EP2633026A2

EP2633026A2 - Novel strains of microalgae of the botryococcus genus, and method for cultivating said microalgae in a mixotrophic mode

Info

Publication number: EP2633026A2
Application number: EP11832134.8A
Authority: EP
Inventors: Pierre Calleja
Original assignee: Fermentalg SA
Current assignee: Fermentalg SA
Priority date: 2010-10-28
Filing date: 2011-10-27
Publication date: 2013-09-04
Also published as: FR2966840A1; FR2966840B1; WO2012056187A3; US20130210095A1; WO2012056187A2; BR112013010341A2; CA2812579A1

Abstract

The invention relates to novel strains of microalgae which belong to the Botryococcus genus and which can grow in a mixotrophic mode, as well as to a cultivation method which comprises providing light in the form of flashes for the production of lipids and hydrocarbons, in particular in the form of botryococcenes, which are useful in the production of biofuel.

Description

New microalgae strains of the genus Botryococcus and method of cultivation in mixotrophic mode of said microalgae

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.

Preamble

Unicellular algae are photosynthetic microorganisms with an autotrophic character, that is, they have the ability to grow autonomously by photosynthesis.

Most unicellular algae species found in freshwater or oceans are strictly autotrophic, that is, they can only grow by photosynthesis. For these, the presence in their environment of carbon substrates or organic materials is not favorable to them and even tends to inhibit their growth.

However, a number of unicellular algae species, of very diverse families and origins, are found not to be strictly autotrophic. 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 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.

This particularity of so-called "mixotrophic" algae seems to be linked to their metabolism, which allows them to operate simultaneously photosynthesis and fermentation. Both types of metabolism coexist with one effect overall positive on the growth of algae [Yang C. et al. (2000) Biochemical Engineering Journal 6: 87-102].

At present, the classification of algae is still largely based on morphological criteria and the nature of photosynthetic pigments that contain their cells. As a result, it is not very indicative of the autotrophic, heterotrophic or mixotrophic nature of algae, whereas these cover a very large diversity of species and forms [Dubinsky et al. 2010, hydrobiologia, 639: 153-171]. As a result, a strain is considered to be mixotrophic since, experimentally, it can be proved that it has the capacity to grow by photosynthesis in a minimum medium, in which a carbon substrate, such as glucose, is added. acetate or glycerol. If this supplementation of carbon substrate does not give rise to a growth inhibition during the illuminated phase, then the strain can be considered to have a mixotrophic character.

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.

Under favorable conditions, 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].

It is also known that certain 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 . They can be converted by cracking and refining into kerosene or gas oil. Different groups of 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, reputedly highly pigmented, is generally carried out under conditions of high brightness, between 500 and 1500 μιηοΙ. 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.

A mutant strain of this strain, 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.

However, the lipid yields obtained using these algae are currently insufficient to be able to consider a profitable production of hydrocarbons on an industrial scale. Indeed, for the energy balance of the exploitation of microalgae of the genus Botryococcus to be satisfactory, it would be advisable to reduce the energy brought to the cultures in the form of light, while increasing the quantity and the quality of the lipids or the hydrocarbons convertible into bio-fuels.

To achieve this goal, 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. In particular, 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. In addition, 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. .

Without being bound by the theory, 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.

Moreover, according to him, it is when the strains pass from one type of metabolism to another, according to the variations of luminous intensity, that they tend to store lipid reserves, in particular in the form of hydrocarbons.

The implementation of the selection process according to the invention described hereinafter, applied more particularly to strains of microalgae of the genus Botryococcus, made it possible to isolate three new strains of the species Botryococcus braunii and another of the species Botryococcus sudeticus, particularly suitable for the production of lipids and hydrocarbons.

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.

These three strains were the subject of three strains of a strain deposit under the Budapest Treaty on October 20, 2010, with the CCAP (Culture Collection of Algae and Protozoa, Scottish Association for Marine Science, Dunstaffnage Marine Laboratory, Oban, Argyll PA371QA, Scotland, United Kingdom).

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.

These 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.

The various aspects and advantages of the invention are detailed below.

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 (Δ). 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 (Δ). 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 (Δ). 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.

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.

Without being bound by the theory, 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.

By discontinuous illumination, it is necessary to hear an illumination punctuated by periods of darkness. 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.

According to a preferred aspect of the invention, 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.

According to another embodiment of the invention, 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.

According to the invention, 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.

According to the invention, it is also possible to carry out a light supply combining continuous and discontinuous illumination phases.

In particular, 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, discontinuous or variable, 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 μιηοΙ. m ^"2. s ^{" 1} . In other words, 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. Preferably, 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 .

It is recalled that 1 pmol. m ^"2 , s ^{" 1} corresponds to 1 μΕ m ^"2 , s ^{" 1} (Einstein), unit used in the examples of the present application.

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 is characterized in that it comprises one or more of the following steps:

the cultivation of different strains of the genus Botryococcus in the dark with discontinuous or variable light input over time, the intensity of which in micromoles of photons preferably varies by an amplitude equal to or greater than 50 pmol. m ^"2 , s ^{" 1} at least once an hour; the maintenance of said culture over several generations;

the isolation of the strain or strains whose cell number has increased the most during said generations.

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.

To carry out the screening of strains, different 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).

Alternatively, 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. In this case, 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.

For the purposes of the present invention, 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).

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.

Preferably, 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.

As indicated by the examples of the present application, 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:

- Botryococcus braunii strain 827, deposited under the number CCAP 807/5.

- Botryococcus braunii strain 828, deposited under the number CCAP 807/6.

These two strains have the characteristic of being mixotrophic but are not heterotrophic, that is to say that they are cultivable in a minimum medium supplemented with carbon substrate, in the presence of a luminous contribution, but that they are not in the absence of light. This behavior is observed, in particular, when the carbon substrate added in the culture medium is sucrose, lactose or glycerol. To the knowledge of the inventor, this is the first time that strains of Botryococcus have this feature.

- Botryococcus sudeticus strain 841, deposited under the number CCAP 807/4.

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.

As observed by the applicant, the fact that the strains thus selected have good aptitude to grow in mixotrophic mode, in the presence of a discontinuous light, predisposes said strains to a higher production of lipids and hydrocarbons, especially botryococcenes.

Nevertheless, 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. In fact, 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.

The following examples are intended to complete the description and illustrate the invention. They can not confer any limitation on the invention. Example 1

1 - Strains: 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 Protistenkunde, 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].

2- Culture conditions:

Several isolates of Botryococcus braunii and Botryococcus sudeticus were first cultured at 22 ° C in autotrophic (200 μl 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). ₃ 1 1 .4g / L, ZnS0 ₄ .7H ₂ 0 22g / L MnCl ₂ 4H ₂ O 5.06g / L FeS0 ₄ 7H ₂ O 4.99g / L, COCl ₂ 6H ₂ 0 1 .61 g / L, CuS0 ₄ 5H ₂ O 1 .57g / L, Mo ₇ O ₂ (NH4) ₆ 4H ₂ 0 1 .1 g / L EDTA 50 g / L), 2.42g / L Trizma base, pH adjusted between 7.2 and 7.4 by HCl, 1 .2mg / L Vitamin Bi and 0.01 mg / L Vitamin Bi ₂ (added extemporaneously)].

Cultures were carried out in mixotrophic mode (200 μl) in continuous and discontinuous light, as well as in heterotrophic mode (control at 0 μl 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 heterotrophic and / or mixotrophic nature of the Botryococcus strains was evaluated by culturing the microalgal strains in an MM + carbon substrate medium in 24-well microplates (V = 2mL). Growth control in autotrophy (MM) has been systematically performed to serve as a reference for mixotrophic and heterotrophic cultures.

The 24-well microplates were placed in an incubation chamber (SANYO MLR-351 H) at 22 ° C, 60% humidity and 200 μΕ light intensity for autotrophic and mixotrophic cultures and incubation chamber (BINDER). KB53) at 22 ° C, 60% humidity and in the dark (ΟμΕ) for heterotrophic cultures.

Cell growth was evaluated by comparing turbidity and / or chlorophyll content with reference to autotrophy. Biweekly monitoring was provided for 2 weeks for autotrophy and mixotrophy cultures and for 2 to 3 weeks for heterotrophic cultures. Mobility and pigmentation of microalgae grown in autotrophy / mixotrophy (auto / mixo) and heterotrophy were observed and compared using a 10X and 32X binocular microscope.

For the cultures carried out in mixotrophic mode in discontinuous light, the contribution of light consisted of flashes at the rate of 30 flashes of 30 seconds per hour.

Strains whose culture in discontinuous light was more favorable than in continuous light were selected. Among these strains, 4 were more particularly studied: 3 strains of Botryococcus braunii (827, 828 and 829) and a strain of Botryococcus sudeticus (841).

3 - Properties of the four strains of the genus Botryococcus selected:

The effect of light (autotrophic and mixotrophic columns) and carbon substrates (mixotrophic and heterotrophic columns) such as glucose (Glc 5g / L), acetate (Ac 1g / L), sucrose (Sac 10g / L) ), lactose (Lac 10g / L) and glycerol (Gly 5g / L) on the growth of 4 strains of the genus Botryococcus was evaluated by microplate screening in 24 wells on MM liquid medium (see table below). Growth monitoring was carried out for 3 to 4 weeks bi-weekly macroscopic observation of cultures and microscopic binocular (10X and 32X objectives).

Table 1: Growth of Selected Strains in Mixotrophy

inhibited growth; +: moderate growth;

++: significant growth; +++: strong growth

Of the 4 strains tested of the genus Botryococcus, 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 β. braunii 829 are strictly mixotrophic at 200 μΕ in the presence of sucrose, and B. sudeticus strain 841 is strictly mixotrophic at 200 μΕ in the presence of acetate. Indeed, the two strains B. braunii 827 and B. sudeticus 841 show a significant growth at 0 μΕ in the presence of 10 g / l of sucrose, growth greater than that in autotrophy. There is also an increased growth at 200 μΕ of light intensity of 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 μΕ of light intensity).

4 - Conclusion:

This study has highlighted new strains of Botryococcus braunii and Botryococcus a mixotrophic character vis-à-vis certain carbon substrates. The addition of carbon substrates such as sucrose and acetate significantly improves the respective growth of these strains.

Example 2

1 - Culture of Botryococcus strains in bioreactor according to the Mixo / flash process

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 stirring speed and the aeration rate were regulated for a minimum of 100 rpm and a maximum of 250 rpm with Qmini = 0.5 vvm / Qmaxi = 2 vvm respectively. 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 μΕ. 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 μE. 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).

Quantitation of total lipids was performed on cell samples (10 ⁷ cells / mL) extracted from 5-day old cultures. The lipids were extracted according to the lipid extraction methods described by Bligh, EG and Dyer, WJ [A rapid method of total lipid extraction and purification (1959) Can. J. Biochem. Physiol 37:91 1 -917].

3 - Results Evolution of the biomass:

The biomass measurements made in the various samples taken daily from the crops for 15 days were plotted in the graphs of Figures 1 to 3 for each of strains 828, 829 and 841. The graphs make it possible to compare the evolution of dry biomass. by volume of culture in the different modes of autotrophic, mixotrophic (continuous light) and mixotrophic with flash.

These measurements show that, for each of the strains, a substantial gain in biomass is obtained in flash mode. The increase is of the order of 30% compared to the mixotrophy in continuous light and of the order of 120% compared to the autotrophic diet.

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).

The results are shown in diagrammatic form in Figure 4, for both analyzed strains 828 and 841. These results show an increase in the content of fatty acid strains by a factor of 4 to 5 when the microalgae are cultivated in mixotrophic mode compared to the cultures in autotrophic mode. The content obtained in mixotrophic mode with flash and without flash is comparable after 5 days.

Claims

1. Process for the cultivation of microalgae of the genus Botryococcus for the production of lipids or hydrocarbons, characterized in that the steps comprising:

the cultivation of one or more strains of microalgae of the genus Botryococcus in the dark in the presence of a discontinuous or variable light input over time, the intensity of which in micromoles of photons varies by an amplitude of more than 10 μιτιοΙ. m ^"2 , s ^{" 1} , at least once an hour;

the maintenance of said culture over several generations in the presence of a carbon substrate in the culture medium;

harvesting the cells loaded with hydrocarbons or lipids.

2. Method according to claim 1, characterized in that the light input is discontinuous.

3. Method according to claim 1 or 2, characterized in that the light input varies more than 40, preferably more than 50 μιτιοΙ. m ^"2 ^{s -1.}

4. Method according to any one of claims 1 to 3, characterized in that it further comprises the recovery of lipids or hydrocarbons contained or excreted by microalgae.

5. Process according to claim 4, characterized in that the hydrocarbons contained or excreted by the microalgae comprise botryococcenes.

6. Method according to any one of claims 1 to 5, characterized in that the culture medium is a minimum medium comprising a carbon substrate.

7. Method according to any one of claims 1 to 6, characterized in that the carbon substrate comprises acetate, glucose, cellulose, starch, lactose, sucrose or glycerol.

8. Method according to any one of claims 1 to 7, characterized in that the light input is in the form of flash.

9. Method according to claim 8, characterized in that said flashing consists of successive phases of illumination of a duration of between 5 seconds and 10 minutes, preferably between 10 seconds and 2 minutes, more preferably between 20 seconds and 1 minute. minute.

10. Method according to any one of claims 1 to 9, characterized in that the microalgae are selected from the species Botryococcus braunii.

11. Method according to any one of claims 1 to 10, characterized in that the microalgae are selected from the species Botryococcus sudeticus.

12. Microalga cultivable according to the method of any one of claims 1 to 11, corresponding to one of the following strains deposited with the CCAP:

- Botryococcus sudeticus strain deposited under No. CCAP 807/4;

- Botryococcus braunii strain deposited under No. CCAP 807/5; or

- Botryococcus braunii strain deposited under No. CCAP 807/6.