the duration of the flashes is 20 seconds with an intensity of 75 ⁇ m "2 , s " 1 .
• the flashes have a duration of 30 seconds and an intensity of 150 ⁇ m "2 , s " 1 .
• the luminous intensity of the flash system used in mixotrophy is equal to that used in autotrophy (control).

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• 2011
  • 2011-06-21 FR FR1155443A patent/FR2976951A1/fr not_active Withdrawn
• 2012
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