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
  • 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 new strain of microalgae of the genus Odontella, capable of growing in heterotrophy and mixotrophy, as well as to a method for selecting and culturing said microalgae, allowing production of polyunsaturated fatty acids, in particular EPA and DHA, in heterotrophic or mixotrophic mode.
• This new strain is found to be particularly useful for producing ⁇ and DHA, especially in a culture process carried out in mixotrophic mode, in which the light input takes the form of flashes.
• 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.
• 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.
• mixotrophs can be grown both in the presence of light and organic matter.
• microalgae is still based largely on morphological criteria and on the nature of the photosynthetic pigments contained in their cells. As a result, it is not very indicative of the autotrophic, heterotrophic or mixotrophic nature of the different species of algae, whereas these cover a very large diversity of species and forms [Dubinsky et al. 2010, Hydrobiologia, 639: 153-171].
• 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 relatively constant biochemical composition biomass.
• lipids from microalgae do not have an unpleasant smell and contain little or no cholesterol.
• the lipids produced by microalgae have a generally simpler fatty acid profile than that of fish oils, which limits the separation steps of the fatty acids of interest.
• the main microalgae producing EPA and DHA are marine species belonging to different phyla. However, of the hundreds of thousands of species covered by these phyla, only a small number of species taxonomically distant from each other have high levels of EPA and DHA.
• the species capable of producing significant amounts of EPA and / or DHA most often mentioned are those belonging to the genera Schizotrium sp., Crypthecodinium sp. (Dinophyceae), Phaeodactylum sp. (Bacillariophyceae, Naviculales) and Odontella sp. (Bacillariophyceae, Coscinodiscophyceae).
• Microalgae of the genus Odontella are unicellular algae of large size, whose length can wait 35 to 50 microns. They have a silica frustule composed of two symmetrical valves. These are ubiquitous and cosmopolitan microalgae of the neritic zone, not forming dense planktonic populations, and which are often associated with various species of benthic macroalgae in coastal zones. In the natural state, the species Odontella aurita accumulates between 1.6% to 3.4% by total dry weight of EPA, which represents on average 21% of the total fatty acids produced by this microalgae.
• Odontella aurita is usually grown in autotrophic mode in outdoor ponds for use in animal feed, particularly for feeding fish larvae and crustaceans [Pulz and Gross (2004). Applied to biotechnology of microalgae, Appl. Microbiol. Biotechnol. 65 (6): 635-648].
• microalgae in autotrophic mode in open basins is not very suitable for industrial exploitation of these. Indeed, in view of the intensive exploitation of microalgae, the production of biomass must be able to be carried out in large quantities, in closed and large photo-bioreactors. However, it is difficult under such conditions to provide satisfactory illumination to all cells in the culture medium, particularly when the density of microalgae becomes important.
• Odontella would be to practice cultures in heterotrophic mode, that is to say in the absence of light, with a supply of energy in the form of carbon substrates, or in mixotrophic mode, that is to say, in the presence of a light input of less intensity, with also a contribution of organic substrate.
• This new strain of Odontella was isolated from the environment and cultured by the inventor, and more particularly, according to a process developed by it, consisting of cultivating microalgae under conditions of mixotrophy, in the presence of discontinuous illumination, especially in the form of flashes.
• the close alternation of lighted phases and dark phases, generally perceived as stressful for microalgae, has allowed, surprisingly, to obtain from this strain a high production of polyunsaturated fatty acids.
• the implementation of such a strain, according to the invention makes it possible to envisage an industrial production of polyunsaturated fatty acids in fermentors benefiting from a reduced light input, and thus, to save energy and energy. surface compared to existing autotrophic culture methods.
• Odontella strain, FCC 675 which is the first of these strains to be selected according to the present invention, was deposited according to the provisions of the Budapest Treaty to the CCAP (Culture Collection of Algae and Protozoa, Scottish Association for Marine Science, Dunstaffnage Marine Laboratory, Oban, Argyll PA371QA, Scotland, United Kingdom), May 27, 2011, and was granted accession number CCAP 1054/5.
• the subject of the present invention is therefore an isolated microalga, of the genus Odontella (Phylum: Bacillariophyta, Class: Coscinodiscophyceae, Family: Eupodicaceae) [ITIS, Catalog of Life, 2010], characterized in that it is cultivable in heterotrophic or mixotrophic mode. .
• this microalga is cultivable in the heterotrophic mode by its capacity to multiply in the dark in a conventional culture medium, usually used for the cultivation of Odontella in autotrophic mode, preferably a mineral medium, in which a carbon substrate is added.
• mineral medium is meant a culture medium consisting of an aqueous solution, often based on seawater, in which inorganic compounds are dissolved, as well as possibly vitamins and some amino acids.
• An environment Suitable mineral for the cultivation of Odontella is, for example, the medium f / 2 [Guillard, RRL (1975) Culture of phytoplankton for feeding marine invertebrates. pp 26-60.
• this microalga is cultivable in mixotrophic mode is appreciated by the capacity of the microalga to multiply in the presence of a light supply, in a culture medium similar to that described above, that is to say a medium usually used for the cultivation of Odontella in autotrophic mode, but in which a carbon substrate is added.
• the intensity of the light input is greater than or equal to 5 ⁇ , preferably between 5 and 300 ⁇ , more preferably between 10 and 200 ⁇ , and even more preferably between 20 and 150 ⁇ .
• a carbon substrate is brought to the culture.
• the carbon substrate comprises or consists of, in pure form or as a mixture, generally one or more of the following compounds: starch, glucose, xylose, arabinose, lactose, lactate, cellulose and its derivatives, sucrose, acetate and / or glycerol.
• Products derived from the biotransformation of starch for example from maize, wheat or potato, especially starch hydrolysates, which consist of small molecules, can form exploitable carbon substrates for cultivation of microalgae in heterotrophic or mixotrophic mode.
• the mixotrophic or heterotrophic culture of this microalga is preferably carried out 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. .
• Those skilled in the art know how to determine the maximum concentrations of the carbon substrate to be used.
• the cultivation in mixotrophic or heterotrophic mode of this microalgae can be carried out in the presence of 10-200 mM, preferably between 20 and 50 mM of 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. Those skilled in the art can determine the amounts of the carbon substrate to be added to the culture to maintain a constant concentration of the carbon substrate in the culture medium.
• the culture can be carried out with cumulative concentrations of carbon substrate 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. mM.
• arabinose and xylose are preferred carbon substrates for the odontella culture in the heterotrophic mode according to the invention.
• Acetate and sucrose constitute preferred carbon substrates for the cultivation of Odontella in the mixotrophic mode according to the invention.
• the subject of the invention is thus a process for cultivating or selecting a microalgae of the Odontella genus of heterotrophic or mixotrophic nature according to the invention, characterized in that it comprises the following stages:
• the cultivation method according to the invention is intended in particular to produce polyunsaturated fatty acids, more particularly ⁇ and DHA, which accumulate in the microalgae thus selected or produced.
• Such a culture method is particularly advantageous when a variable or discontinuous light input is implemented, in other words, when the light flux brought to the algae 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 (d mol (photons) / dt ⁇ 0) .
• the invention relates to a method for cultivating microalgae of the genus Odontella, characterized in that said algae are cultivated in the dark with a discontinuous or variable light input over time, the intensity of which in micromoles of photons vary by an amplitude equal to or greater than 10 pmol. m “2 , s " 1 , preferably equal to or greater than 50 pmol. m “2 , s " 1 , more preferably equal to or greater than 100 pmol. m “2 , s " 1 , one or more times per hour, advantageously more than once per hour.
• 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 (pmol.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 pmol. m “2 , s " 1 , more preferably greater than or equal to 50 pmol. nor "2 , s " 1 .
• the light intensity reaches, each hour, preferably several times in the hour, a high and low value, whose difference is equal to or greater than that indicated above.
• said luminous intensity successively reaches 50 pmol. m “2 , s “ 1 and 100 pmol. m “2 , s " 1 every hour, more preferably the values 0 and 50 pmol. m “2 , s 1 , more preferably still values 0 and 100 ⁇ . 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 cultures can be carried out in a fermenter in which the culture medium circulates regularly to reach an illuminated part of this fermenter.
• a fermenter can take, for example, the shape of a device equipped with a circular pipe, part of which is transparent and illuminated from the outside. The culture medium and the algae in suspension, actively circulating through the illuminated part of such a device, are thus periodically in contact with the light.
• Odontella FCC 675 A particular strain of Odontella FCC 675, isolated, selected and grown by the applicant, was filed at CCAP on May 27, 2011, under the number CCAP 1054/5. According to current taxonomic analyzes, it belongs to the species Odontella aurita. Nevertheless, given its affiliation with other species of Odontella, the invention relates to any species of microalgae of the genus Odontella having a mixotrophic character, as described in the present application.
• strains selected according to the process of the invention have good aptitude to grow in heterotrophic or mixotrophic mode, predisposes these strains to a higher production of polyunsaturated fatty acids, in particular EPA and DHA.
• the cultivation method according to the invention therefore makes it possible to select Odontella strains of a mixotrophic nature, similar to that isolated by the applicant and deposited with CCAP, having a high yield of polyunsaturated fatty acids.
• strains of Odontella 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 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 and DHA).
• 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 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 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 invention also relates to a process for enriching microalgae of the genus Odontella in polyunsaturated fatty acids, characterized in that it comprises the selection and cultivation of microalgae of the genus Odontella, in mixotrophic or heterotrophic mode, more particularly according to culture methods described above.
• the invention also aims to produce lipids, in particular polyunsaturated fatty acids, via the cultivation of microalgae of the Odontella genus with a heterotrophic or mixotrophic nature, then the recovery of the microalgae thus cultivated to extract the lipid content, in particular ⁇ and / or DHA.
• microalgae are preferably cultivated or selected according to the methods referred to above.
• the cultivation of Odontella strains according to the invention in mixotrophic or heterotrophic mode generally makes it possible to increase the total biomass by more than 20%, more often by more than 30%, and sometimes even by more than 40% with respect to the cultivation of the same strain of Odontella in autotrophic mode.
• the content of total lipids in EPA and DHA in the total lipids extracted from the strains of Odontella thus cultivated represents more than 10%, generally more than 30%, very often more than 40%, or even more than 50% of the total cellular lipids, in dry weight.
• ⁇ and DHA are known to those skilled in the art and are, for example, described by Bligh, E.G. and Dyer, W.J. [A rapid method of total lipid extraction and purification (1959) Can. J. Biochem. Physiol. 37: 91 1-917].
• the EPA and DHA thus extracted can be used as additives in nutritional compositions, such as infant formula, or in cosmetic or therapeutic compositions.
• microalgae selected, cultured or enriched in polyunsaturated fatty acids according to the method of the invention, can be used directly, in hydrated or dehydrated form, or after processing, as a food supplement, particularly in fish farming, or as a an ingredient in the composition of cosmetic or therapeutic products.
• the cultures are made in 2L fermenters (bioreactors) which are 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 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 pre-culture carried out on a shaking table (140 rpm) in a thermostatically controlled enclosure (22 ° C.) and continuously lit at 100 ⁇ m- 2 s- 1 .
• Pre-cultures and cultures in bioreactors are performed in f / 2 medium.
• the organic carbon used for the bioreactor mixotrophic culture is sodium acetate at concentrations between 20 mM and 50 mM.
• the organic carbon substrate is added to the culture medium in "fed-batch" mode.
• the culture conditions in heterotrophy are identical to those of the mixotrophy in the absence of light.
• 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).
• the quantification of total lipids 10 7 cells / ml were extracted.
• the 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].

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