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• • 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
  • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
  • C12P5/007—Preparation of hydrocarbons or halogenated hydrocarbons containing one or more isoprene units, i.e. terpenes
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  • 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
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  • 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
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
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  • 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
  • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
• • 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
• • 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 method for culturing microalgae of the Botryococcus genus using a discontinuous or variable light input over time, in particular in the form of flashes, as well as to new strains of microalgae of the Botryococcus genus which are particularly adapted the production of hydrocarbons in mixotrophic mode.
• Unicellular algae are photosynthetic microorganisms with an autotrophic character, that is, they have the ability to grow autonomously by photosynthesis.
• heterotrophic are able to develop in the total absence of light, by fermentation, that is to say by exploiting the organic matter.
• mixotrophs Other species of algae, for which photosynthesis remains essential for their development, are able to take advantage of both photosynthesis and the organic matter present in their environment. These intermediate species, called mixotrophs, can be grown both in the presence of light and organic matter.
• Unicellular algae 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 can thus accumulate up to 80% of their dry weight in fatty acids and, as a result, offer a credible alternative to the cultivation of land-based oil plants, in particular for the production of biofuels [Li, Y and al., 2008 Biotechnol. Prog., 24: 815-820].
• microalgae strains of the genus Botryococcus (Chlorophyta, Chlorophyceae, Chlorococcales, Disctyosphaericae) [ITIS, Catalog of Life, 2010] are capable of producing hydrocarbons in a significant quantity, in particular n-alkadienes, trienes, methylated squalenes, triterpenoids, tetraterpenoid and lycopadienes.
• These strains produce, in addition, particular hydrocarbons with long carbon chains, grouped under the name of botryococcenes.
• These hydrocarbons consist mainly of unbranched isoprenoid triterpenes of formula C n H 2n-10 .
• Botryococcus strains A, B and L are distinguished according to the lipid profile constituting the botryococci [Metzger, P et al. (2005) Applied Microbiology and Biotechnology 6 (25): 486-496].
• 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 .
• botryococcenes 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.
• the applicant has selected new strains of Botryococcus from his personal collections. Amongst these strains, those with the capacity to grow both in mixotrophic mode and in the presence of discontinuous illumination, especially in the form of flashes, were searched for.
• the close alternation of lighted phases and dark phases, generally perceived as stressful for micro-algae such as Botryococcus, has, unexpectedly, allowed the isolation of new strains of Botryococcus with a greater ability to evolve under mixotrophy and to adapt to light fluctuations.
• These new strains of Botryococcus able to withstand repeated changes in light intensity, are particularly suitable for the production of lipids and hydrocarbons in mixotrophic mode.
• they are content with a discontinuous light input, whose intensity is generally lower than that required by cultures in autotrophic mode, or whose illumination is permanent.
• part of the energy consumed by algae in this system comes from supplementation of the culture medium with carbon substrates, such as glycerol or acetate, which can come from by-products from various industries. .
• the inventor hypothesizes that the selection of the strains by flashing, makes it possible to isolate strains having a mixed metabolism, that is to say more able to simultaneously practice photosynthesis and the fermentation.
• the three strains of Botryococcus braunii are original in that one (827) appeared to be both mixotrophic and heterotrophic, which is not the case with the other known mixotrophic strains of Botryococcus braunii and the other two (828). and 829) as strictly mixotrophic (total inhibition of growth in heterotrophic) in the presence of glycerol, sucrose and lactose in their culture medium. These last two strains, 828 and 829, seem to have the same characteristics.
• the strain of Botryococcus sudeticus 841 for its part, has a strict mixotrophy in the presence of glucose and acetate in the culture medium, which distinguishes it from other strains and strains of the state of the art.
• the two strains of Botryococcus braunii, 827 and 828 received the CCAP 807/5 and CCAP 807/6 deposit numbers, respectively.
• the strain of Botryococcus sudeticus, 841 has received the deposit number CCAP 807/4.
• strains are found to be particularly suitable for the production of lipids in mixotrophic culture conditions, especially when they are cultivated in the presence of a light input whose intensity is variable or discontinuous.
• Figure 1 Graph showing the evolution of the biomass of microalgae (g / L) in culture, in f / 10 culture medium, supplemented with 10% of soil extract over time (days) for the different modes of culture: autotrophy, ()) mixotrophy ( ⁇ ) and flash mode mixotrophy according to the invention ( ⁇ ).
• the cultures are carried out using strain 828, as described in Example 2.
• Figure 2 Graph showing the evolution of the biomass of microalgae (g / L) in culture, in f / 10 culture medium, supplemented with 10% of soil extract over time (days) for different culture modes: autotrophy, ( ⁇ ), mixotrophy ( ⁇ ) and mixotrophy in flash mode according to the invention ( ⁇ ).
• the cultures are carried out using strain 829, as described in Example 2.
• Figure 3 Graph showing the evolution of the biomass of microalgae (g / L) in culture, in f / 10 culture medium, supplemented with 10% of soil extract over time (days) for the different modes of culture: autotrophy ( ⁇ ), mixotrophy ( ⁇ ) and mixotrophy in flash mode according to the invention ( ⁇ ).
• the cultures are carried out using strain 841, as described in Example 2.
• Figure 4 Diagram showing the fatty acid content of strains 828 and 841 after 5 days of culture according to the different culture modes (% fatty acids / dry matter) in autotrophy (auto), mixotrophy (mixo) and mixotrophy in flash mode ( mixo flash). detailed description
• the present invention therefore relates to a method for screening or selection strains of unicellular algae (microalgae), including the genus Botryococcus, capable of ensuring a high production yield of lipids and hydrocarbons.
• microalgae unicellular algae
• Botryococcus the genus Botryococcus
• the selected algae are both capable of growing in mixotrophic mode, thus of using one or more carbonaceous substrates as energy source, and of taking advantage, by photosynthesis, of a variable or discontinuous light input. Algae with these properties are considered to have higher lipid and hydrocarbon production potential than the others.
• the subject of the invention is also a process for the cultivation of microalgae of the genus Botryococcus, using a variable or discontinuous light supply under conditions similar to those used for the selection of microalgae.
• This method is characterized in that the light flux supplied to the algae in culture is variable or discontinuous over time. Contrary to popular belief, it appeared that a variable or discontinuous illumination of crops, especially in mixotrophic mode, had a favorable impact on the development of algae and allowed, in particular, to increase the production of lipids by the latter.
• the inventor believes that a discontinuous or variable light input has the effect of causing stress in the algae favorable to lipid synthesis. Indeed, it is common in nature that algae 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. It is provided, for example, in the form of flashes, that is to say on short periods of time.
• 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, that is to say that the illumination is not interrupted by dark phases, but 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), in a controlled and controlled way.
• the invention relates to a method for cultivating unicellular algae, 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 varies by an amplitude equal to or greater than 10 pmol. m “2 , s " 1 several times an hour, preferably equal to or greater than 40 pmol. m “2 , s " 1 , more preferably equal to or greater than 50 pmol. 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 (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 ⁇ . m “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. RRI 2 s "1, more preferably the values 0 and 100 pmol. 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 selection and culture method according to the invention is more particularly applicable to microalgae of the genus Botryococcus in order to select high yield lipid strains.
• the cultivation method according to the invention aims to increase the production of lipids and / or hydrocarbons, in particular via the recovery of lipids and hydrocarbons contained or excreted by microalgae, more particularly botryococcal type hydrocarbons.
• strains of microalgae in particular of the genus Botryococcus, 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 the production of lipids and hydrocarbons in terms of quality (lipid profile) and quantitative (lipids or total hydrocarbons produced).
• the microalgae can be selected in a fermentor from a pool of microalgae diversified and which we seek to select the variants favored by the mode of selection 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 cultivation method according to the invention is characterized more particularly in that the culture of the strains is carried out over several generations, preferably in a mixotrophic mode, and in that the cells loaded with lipids or hydrocarbons are harvested.
• a species of algae is considered to be mixotrophic, since it can be grown in the light, in a minimum medium (for example MM or f / 10 supplemented with 10% of soil extract) in which a carbon substrate is added at a rate of, for example, a concentration of carbon or glycerol, equivalent or greater than 5 mM, without observing growth inhibition, it is that is to say without noting a loss of biomass in dry weight relative to a culture carried out in the same minimum medium lacking a carbon substrate (that is to say in autotrophic mode).
• a minimum medium for example MM or f / 10 supplemented with 10% of soil extract
• a carbon substrate is added at a rate of, for example, a concentration of carbon or glycerol, equivalent or greater than 5 mM
• Preferred carbon substrates include acetate, glucose, cellulose, starch, lactose, sucrose, and glycerol.
• Products derived from the biotransformation of starch for example from maize, wheat or potato, in particular starch hydrolysates, which consist of small molecules, constitute carbon substrates of choice.
• the microalgae are selected from the species Botryococcus braunii and Botryococcus sudeticus. The membership of one or the other of these species being established on the usual criteria of classification of microalgae.
• the invention also relates to microalgae strains with a high hydrocarbon and / or lipid yield, which can be selected according to the method of the invention, characterized in that they are mixotrophic and capable of growing in discontinuous or variable light.
• the implementation of the method according to the invention more particularly made it possible to isolate new strains of the genus Botryococcus.
• These strains which were deposited in the Culture Collection of Algae and Protozoa (CCAP) collection, on October 20, 2010, according to the provisions of the Budapest Treaty, are as follows:
• This strain of the species sudeticus has the characteristic of being mixotrophic. To the applicant's knowledge, this is the first strain of this species described as being mixotrophic. Moreover, as for the preceding strains, this strain has the characteristic of being mixotrophic without being heterotrophic. This is particularly observed when the carbon substrate added in the minimum culture medium is glucose or acetate.
• the culture method according to the invention is applicable to any strain of the genus Botryococcus cultivable in a mixotrophic condition and is not limited only to the use of the new strains described in the present application.
• the inventors have been able to observe a productivity gain in the crops, particularly in terms of biomass, in all the strains of Botryococcus previously identified as being able to grow under mixotrophic conditions compared to the same cultures produced in the autotrophic mode.
• Botryococcus strains were selected from a collection of applicant strains consisting of strains taken from freshwater, isolated and characterized according to common criteria [Komarek, J. et al. (1992) P. Morphological differences in natural populations of the genus Botryococcus (chlorophyceae). Archiv fur Protistenischen, 141 (1-2): 65-100] [Dayananda C. et al. (2007) Isolation and characterization of hydrocarbon producing green alga Botryococcus hraunii from Indian freshwater bodies. Elect. J. Biotechnol., 10: 1-14].
• Botryococcus braunii and Botryococcus sudeticus were first cultured at 22 ° C in autotrophic (200 ⁇ l light) in Minimum Medium (MM) liquid [50 mL / L Beijerink Solution (NH 4 CI 8g / L, CaCl 2 ( 1 g / L, MgSO 4 2 g / L), 1 mL / L Buffer Phosphate (K 2 HPO 4 106 g / L KH 2 PO 4 53 g / L), 1 mL / L solution of trace elements (B0 3 H).
• MM Minimum Medium
• Cultures were carried out in mixotrophic mode (200 ⁇ l) in continuous and discontinuous light, as well as in heterotrophic mode (control at 0 ⁇ l of light) at 22 ° C on MM medium supplemented with carbon substrates: acetate 1 g / L, glucose 5g / L, lactose 10g / L, sucrose 10g / L or glycerol 5g / L.
• the 24-well microplates were placed in an incubation chamber (SANYO MLR-351 H) at 22 ° C, 60% humidity and 200 ⁇ light intensity for autotrophic and mixotrophic cultures and incubation chamber (BINDER). KB53) at 22 ° C, 60% humidity and in the dark ( ⁇ ) for heterotrophic cultures.
• SANYO MLR-351 H incubation chamber
• BINDER incubation chamber
• KB53 at 22 ° C, 60% humidity and in the dark ( ⁇ ) for heterotrophic cultures.
• strains whose culture in discontinuous light was more favorable than in continuous light were selected.
• 4 were more particularly studied: 3 strains of Botryococcus braunii (827, 828 and 829) and a strain of Botryococcus sudeticus (841).
• the two strains B. braunii 827 and B. sudeticus 841 have a strict heterotrophic character in the presence of sucrose.
• the other 2 strains B. braunii 828 and ⁇ . braunii 829 are strictly mixotrophic at 200 ⁇ in the presence of sucrose, and B. sudeticus strain 841 is strictly mixotrophic at 200 ⁇ in the presence of acetate.
• the two strains B. braunii 827 and B. sudeticus 841 show a significant growth at 0 ⁇ in the presence of 10 g / l of sucrose, growth greater than that in autotrophy.
• strains B. braunii 828 and B. braunii 829 when adding 10 g / L of sucrose in the culture medium and increased growth of the strain B sudeticus 841 when adding 1 g / L of acetate in the culture medium, compared to their growth in autotrophy (200 ⁇ of light intensity).
• the cultures of each of the strains isolated in Example 1 (828, 829 and 841) were carried out in fermenters (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 was set at 23 ° C.
• Stirring was carried out by means of 3 stirring rods placed on the shaft according to the Rushton configuration (three-bladed pumping propellers).
• the bioreactor is equipped with an external lighting system surrounding the transparent tank. The intensity as well as the light cycles are controlled and regulated by a dedicated automaton supervised by a computer station.
• the contribution of light in the bioreactor cultures was obtained from the LED lamps distributed around the outer wall of the fermenters.
• a clock triggers these LEDs for illumination times or pulses between 8 and 50 ⁇ .
• the luminous intensity of the flash system used in mixotrophy is equal to that used in autotrophy (control).
• the reactors were inoculated using a pre-culture performed on a shaking table (140 rpm) in a thermostatically controlled enclosure (22 ° C.) and continuously lit at 100 ⁇ E.
• Pre-cultures and cultures in bioreactors were performed in the middle f / 10 supplemented with 10% soil extract and 10 mM NaHCO 3.
• the carbonaceous substrate used for the bioreactor mixotrophic culture is sodium acetate at concentrations of between 20 mM and 50 mM. 2 - Crop monitoring
• the total biomass concentration was monitored by measuring the dry mass (filtration on GFC filter, Whatman, then drying in a vacuum oven, 65 ° C and -0.8 bar, for 24 hours minimum before weighing).
• Lipid content The ratio of the quantity of fatty acids present in the cells after 5 days of culture, relative to the total dry matter, was established for each culture mode: autotrophy (auto), mixotrophy (mixo) and mixotrophy in flash mode (mix-flash).

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