WO2014003530A1

WO2014003530A1 - Method for increasing the potential for biofuel production from microalgae by using bio-modulators

Info

Publication number: WO2014003530A1
Application number: PCT/MA2013/000020
Authority: WO
Inventors: Imane WAHBY; Iman BENNIS; Hicham EL ARROUSSI; Redouane BENHIMA
Original assignee: Moroccan Foundation For Advanced Science, Innovation & Research (Mascir)
Priority date: 2012-06-28
Filing date: 2013-06-28
Publication date: 2014-01-03
Also published as: WO2014003530A4; MA34793B1

Abstract

The present invention proposes a method for cultivating and processing microalgae isolated from marine and extremophilic environments for optimal biofuel production. The species of microalgae selected can be species of the genus Dunaliella (D. bardawil, D. salina, D. acidophilo, D. biolecta, D. lateralis, D. maritima, D. minuta, D. parva, D. peircei, D. polymorpha, D. primolecta, D. pseudosalina, D. quartolecta, D. tertiolecta, D. viridis and others). For the purpose of cultivating microalgae for the production of biofuel, the optimal conditions for the production of biomass and lipids are different. This invention proposes a method which makes it possible to simultaneously stimulate the accumulation of biomass and intracellular lipids (more than 60% by dry weight), essentially the neutral lipids sought for the production of biodiesel, by reinforcing the action of natural bio-modulators or chemical analogues (auxins, cytoquinines, gibberellins, NaCI) in hypersaline and alkaline conditions. Another aspect of the invention is the application of ad hoc changes to the pH of the cultivation media causing the spontaneous precipitation of cells loaded with lipids once the cycle of growth and storage of lipids intended for the production of biodiesel is complete. This method makes it possible to recover microalgal biomass quickly and passively, requiring little energy and without the step of centrifugation or filtration, in order to extract the intracellular lipids and transform same into biodiesel. This method ensures very selective cultivation conditions which only a predetermined number of species has a similar response to and/or is able to tolerate, which limits the risks of contamination by other microalgae, bacteria or fungi, this frequently being a problem in open cultivation systems such as ponds. This offers a system for cultivating microalgae that is advantageous and applicable on a large scale for the biofuel application and others.

Description

Method for increasing the biofuel production potential from microalgae using bio-modulators

Field of the invention

The invention provides a method and composition for increasing the potential of a microalgal culture for the production of biofuel, and other high value-added products, using bio-modulators under selective culture conditions.

State of the art:

Due to the decline in global oil reserves and increasing global awareness of the development of renewable energy, the exploitation of alternative energy sources alternative to conventional fuels is attracting more and more global attention. Among these forms of clean energy are solar, wind and biofuels that come from biological sources that fix carbon such as photosynthetic organisms. Thus, the use of liquid biofuels in the transport sector has experienced strong growth due, mainly, to policies focused on energy security and the reduction of greenhouse gas emissions. Given the technical and socio-economic constraints related to the use of first and second generation biofuels (based mainly on the use of food sources such as plants for the production of biodiesel), third generation biofuels obtained from microalgae , seem to be the only source of transportation biofuels that could meet global demand (Chisti 2007, Biotechnol Adv, 25: 294-306).

[0003] Microalgae cover a diverse group of uni- or multicellular microorganisms, most of which are photosynthetic organisms. They incubate prokaryotes (cyanobacteria or chloroxybactria) and eukaryotes such as green algae (Chlorophyta), red algae (Rhodophyta) and diatoms (Bacillariophyta). The benefits of using microalgae for biofuel production are: (1) their rapid growth; they can double their biomass in 3 to 3.5 hours during the exponential phase. (2) several species have a high lipid content (20-60%) per dry weight of the cell. (3) the production of one kg of microalgae biomass consumes 1.83 kg of C0 ₂ per fixation, which would contribute to the reduction of greenhouse gas emissions. (4) the cultivation of microalgae uses resources that can not be used in agriculture, such as non-arable land or seawater (for marine species) or wastewater, posing no threat to agricultural resources.

[0004] Therefore, based on the photosynthetic efficiency and growth rate of microalgae, the theoretical calculations indicate that the annual production of algal oils could exceed 30,000 L, or 200 barrels per hectare in crops. massive microalgae. 100 times higher than some oil crops such as soybeans; the main source currently used for biodiesel production in the United States. Despite all these advantages, some handicaps delay the advancement of this type of project towards the development of profitable production processes on a large scale. These critical points include: the cultivation method, the productivity of microalgae and the method of harvesting biomass. In this sense, the use of biotechnology seems to be the most promising way to improve the yield of oilseed microalgae and to develop a viable third generation biodiesel industry.

The yield of microalgae used for the production of biofuels is essentially evaluated in terms of growth and quantity and quality of lipids accumulated by the microalgal cell. However, for microalgae, the optimal conditions of biomass production and those of lipid production are different. Griffiths and Harrison (2009) J. App. Phycol. 21: 493-507 mentioned that there is no correlation between lipid content and lipid productivity: some lipid-rich species (greater than 50% by dry weight) have a productivity of less than 20 mg / L /day. Similarly, species with low lipid content (10% by dry weight) can show considerable productivities (more than 50 mg / L / day).

[0006] A profitable biofuel production process involves the optimization of both aspects: growth (biomass) and lipid content. Productivity can be improved by modifying the physicochemical parameters such as temperature, light intensity, pH and nutritional composition of the growth medium as Microalgae are very sensitive to environmental conditions. Thus, in the face of adverse conditions, certain microlagues can respond with an increase in lipid reserves. Another method for improving the yield of microalgae is the modification of the culture conditions, for example the change in trophic conditions (US Patent Publication 2009/0148928), or nutritional stress (US Patent Application No. 2012/0088279). Rosenberg et al., Curr Opin Biotech 19: 430-436 (2008), but these methods can cause a repression of growth and hence a decrease in productivity, and in others they are difficult to apply on a large scale to production. As alternatives, several studies have shown the effectiveness of working in two phases to increase the productivity of the third-generation biofuel production process: a first phase to stimulate microalgae growth, and hence biomass, followed by a second phase phase of lipid accumulation by application of stress, for example the application of phytohormones or biochemical / biological modulators to promote the growth of microalgae followed by a nitrogen deficiency phase to stimulate lipid production in microalgae species that respond to this type of stress (US Patent Appl. Publ. 2010/021002). The choice of the type of stress and its period of application determines the effectiveness of the process and depends closely on the requirement of the microalgae. With regard to the halophilic microalgae, which respond to salt stress by an increase in their reserve lipid content (sought for biofuel production), the use of phytohormones to stimulate growth has already been described, however this type of stress essentially stimulates the accumulation of biomass. The combination of this type of bio-modulators with osmotic stress by increasing salinity, at the end of the active growth phase, would significantly increase the productivity of the halophilic species. Description of the invention

The invention provides a biofuel production process from microalgae optimized both for the production of biomass and lipids and for passive recovery of biomass once the microalgae are loaded with lipids. This allows the increase of the productivity of these microalgae and the decrease of the cost of production as the biomass harvest is done passively.

[0008] A microalgae culture refers to one or more species of microalgae living in an environment that ensures their growth and spread. Crop components typically include water, C0 ₂ , minerals and light (for autotrophic species). The culture temperature is also controlled.

[0009] A first aspect of the invention provides a method and composition for the production of biofuel from microalgae. The culture medium is based on enriched seawater with non-stressful levels of nutrients, mainly nitrogen, phosphorus and potassium, iron and other microelements, chelators and buffers for pH control (Tris, MES). , HEPES, MOPS, mono-, di- or tri-basic phosphate, etc.). The growth temperature is between 20 and 35 ° C depending on the species, for non-thermophilic microalgae object of this invention.

In certain embodiments, the microalgae species chosen may be oleaginous species and others, belonging to the classes Chlorophyceae, Cyanophyceae, Bacillariophyceae, Xantophyceae, Chrysophyceae. Oleaginous microalgae are the species that can, under known conditions, accumulate a considerable lipid level with respect to their biomass. For example, oleaginous microalgae are capable of accumulating lipids up to at least 10%, at least 20%, at least 30%, at least 40%, at least 50% of their biomass. These microalgae may belong to the genera Dunaliella, Chlorella, Nannochloropsis, Haematococcus, Skeletonemas, Melosira, Thalassiosira, Nitzschia, Navicula, Tetraselmis, Nannochloris. One of the recommended genera for biofuel production is Dunaliella which belong to the class of Chlorophyceae, Order Volvocales, family Dunaliellaceae, and contains the following species: D. bardawil, D. salina, D. acidophila, D. biolecta, D lateralis, D. maritima, D. minuta, D. parva, D. peircei, D. polymorpha, D. primolecta, D. pseudosalina, D. quartolecto, D. salina, D. tertiolecta, D. viridis and others capable of producing lipids up to at least 10%, at least 20% of the biomass.

In some embodiments, the microalgae species may be Dunaliella tertiolecta.

A second aspect of the invention provides a method for selectively accelerating the growth of microalgae using plant growth factors as bio-modulators. These named type I bio-modulators include at least one, two, three, or more molecules belonging to the auxin family, cytokinins, gibberellins, abscisic acid or ethylene.

Growth factors such as phytohormones or plant hormones, plant growth regulators and synthetic molecules having a similar effect, are involved or control, among others, cell division, growth and metabolism of plant cells. The concentration and combination of these phytohormones determines their mode of action.

In certain embodiments, the auxins may be 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), acid 1-naphthalene acetic acid (AIMA), indole acetic acid (AIA), indole-3-butyric acid (AIB).

In some embodiments, the cytokinins may be zeatin, thidiazuron (TDZ), benzylaminopurine (BAP), isopentenyladenine (IPA), kinetin.

In some embodiments, gibberellins include GA ₃ .

In some embodiments, other bio-modulators such as signaling molecules may be used. These molecules include jasmonates and salysilic acid at concentrations of 10 ^{~ 2} , 10 ^"3 , 10 ^{" 4} , 10 ^"5 , 10 ^{" 6} , 10 ^"7 , 10 ^{" 8} M in the culture medium.

In certain embodiments, the ratio (w / w) of the bio-modulators I in the case of auxin mixture: cytokinin, auxin: gibberellin can range from 1: 2 to 2: 1, preferably 1: 1. . In some embodiments, the addition of this first type of bio-modulators improves the growth of microalgae, 20%, 40%, 60%, 80%, 100% compared to the first culture conditions.

In some embodiments, the bio-modulators I are added in the presence of vitamins B1 (Thiamine), B8 or H (biotin) and B12 (cobalamin or cyano-cobalamin) or similar substances having a similar effect. Vitamins are added alone or in combination at concentrations between 0.005 and 0.05 mg / L of culture medium.

In some embodiments, the addition of bio-modulators I improves the accumulation of the accumulated lipid content per unit of biomass by 40%, 50%, 70% and 90%, 100% compared to the control no. treaty.

Another aspect of the invention is the introduction of a second type of biomodulator, called bio-modulators II, just at the beginning of the storage lipid storage phase (between 6 and 20 days depending on the species and culture conditions). This second type of bio-modulators is osmo-regulators that potentiate the action of the first type of bio-modulators and generates the rapid accumulation of lipids. For biological organisms these mineral salts are involved in: the control of the water balance, the action of hormones and enzymes, the regulation of the acid-base balance (pH), the catalysis of many biological reactions, etc. In some embodiments, the time of addition of the osmo-regulators is decisive for the yield: the early or late addition of the osmo-regulators can decrease the yield and even cause the slowdown of the growth of microalgae.

In some embodiments, bio-modulators II include NaCl, KCl or other osmo-regulators at concentrations between 0.5 and 5 M relative to the culture medium.

In some embodiments, the addition of bio-modulators II improves the total lipid level by 100%, 120%, 130%, 140%, 150%, 180%, 190% and more than 200%. compared to control. The percentage represents the lipid level per unit of biomass.

In some embodiments, the osmo-regulators improve the action of bio-modulators I between 30 and 80% compared to the use of bio-modulators I alone. In certain embodiments, the neutral lipids generally represent the most sought-after lipids for the production of lipid biofuels. The use of bio-modulators I improved the accumulation of neutral lipids by 5%, 20%, 30%, 50%, 90% and 120% and more over control. The combination of the two biomodulators further improved the accumulation of neutral lipids with an increase of between 60%, 70%, 140%, 150% and 170%.

In some embodiments, the bio-modulators II have potentiated the action of the first group of bio-modulators with percentages ranging from 20%, 30%, 50%, 60%, 80% relative to the use. bio-modulators I alone.

In some embodiments, this combination of bio-modulators can be used for the production of other high value-added products such as carotenoids and polyunsaturated fatty acids.

Another aspect of the invention is the introduction of a third bio-modulator, which is an acid or a base or other product having an effect on the pH, in the presence of both bio-modulators I and II. This third bio-modulator is added at the beginning of the production cycle and undergoes a modification (increase in concentration) 4 days after the addition of bio-modulator II, which causes the precipitation of the biomass and facilitates its harvest. At the end of the storage lipid accumulation phase (and other types of lipids) in the microalgal cells, the biomass is separated from the culture medium by different methods alone or in combination. These methods can be centrifugation, pressure filtration, mechanical filtration or others, used alone or in combination. On an industrial scale, in view of the large volumes used, the centrifuges or filtrating apparatuses are of high capacity (US Patent Appl Publ No. 20040121447, US Pat No. 6,524,486) which makes the harvesting process very expensive. . Other methods of separating the microalgal biomass from the culture medium by including chemicals may be used. These substances have a flocculating or coagulating effect and may be aluminum sulphate, polyacrylamide or others. However, the addition of these substances to the biomass can change its composition and negatively affect the next step which is the extraction of lipids. In addition, on an industrial scale the cultures are generally carried out in continuous mode and the elimination of these substances from the medium after the recovery of biomass can be very complicated, hence the difficulty of applying these methods of biomass recovery on an industrial scale.

The proposed method is based on point changes in the concentration of bio-modulator III in the culture medium to maintain the microalgae in suspension during the biomass and lipid accumulation phases and to cause the sedimentation of the charged cells. in lipids. These pH changes vary between 6 and 11. A large number of acids can be used to modulate the pH including hydrochloric acid, lactic acid, acetic acid, sulfuric acid. Similarly, a large number of bases can be used to modulate the pH including potassium hydroxide, sodium hydroxide, calcium hydroxide. The advantage of this method on an industrial scale is that it is low cost and reversible: a re-modification of the pH after the harvest makes that the young microalgae, used for a new production cycle, are maintained in suspension (natural state) until the accumulation of lipids, and so on.

Another aspect of the invention provides a method of cultivating microalgae and producing lipids suitable for industrial production of biofuel from microalgae. The use of selective conditions increases productivity and limits contamination by other species, especially for open-water crops. The possibility of the process of passively harvesting the biomass loaded with lipids makes it easier to harvest biomass and to reduce the cost of harvesting biomass by existing techniques such as centrifugation or filtration.

When the microalgae biomass is harvested, bioproducts (such as lipids) are released by mechanical methods (press, glass beads, heat shock or osmotic, trituration, ultrasound or other known methods), chemical (solvents, enzymes, surfactants or others). The separation of lipids from the remainder of the biomass can be done using a mixture of solvents such as hexane, methanol, ethanol, isopropanol, chloroform, dichloromethane, generally coupled with a mechanical method such as centrifugation. The isolated product is therefore treated in different ways depending on the purpose. For example, for the production of biodiesel from lipids extracted from biomass, one can apply a method which is transesterification as the example of the process used in US Pat. No. 5,354,878. The standard transesterification protocol involves an alkaline or acid catalyst to convert triglycerides (and other lipids) into fatty acid esters (biodiesel) and glycerol.

Brief description of the Figures

FIG.l. Explain the process developed. The active or exponential growth phase represents the biomass accumulation phase, it can begin directly after the microalgae culture or be preceded by a latency phase during which the microalga adapts to its new environment. The bio-modulator I is introduced into the medium during this phase, generally the same day of the launching of the culture. The active growth phase is followed by a stationary phase where growth stops and begins the accumulation of biomolecules like the reserve lipids sought for the production of biofuels. Type II biomodulators are introduced at the beginning of this phase. At the end of the stationary phase the third type of biomodulators is added to increase the pH of the culture medium and precipitate microalgae to facilitate their harvest.

FIG.2. Shows a copy of the growth curve of Dunaliella tertiolecta in the presence and absence of bio-modulators I. Growth monitoring is performed by measuring the optical density of the cultures.

FIG.3. Shows a copy of the combined effect of bio-modulators I and II on lipid production by Dunaliella tertiolecta compared to untreated control. Lipid analysis is performed gravimetrically after 14 days of start of culture.

FIG. 4. Shows a copy of the combined effect of bio-modulators I and II on neutral lipid production by Dunaliella tertiolecta compared to untreated control and application of bio-modulators I alone. Lipid analysis is performed by flow cytometry after 4 days of treatment.

Examples:

After the detailed description of the invention, the specific examples given below are given to illustrate certain aspects of the invention. Example 1. Effect of the addition of bio-modulators I on the accumulation of biomass

Dunaliella tertiolecta.

The microalgae cultures were carried out (in triplicates and in three independent experiments) in previously sterilized glass bioreactors, containing filtered seawater, sterilized and enriched with the f / 2 growth medium ( Guillard and Ryther 1962, Guillard 1975). All cultures were agitated by bubbling using clean air, and the light intensity was set at 65 μηηοΙ ^ -ιτΓ ² under a continuous lighting regime.

An example of the composition of the medium f / 2 is given in Table 1.

Table 1

Compound Quantity Stock Solution Final Concentration

NalM0 ₃ 1 mL / L 75 g / L dH ₂ 0 8.83 10 ^-4 M

NaH ₂ P0 ₄ H ₂ 0 1 mL / L 5 g / L dH ₂ 0 3.63 10 ^-4 M

FeCl ₃ H ₂ 0 1 ml / L 3.15 g / L dH ₂ 0 1 10 ^-5 M

Na ₂ EDTA 2H ₂ 0 1 ml / L 4.36 g / L dH ₂ 0 1 10 ^-5 M

CuS0 ₄ 5H ₂ 0 1 mL / L 9.8 g / L dH ₂ 0 4 10 ^~ 8M

Na ₂ Mo0 ₄ .2H ₂ 0 1 mL / L 6.3 g / L dH ₂ 0 3 10 ^"8 M

ZnS04 7H ₂ 0 1 mL / L 22.0 g / L dH ₂ 0 8 10 ^"8 M

COCl ₂ 6H ₂ 0 1 mL / L 10.0 g / L dH ₂ 0 5 10 ^"8 M

MnCl ₂ 4H ₂ 0 1 mL / L 180.0 g / L dH ₂ 0 9 10 ^"7 M

To prepare 1 L of seawater enriched with the medium f / 2, 1 L of natural or synthetic seawater is filtered, autoclaved and added with 1 ml of sterile stock solutions of NaNO ₃ , 1 ml NaH ₂ P0 ₄ H ₂ 0 and 1 ml of microelements and 0.5 ml of vitamid solution. The initial pH of the medium is adjusted to 7.2-7.8 before inoculation of microalgae. In this example at least 10 bio-modulators were selected including 2,4-D (1 mg / L) and / or AIA (1 mg / L) and / or 6-BAP (1 mg / L). and / or GA (lmg / L) and 3 vitamins: B1: 0.01 mg / L, B8: 0.005 mg / L and B12 0.05 mg / L (Table 2). Stock solutions are prepared independently, sterilized by filtration (0.2 μιη) before preparing the final formula of bio-modulators I.

Table 2

Elements included in the Stock Solution Final Concentration (L ¹ )

Bio-modulator 1

2,4-1 mg / ml 1 mg acid

dichlorophénoxyacétique

Lindole-3-acetic acid 1 mg / ml 1 mg

6-Benzylaminopurine 1 mg / ml 1 mg

Gibberellic acid 1 mg / ml 1 mg

Vitamin Bl 200 mg / L 0.1 mg

Vitamin B8 1 mg / L 0.0005 mg

Vitamin B12 1 mg / L 0.0005 mg

The growth of microalgae was measured by regular sampling every 2 days to measure the optical density. In Table 3, it is obvious that the addition of group I of bio-modulators caused an increase in biomass accumulation, mainly at concentrations between 0.5 and 1 mg / L of medium during the active growth phase. of Dunaliella teriolecta. This improves its growth for a later application in the production of products derived from microalgae. According to Figure 2, the application of biomodulators I at selected concentrations improves growth between 20%, 30%, 40%, 50% and 60% compared to the untreated contract. In most cases, this improvement was 40% in the case of Dunaliella species. Table 3

Effect of different concentrations of bio-modulators I on the growth of Dunaiiella tertiolecta

Control concentration 0.5 mg / L 1 mg / L 1.5 mg / L of Biomodulators I

T0 0.029 ± 0.001 0.030 ± 0.01 0.030 ± 0.001 0.024 ± 0.006

Growth TO + 2 0.126 0.012 0.189 ± 0.006 0.139 ± 0.009 0.155 0.010 expressed in TO + 4 0.306 ± 0.020 0.361 ± 0.030 0.321 ± 0.010 0.289 ± 0.040 density TO + 6 0.402 ± 0.040 0.523 ± 0.024 0.511 ± 0.022 0.452 ± 0.042 optical TO + 8 0.518 ± 0.050 0.700 ± 0.020 0.710 ± 0.040 0.550 ± 0.043

Example 2. Effect of Biomodulators I and II on Total Lipid Accumulation: Gravimetric Analyzes

The example deals with the effect of combined bio-modulators I and II on the accumulation of lipids in two strains of Dunaiiella tertiolecta. The strains used in the example are D. tertiolecta (SAG 13.86) and D. tertiolecta (MAR 029) isolated from Morocco. The cultures were carried out as described in Example 1. The group I of bio-modulators was initially added to the culture medium and after 8 to 10 days, the group II containing osmo- regulators was added to the medium to potentiate The action of group I. The osmo- regulators (NaCl or KCl) were prepared in concentrated solutions and the necessary volumes were added to the culture medium to have a final concentration of 1 to 5M. In this example, the concentration of osmo-regulators used was 2 M. After 4 days of culture in the presence of the two bio-modulators, the microalgal biomass is harvested (using the method described in Example 4. The total lipids are extracted using a modified protocol from the method of (Bligh and Dyer, 1959) Lipid extraction is done after biomass submission to ultrasound treatment for 15 min for cell disruption. subsequently carried out using a mixture of chloroform: methanol: water (2.1: 0.75 v: v: v) followed by centrifugation for 3 min to separate the organic and aqueous phases The organic phase is recovered and added with sodium chloride (0.9%, 1: 5 v: v) and magnesium sulphate To avoid oxidation of lipids, the protocol includes the use of 1 to 5 ml of BHT (10%). solvents are finally evaporated and the weight of the lip measured ideas. Table 4 shows the improvement of lipids following the treatments. The addition of group I bio-modulators improves lipid accumulation (Figure 3). It is noted that the use of group I of bio-modulators improved the accumulated lipid level by 40%, 50%, 70% and 96% compared to control. The addition of the second group of biomodulators (osmo-regulators) increased the accumulated lipid level by 120%, 140%, 180%, 190% and more than 200% compared to control. Thus, osmo-regulators improved the action of bio-modulators I by between 30 and 60% compared to the use of bio-modulators I alone. In this example, the best combination was to use the bio-modulators I between 0.5 and 1 mg / L and the osmo-regulators with 2 M.

Table 4

Effect of the combination of bio-modulators I and II on lipid accumulation in Dunaliella tertiolecta. Lipids are quantified by gravimetry and expressed in% (g lipid per g of biomass)

Body Treatment

D. tertiolecta 0.5 mg / L 1 mg / L 1.5 mg / L

Bio-modulators 1

(SAG 13.86) 38.33% 43.14% 35.02%

Bio-modulators l + ll 68.31% 69.55% 54.56%

Control 24.39%

D. tertiolecta Bio-modulators 1 48.62% 42.04% 37.13%

(MAR 029) Bio-modulators l + ll 65.56% 62.2% 51.26%

Control 21.39%

EXAMPLE 3 Effect of Biomodulators I and II on Neutral Lipid Accumulation: Flow Cytometry Analyzes

Neutral lipids generally represent the most sought-after lipids for the production of biodiesel. Neutral lipids can be analyzed by different methods including flow cytometry. The use of this method for the analysis of the lipids of microalgae has been previously developed (Doan et al., Bio Bioener, 35: 2534-2544, Lopes da Silva et al., 2009 Appl Biochem Biotechnol, 159: 568). 578, Alonzo and Mayzaud 1999. Marine Chem, 67: 289-301). Visualization of lipids in the cytometer is possible following a labeling with a fluorescent marker called red Nile which, after excitation by a laser at 488 nm, emits an intense yellow fluorescence (emission length 560-640 nm) when it is associated with neutral lipids. When Nile red is dissociated in polar lipids it emits a red fluorescence at more than 650 nm (these emission lengths are specific to the equipment used: in this case it is a FACS Calibur System, Becton Dickinson). For the labeling of the microalgae cells, 1 ml of culture is added with 50 μl of a stock solution of NR (prepared in acetone) and the mixture is incubated for 2 to 10 min at 37 ° C. in the dark. The sample is then analyzed in the cytometer using the excitation and emission wavelengths mentioned.

The analysis of the accumulation of neutral lipids following the biomodulator I and I + 11 treatments was performed every 2 days. Table 5 shows the positive effect of the combination of the two bio-modulators I and II on the accumulation of neutral lipids by two strains of Dunaliella tertiolecta. The strains used in the example are D. tertiolecta (SAG 13.86) and D. tertiolecta (MAR 029) isolated from Morocco. It is found that the use of bio-modulators I improved the accumulation of neutral lipids by 5%, 20%, 30%, 50%, 90% and 120% compared to the control, depending on the treatment and the strain. The best response was obtained using a 1 mg / L biomodulator I concentration and the MAR029 strain generally showed a better response to treatment. The combination of both bio-modulators significantly improved lipid accumulation with an increase of between 60%, 70%, 140%, 150% and 170%. Compared to the control. It is clear that osmo-regulators have potentiated the action of the first group of bio-modulators with percentages ranging from 20%, 30%, 50% and more than 60% compared to the use of bio-modulators alone. (Figure 4).

Arbitrary unit of fluorescence: F / F _A

F: fluorescence of the sample after marking

F _A : auto-fluorescence of the sample before marking Table 5

Effect of the combination of bio-modulators I and II on the accumulation of neutral lipids in Dunaliella tertiolecta. Lipids are analyzed by flow cytometry and expressed in

arbitrary fluorescence unit

Body Treatment

D. tertiolecta 0.5 mg / L 1 mg / L 1.5 mg / L

Bio-modulators I

(SAG 13.86) 16.9 20.9 18.8

Bio-modulators l + ll 26.7 26.1 25.6

Control 15.9

D. tertiolecta Bio-modulators 1 31.6 35.5 24.9

(MAR 029) Bio-modulators l + ll 39.1 44.7 40.5

Control 16.3

Example 4. Passive recovery of biomass

After accumulation of lipids, the micoalgal biomass must be recovered when the maximum lipid is accumulated, otherwise these lipids can be consumed by the cell to meet these own energy needs. Different methods can be used to separate the biomass from the culture medium such as centrifugation or filtration. Example 4 offers evidence that the method developed makes it possible to maintain the cells in suspension during the phase of growth and accumulation of lipids (periodic pH changes between 6 and 8). Once the cells loaded with lipids, spot changes in pH between (7 and 11) allowed them to precipitate. The maximum pH to be applied in combination with bio-modulators II is 10.5. Different pH-fluctuation submission times were applied (30 min to 2 h) with total precipitation of the lipid-rich biomass at 2 hours of the last sudden change in pH. Table 6 shows the progressive effect of the last change in pH on the precipitation of lipid-loaded cells, this pH change is applied 4 days after treatment with the osmo-regulators. The positive control is a biomass for which this last change in pH has not been applied. The results obtained were compared with the positive control and with a biomass of the same age and even percentage of lipids (70%) maintained at pH between 6 and 8 (depending on the state of the culture) and centrifuged for 5 min at 5000 rpm.

Table 6

Effect of last modification of pH on the precipitation of microalgal cells treated with bio-modulators I and II. The results are expressed in optical density

Body Treatment

D. tertiolecta 30 min l h 2 h

(SAG 13.86) KOH NaOH KOH NaOH KOH NaOH pH = 9.5 0.311 0.287 0.090 0.086 0.057 0.048 pH = 10 0.251 0.312 0.123 0.097 0.046 0.051 pH = 10.5 0.227 0.230 0.173 0.187 0.010 0.020

Control no 0.521

treaty

biomass

centrifuged

The increase in pH caused rapid precipitation of cells loaded with lipids essentially after 2 hours of maintaining this pH value. Recovery of 90-100% of treated Dunaliella tertiolecta cells was observed without any additional treatment, and at the end the OD was equivalent to that obtained after centrifugation recovery.

Claims

claims

A method for increasing the biofuel production potential from microalgae, characterized in that selected microalgae undergo a growth phase in a culture medium enriched by bio-modulators I and exposed to light followed by a stress phase by increasing the salinity of the medium (bio-modulators II), finally, the phase of passive biomass recovery by occasional changes in the pH values (bio-modulators III) of the medium of culture.

2. Process for increasing the biofuel production potential from microalgae according to claim 1, characterized in that the microalgae used are halophilic species and / or belonging to the classes Chlorophyceae, Cyanophyceae, Baciilariophyceae, Xantophyceae , Chrysophyceae. These microalgae may belong to the genera Dunaliella, Chlorella, Nannochloropsis, Haematococcus, Skeletonemas, Melosira, Thalassiosira, Nitzschia, Navicula.

3. Process for increasing the biofuel production potential from microalgae, according to claims 1 and 2, characterized in that the micro-alga used is Dunaliella tertiolecta.

4. Process for increasing the biofuel production potential from microalgae, according to claims 1 to 3, characterized in that the biomodulator I is added to the culture medium during the active growth phase.

5. Process for increasing the biofuel production potential from microalgae, according to claims 1 to 4, characterized in that the Bio-modulator I is a phytohormone of the type:

An auxin (2,4-Dichlorophenoxyacetic acid 2,4-D, naphthalene acetic acid ANA, indole acetic acid AIA, indole butyric acid AIB), • A cytokine (zeatin, thidiazuron TDZ, benzylaminopurine BAP, isopentenyladenine IPA, kinetin),

• Gibberellin (gibberellic acid GA _S ),

A compound of structure or function similar to an auxin, a compound of structure or function similar to a cytokinin, a growth factor, a growth promoter,

• Molecules involved in cell signaling (salicylic acid SA, jasmonic acid JA, methyl jasmonate MeJa).

6. Process for increasing the biofuel production potential from microalgae, according to claims 1 to 5, characterized in that the concentration of the phytohormones, used alone or in combination, is in the range [0.01 - 2] mg / L.

7. Process for increasing the biofuel production potential from microalgae, according to claim 1, characterized in that in the growth phase vitamins B1, B8, B12 or substances having a similar effect as a nutrient are used. .

8. Process for increasing the biofuel production potential from microalgae according to claim 1, characterized in that the salt stress phase is obtained by introducing at the end of the active or exponential growth phase of a second group of bio-modulators (bio-modulator II) which potentiates the action of the first group and stimulates the rapid accumulation of lipids in the micro-algal cells. This second group of bio-modulators represents mineral salts having an osmo-regulating role such as NaCl, KCl or others.

9. Process for increasing the biofuel production potential from microalgae, according to claims 1 and 8, characterized in that the duration of the salt stress is between the beginning and the end of the stationary phase.

10. Process for increasing the biofuel production potential from microalgae according to claim 9, characterized in that the duration of the salt stress is 4 days.

11. Process for increasing the production potential of biofuel from microalgae, according to claims 9 and 10, characterized in that the concentration of bio-modulator II in the culture medium during the salt stress phase is between 0.5 and 5 M.

12. Process for increasing the biofuel production potential from microalgae according to claim 1, characterized in that the passive recovery of the biomass is done by spot changes in the pH values of the culture medium by the addition of a bio-modulator III of the genus NaOH or KOH.

13. Process for increasing the biofuel production potential from microalgae according to claim 12, characterized in that the addition of the biomodulator III is carried out between 2 to 10 days after the addition of the biomodulator II.

14. Process for increasing the biofuel production potential from microalgae according to claim 12, characterized in that the biomodulator III is kept in contact with the culture medium between 15 minutes and 2 hours before the recovery of the microalgae. the biomass.

15. Process for increasing the biofuel production potential from microalgae according to the preceding claims, characterized in that the said process is used to produce high value-added molecules such as carotenoids and polyunsaturated fatty acids.