METHOD FOR CULTIVATING UNICELLULAR RED ALGAE (URA) ON A MIXTURE OF SUBSTRATES

Abstract

METHOD FOR CULTIVATING UNICELLULAR RED ALGAE (URA) ON A MIXTURE OF SUBSTRATES. The present invention relates to the cultivation of URA for the production of biomass for the production of products of interest, such as dried biomass or compounds or mixtures of compounds of interest extracted from the biomass produced, in particular food pigments or colorings. The invention relates more particularly to the industrial production of this biomass, which must meet an economic equilibrium of profitability, with, on the one hand, an increase in productivity (quantity of biomass and compounds of interest in the biomass) and, on the other hand, an economically acceptable production cost.

Description

Title of the invention: METHOD FOR CULTIVATING UNICORN RED ALGAE (URA) ON A MIXTURE OF SUBSTRATES FIELD OF THE INVENTION

[0001] The present invention relates to the cultivation of ARU for the production of biomass for the production of products of interest, such as dried biomass or compounds or mixtures of compounds of interest extracted from the biomass produced, in particular food pigments or colorings. The invention relates more particularly to the industrial production of this biomass, which must meet an economic balance of profitability, with on the one hand an increase in productivity (quantity of biomass and compounds of interest in the biomass) and on the other an economically acceptable production cost. STATE OF THE ART

[0002] Phycocyanins are pigments produced by single-celled microorganisms that can be used to color food. Today, they are mainly produced by the cultivation of cyanobacteria, in ponds and in autotrophy, with a low yield in biomass produced. To increase the production of these phycocyanins, we have sought species that produce phycocyanins and can be cultivated under fermentation conditions in a bioreactor in order to increase the quantity of biomass produced.

[0003] Unicellular red algae (URA) belonging to the class of cyanidiophyceae such as Galdieria sulphuraria are capable of growing in autotrophic, mixotrophic and heterotrophic conditions. The addition of one or more carbon sources in the culture medium makes it possible to significantly increase the growth rate. These microalgae are capable of consuming a large number of carbon metabolites, in total more than 30 have been counted (glucose, glycerol, pentose, etc.) (Gross et al., 1995; Oesterhelt et al., 1999 - 2007). However, despite faster growth, the cellular content of phycocyanins is much lower in heterotrophic and mixotrophic conditions than in autotrophy. The addition of a carbon source to the culture medium - in the presence or absence of light - strongly represses the production of phycocyanins (Stadnichuk et al., 1998).

[0004] To compensate for this decrease in intrinsic phycocyanin production, culture methods have been developed to increase cell density in fermenters and the amount of dry matter produced. By increasing the dry matter, the amount of phycocyanin produced is also increased (WO 2017 / 050917, WO 2017 / 093345). Depending on the ARUs used, it is also possible to produce phycocyanins with new properties, such as having better stability at acidic pH (WO 2017 / 050918, FR 1752674 filed on March 30, 2017).

[0005] An improvement in these production processes would make it possible to combine the advantages of industrial production by fermentation on carbon substrate, while reducing the repression of the production of phycocyanins associated with the carbon source. Statement of the invention

[0006] The present invention relates to a novel method for cultivating unicellular red algae (URA) for the production of a biomass rich in phycocyanins, comprising the steps (i) of cultivating said URA in mixotrophic or heterotrophic mode on a culture medium comprising a carbon source comprising glucose, and (ii) of recovering the biomass, characterized in that a quantity of glycerol sufficient to increase the production of phycocyanin compared to the culture without glycerol is added to the culture medium.

[0007] The invention also relates to a process for preparing phycocyanins which comprises the production of a biomass according to the invention and a step (iii) of extracting the phycocyanins from the previously recovered biomass. DESCRIPTION OF FIGURES

[0008] Figure 1 represents the growth and phycocyanin production monitoring curves under the culture conditions of Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0009] The present invention relates to a new method for cultivating unicellular red algae (URA) for the production of a biomass rich in phycocyanins, comprising the steps (i) of cultivating said URA in mixotrophic or heterotrophic mode on a culture medium comprising a carbon source comprising glucose, and (ii) of recovering the biomass, characterized in that a quantity of glycerol sufficient to increase the production of phycocyanin compared to the culture without glycerol is added to the culture medium.

[0010] The ARUs used to produce biomass by fermentation and the fermentation methods, in particular for the production of phycocyanins, are well known to those skilled in the art. Mention will be made in particular of patent applications WO 2017 / 050917, WO 2017 / 093345, WO 2017 / 050918 and FR 1752674 filed on March 30, 2017.

[0011] The ARUs are in particular chosen from the subdivision of Cyanidiophytina, in particular from the class of Cyanidiophyceae, more particularly from the order of Cyanidiales, even more particularly chosen from the families of Cyanidiaceae or Galdieriaceae. Preferably, the ARUs are chosen from the genera Cyamdioschyzon, Cyanidium or Galdieria. More preferably, the ARUs used in the method according to the invention are chosen from the species Cyamdioschyzon merolae 10D, Cyamdioschyzon merolae DBV201, Cyanidium caldarium, Cyanidium daedalum, Cyanidium maximum, Cyanidium partitum, Cyanidium rumpens, Galdieria daedala, Galdieria maxima, Galdieria partita or Galdieria sulphuraria.

[0012] The culture media used in the method according to the invention are well known to those skilled in the art, in particular described in patent applications WO 2017 / 050917, WO 2017 / 093345, WO 2017 / 050918 and FR 1752674 filed on March 30, 2017.

[0013] These culture media comprise a carbon source comprising glucose. This may be glucose or glucose in a complex form such as lactose, fructose or polysaccharides comprising glucose.

[0014] This carbon source can come from the sugar industry, from beet or cane, from starch hydrolysates of starchy plants such as corn, wheat, potato, or from the dairy industry such as milk permeate (WO 2017 / 093345), rich in lactose, used alone or in a mixture.

[0015] Advantageously, the carbon source comprising glucose is chosen from glucose and lactose.

[0016] The culture medium generally comprises the carbon source comprising glucose in an amount of between 0.05 g / L and 200 g / L, advantageously between 1 g / L and 150 g / L, very advantageously between 10 g / L and 80 g / L.

[0017] The culture medium may comprise other elements well known to those skilled in the art in the field of microalgae culture by fermentation, in particular a source of phosphorus and / or a source of nitrogen, and / or a source of sulfur.

[0018] According to the invention, the sources of phosphorus can be chosen from the following species: phosphoric acid, phosphorus salts, advantageously sodium hydrogen phosphorus (Na2HPO4), or sodium dihydrogen phosphorus (NaH2PO4), or potassium dihydrogen phosphorus (KH2PO4), or potassium hydrogen phosphorus (K2HPO4), or any mixture, in any proportion of at least two of these sources.

[0019] The medium may also include macroelements and microelements which promote the cultivation of ARU.

[0020] The lighting conditions for the mixotrophic cultivation of ARUs are also known to those skilled in the art, in particular described in patent applications WO 2017 / 050917, WO 2017 / 050918, WO 2017 / 093345 and FR 1752674 filed on March 30, 2017.

[0021] The lighting may be continuous or discontinuous, in particular discontinuous in the form of flashes. According to a particular embodiment, the lighting is carried out with blue light (WO 2017 / 050917), more particularly in the form of radiation having a narrow spectrum of wavelength between 400 and 550 nm, advantageously a narrow spectrum of wavelength between 420 nm and 500 nm, advantageously between 430 and 480 nm, very preferably centered on 455 nm.

[0022] The invention is characterized by the fact that glycerol is added to the culture medium in a quantity sufficient to increase the production of phycocyanins compared to the culture without glycerol.

[0023] This addition can be made to the culture medium at the start of culture, or even to culture media, once the biomass has reached a determined density, for example greater than 20 g / L of dry matter in the culture medium.

[0024] The culture step (i) can therefore be split into two sub-steps, (ia) growth to produce biomass with a carbon source essentially comprising glucose in a simple or complex form as defined previously and (ib) accumulation with the addition of glycerol to promote the production of phycocyanin.

[0025] Industrial glycerol sources that can be integrated into production processes have no restrictions on purity or refining (generally between 80% and 100%). Preferably, they are food grade. Glycerol source suppliers are known, in particular players in the biodiesel industry such as the Avril group with Oléon (Glycérine 4808 / 4808K, Glycérine 4827 / 4827K, etc.), or the Cargill company, which also offers a wide range of products based on refined glycerin between 86.5% and 99.7% with kosher, halal, RSPO, E442 or GMO-free certifications, in particular.

[0026] The quantity of glycerol sufficient to increase the production of phycocyanin compared to the culture without glycerol will be easily determined by a person skilled in the art by a simple experiment in fermenters allowing the quantity of phycocyanin produced with or without glycerol to be compared as shown in the examples.

[0027] The desired increase in phycocyanin production will preferably be at least 0.15 mg / g / h, more preferably at least 0.30 mg / g / h, even more preferably at least 1 mg / g / h; or at least 3.6 mg / g, more preferably at least 7.2 mg / g, even more preferably at least 24 mg / gX.

[0028] In general, the amount of glycerol added to the culture medium is sufficient to have a glycerol / carbon source comprising glucose (hereinafter identified as Gly / Glu) weight ratio of at least 1 / 15. Preferably the Gly / Glu ratio is at least 1 / 14, more preferably at least 1 / 10, even more preferably at least 1 / 5.

[0029] The quantity of glycerol used can go up to Gly / Glu ratios of 2 / 1 or more. However, the interest being to ensure optimum profitability between the cost of raw materials and the production of phycocyanin, it will be advantageous not to exceed a Gly / Glu ratio of 1 / 1, or even preferably 2 / 1.

[0030] According to a preferred embodiment of the invention, the glycerol is added to the culture medium for a Gly / Glu weight ratio of 1 / 10 to 1 / 1, preferably of 1 / 7 to 1 / 2, more preferably of approximately 1 / 5 to approximately 1 / 3.

[0031] The method according to the invention may further comprise a step of recovering the biomass. Said recovery of the biomass may be carried out by any technique allowing the recovery of the biomass, in particular filtration methods, gravimetric or under reduced pressure, decantation, or even precipitation methods followed by gravimetric filtration.

[0032] The invention also relates to the biomass capable of being obtained by any of the variants of the process according to the invention.

[0033] According to the invention, the term "biomass" advantageously means a set of microorganism cells produced by the culture thereof, cells which may or may not have retained their physical integrity. It is therefore understood that said biomass may comprise a quantity of degraded microorganism cells ranging from 0% to 100%. By "degraded" is meant that the physical integrity of said microorganism cells may have been altered, such as lysed microorganisms, resulting for example from a homogenization or enzymatic lysis process. Once produced, this biomass may be raw, just separated from its culture medium, dried or not, degraded or not.

[0034] The biomass, depending on whether it is dried or not, totally or partially, can comprise a humidity level of 1% to 90%.

[0035] According to a first embodiment, the biomass has a humidity level of 70% to 90%, preferably 80% to 85%. This is particularly the case when it is essentially made up of industrial, optimized and cultivated microorganisms, after filtration of the fermentation must to separate the cultivated microorganisms from the culture medium, before drying.

[0036] According to another embodiment of the invention, the biomass is dried, totally or partially, and has a moisture content of 1% to 10%, preferably 2% to 7%. According to the invention, said biomass may have an ARU density of between 20 and 200 g / l of dry matter, preferably between 90 and 150 g / l of dry matter.

[0037] According to the invention, said biomass may have a quantity of proteins of between 25% and 60%, or even up to 70%, preferably between 30% and 55%, more preferably between 40% and 50% of the weight of dry matter. The dosage of nitrogen and The calculation of crude protein content is carried out using the block digestion and steam distillation method (NF EN ISO 5983-2).

[0038] According to the invention, said biomass may have an intracellular content of phycobiliproteins (phycocyanin and allophycocyanin) of between 1 and 250 mg / g of dry matter, preferably between 20 and 150 mg / g of dry matter.

[0039] According to the invention, said biomass may also have an intracellular phycocyanin content of between 0.5 and 100 mg / g of dry matter, preferably between 10 and 40 mg / g of dry matter.

[0040] The biomass may be packaged for storage or for use as such, for example as a food supplement or food for human or animal consumption.

[0041] The cake that can be obtained after extraction of phycocyanin from the ARU biomass that can be obtained by the process according to the invention can be used as a food supplement rich in proteins and carotenoids, in human or animal food.

[0042] According to the invention, said biomass may have an intracellular carotenoid content of between 0.1 and 10 mg / g of dry matter, advantageously between 0.250 and 1 mg / g of dry matter.

[0043] ARUs have significant potential for use in many areas, including, for example, human or animal food, cosmetics and medicine.

[0044] According to the invention, said ARU biomass capable of being obtained according to the invention can be used after harvesting either directly, optionally dried, or after processing. In particular, said biomass can be used in the form of flours used in food compositions or in the form of food supplements.

[0045] The ARU biomass that can be obtained according to the invention can be transformed into flour according to any method known to those skilled in the art. It can thus be envisaged, for example, that the ARUs can be separated from the culture medium, lysed and reduced to fine particles (average diameter of 10 microns), then dried.

[0046] The invention also relates to any use of the ARU biomass that can be obtained according to the invention in any known field of use of ARUs, particularly human or animal food, cosmetics, medicine. In the fields of human or animal food and cosmetics, these are obviously non-therapeutic uses which are intended for healthy animals or human beings.

[0047] The biomass obtained after cultivation of ARUs according to the process of the invention can make it possible to obtain in particular a flour rich in antioxidant agents, in particular in carotenoids (particularly zeaxanthin and B-carotenes) in contents of between 0.1 and 10 mg / g of dry matter, advantageously between 0.25 and 1 mg / g of dry matter, including in particular zeaxanthin in a content of between 0.05 and 5 mg / g of dry matter, advantageously between 0.1 and 1 mg / g of dry matter, and / or B-carotene in a content of 0.05 and 5 mg / g of dry matter, advantageously between 0.1 and 1 mg / g of dry matter, meeting a need particularly in the food industry, because they are more palatable, have a better taste, provide antioxidants in significant quantities and can be used in animal or human food.

[0048] The invention therefore relates to a flour capable of being obtained after transformation of the biomass into ARUs capable of being obtained by the process according to the invention.

[0049] Whatever the form of use of the product capable of being obtained by the process according to the invention (native or transformed biomass), said product can be used pure or mixed with other ingredients conventionally used, particularly in non-therapeutic uses in food or cosmetics.

[0050] The invention also relates to any product which may comprise at least some algae biomass which may be obtained according to the invention. The invention also relates to any product which may comprise at least some flour resulting from the processing of algae biomass which may be obtained according to the invention.

[0051] According to the invention, the phycocyanins produced by said biomass can be extracted for use, for example, in food or as a colorant. The extraction of phycobiliproteins, and particularly of phycocyanin, from said biomass can be carried out according to any extraction technique known to those skilled in the art, such as that described by Moon & al. (2014) or by Jaouen & al. (1999) or in applications FR 2 789 399, WO 2017 / 093345 and FR 1752674 filed on March 30, 2017.

[0052] The invention also relates to the use of phycocyanin capable of being obtained according to the process of the invention, in animal or human food, as a food supplement, or even as a colorant, particularly as a food colorant.

[0053] It also relates to a food comprising the phycocyanin obtained by the process according to the invention, in particular a drink, more particularly a carbonated drink. EXAMPLES

[0054] Example 1: Comparison of growth of the G. sulphuraria strain on glycerol, glucose and a glucose-glycerol mixture.

[0055] The growth of the strain is carried out by measuring the absorbance at 800nm ​​over time. The culture medium is that known for this strain (Gross medium), the only difference between the three tested media is the carbon substrate(s) used.

[0056] M&M: Strain: Galdieria sulphuraria (also called Cyanidium caldarium) UTEX#2919

[0057] Culture medium: 30g / L of glucose or 30g / L of glycerol (glycerin 4808 Univar) or 25g / L of glucose + 5g / L of glycerol; 8g / L of (NH4)2SO4; 0.25g / L of KH2PO4; 716mg / L of MgSO4; 44mg / L CaC12; 3ml / L of stock solution of Fe-EDTA (FeSO4 at 6.9g / L and Na2-EDTA at 9.3g / L) and 4ml of stock solution of trace metal (3.09g / L of Na2-EDTA; 0.08g / L CuSO4.5H2O; 2.86g / L H3BO3; 0.04g / L NaVO3.4H2O; 1.82g / L MnC12; 0.04g / L CoC12.6H2O; 0.22g / L ZnSO4.7H2O; 0.017g / L Na2SeO3;

[0058] Culture conditions: The cultures are carried out on a shaking table (140 rpm) in a thermostatically controlled enclosure at 37°C, in the medium as described above in the presence of a light source.

[0059] Culture monitoring: Growth monitoring shows that similar DOs are achieved regardless of the carbon source(s) used, the dry masses and associated phycocyanin dosages after 312 hours of culture are described in the table. Substrate(s) used Dry mass (g / L) Phycocyanin (mg / gX ) Productivity (mg / L / h) Glucose 18.8 14.17 #0.51 0.853 Glycerol 19.7 42.02 # 0.95 2.65 Glucose + Glycerol 17.47 51.64 #0.04 2.9

[0060] The addition of glycerol to the medium activates the production of phycocyanin despite the presence of glucose known as a phycocyanin inhibitor.

[0061] Example 2: Comparison of growth of the G.sulphuraria strain on milk permeate and a milk-glycerol permeate mixture.

[0062] The growth of the strain is carried out by measuring the absorbance at 800nm ​​over time. The culture medium is that known for this strain (Gross medium), the only difference between the two tested media is the carbon substrate(s) used.

[0063] M&M: Strain: Galdieria sulphuraria (also called Cyanidium caldarium) UTEX#2919

[0064] Culture medium: 30g / L of milk permeate or 25g / L of milk permeate + 5g / L of glycerol (glycerin 4808 Univar); 8g / L of (NH4)2SO4; 0.25g / L of KH2PO4; 716mg / L of MgSO4; 44mg / L CaC12; 3ml / L of stock solution of Fe-EDTA (FeSO4 at 6.9g / L and Na2-EDTA at 9.3g / L) and 4ml of stock solution of trace metal (3.09g / L of Na2-EDTA; 0.08g / L CuSO4.5H2O; 2.86g / L H3BO3; 0.04g / L NaVO3.4H2O; l.82g / L MnC12; 0.04g / L CoC12.6H2O; 0.22g / L ZnSO4.7H2O; 0.017g / L Na2SeO3; 0.03g / L (NH4)6Mo7O24, 4H2O).

[0065] Culture conditions: The cultures are carried out on a shaking table (140 rpm) in a thermostatically controlled enclosure at 37°C, in the medium as described above in the presence or absence of a light source.

[0066] Culture monitoring: The dry masses and associated phycocyanin dosages after 168 hours of culture are described in Table 2(A) in the presence of light and in Table 2(B) in the dark. 2(A) Mixotrophy Substrate(s) used Dry mass (g / L) Phycocyanin (mg / gX) Productivity (mg / L / h) Milk permeate 13.6 #0.31 7.983 #0.36 0.65 Milk permeate + Glyc erol 10.93 #0.16 21.153 #0.88 1.38 2(B) Heterotrophy Substrate(s) used Dry mass (g / L) Phycocyanin (mg / gX) Productivity (mg / L / h) Milk permeate 12.03 # 0.07 3.54 #0.11 0.25 Milk permeate + Glyc erol 8.81 #0.37 6.11 #0.15 0.32

[0067] The addition of glycerol to the medium activates the production of phycocyanin despite the presence of the phycocyanin-inhibiting milk permeate and this increase in production is noticeable even in the dark.

[0068] Example 3: Monitoring of a bioreactor culture process of the G.sulphuraria strain on a glucose-glycerol mixture.

[0069] The growth monitoring of the strain is carried out by measuring the dry mass over time and the substrate consumption is identified by HPLC assay. An intracellular phycocyanin assay is also carried out over time. The culture medium is that known for this strain (Gross medium) with 1 / 6 glycerol and 5 / 6 glucose as substrate for the starter culture and a feed composed of glycerol and ¾ glucose for the fed-batch.

[0070] M&M: Strain: Galdieria sulphuraria (also called Cyanidium caldarium) UTEX#2919

[0071] Culture medium:

[0072] Kettle foot: 25g / L glucose + 5g / L glycerol (glycerin 4808 Univar); 8g / L of (NH4)2SO4 ; 0.25g / L of KH2PO4 ; 716mg / L of MgSO4 ; 44mg / L CaC12 ; 3ml / L of Fe-EDTA stock solution (FeSO4 at 6.9g / L and Na2-EDTA at 9.3g / L) and 4ml of trace metal stock solution (3.09g / L of Na2-EDTA; 0.08g / L CuSO4.5H2O; 2.86g / L; NaVO3.4H2O ; l.82g / L CoC12.6H2O ; 0.22g / L ZnSO4.7H2O ; O.017g / L Na2SeO3 0.03.6Mo7O2O).

[0073] Fed-Batch Medium : 375g / L glucose + 125g / L glycerol (Glycerin 4808 Univar); 20g / L of (NH4)2SO4 ; 4.17g / L of KH2PO4 ; 5.96 g / L MgSO4 ; 0.37 g / L CaC12 ; 25ml / L of Fe-EDTA stock solution (FeSO4 at 6.9g / L and Na2-EDTA at 9.3g / L) and 33ml of trace metal stock solution (3.09g / L of Na2-EDTA; 0.08g / L CuSO4.5H2O; 2.86g / L; NaVO3.4H2O ; l.82g / L CoC12.6H2O ; 0.22g / L ZnSO4.7H2O ; O.017g / L Na2SeO3 0.03.6Mo7O2O).

[0074] Culture conditions: The cultures are carried out in a 2L bioreactor at a temperature regulated at 37°C, pH 3, in the presence of a light source.

[0075] Growth monitoring shows that a dry mass of 70g / l is reached after 427h of culture and an intracellular phycocyanin content of 45mg / gX (Figure 1). All of the carbon substrate provided is consumed.

[0076] Example 4: Monitoring of a bioreactor culture process of the G.sulphuraria strain on a milk-glycerol permeate mixture.

[0077] The growth monitoring of the strain is carried out by measuring the dry mass over time and the substrate consumption is identified by HPLC assay. An intracellular phycocyanin assay is also carried out over time. The culture medium is that known for this strain (Gross medium) with 1 / 6 glycerol and 5 / 6 lactose (provided by the milk permeate) as substrate for the starter culture and a feed composed of glycerol and ¾ lactose for the fed-batch.

[0078] M&M: Strain: Galdieria sulphuraria (also called Cyanidium caldarium) UTEX#2919

[0079] Culture medium:

[0080] Base: 25g / L milk permeate + 5g / L glycerol (glycerin 4808 Univar); 8g / L (NH4)2SO4; 716mg / L MgSO4; 3ml / L of stock solution of Fe-EDTA (FeSO4 at 6.9g / L and Na2-EDTA at 9.3g / L) and 4ml of stock solution of trace metal (3.09g / L of Na2-EDTA; 0.08g / L CuSO4.5H2O; 2.86g / L H3BO3; 0.04g / L NaVO3.4H2O; 1.82g / L MnC12; 0.04g / L CoC12.6H2O; 0.22g / L ZnSO4.7H2O; 0.017g / L Na2SeO3;

[0081]

[0082]

[0083]

[0084]

[0085] Fed-Batch medium: 85g / L milk permeate; 28.3g / L glycerol Culture conditions: Cultures are carried out in a 2L bioreactor at a temperature regulated at 37°C, pH 3, in the presence of a light source. Growth monitoring shows that a dry mass of 25g / l is reached after 450 hours of culture and an intracellular phycocyanin content of 14mg / g. Example 5: Variation of PC production as a function of the Gly / Glc proportion in the culture medium. A cross-range of glucose and glycerol was carried out to assess the effect of glycerol on the lifting of inhibition of phycocyanin synthesis linked to the presence of glucose in the culture medium. The total amount of organic carbon in each Erlenmeyer flask is 30 g / l. Glucose is symbolized by the contraction "glc" and glycerol by "gly". The concentration of each element in the medium is indicated in Table 3. PC production according to substrates (mg / gX / h)

[0086] substrate PC productivity (mg / gX / h) MS (gX / L) PC concentration (mg / gX) 30g / l Gly 3.45 19.72 42.02 25 Gly / 5 Glc 5.60 19.97 67.29 20 Gly / 10 Glc 4.80 17.82 64.58 15 Gly / 15 Glc 3.98 16.60 57.53 10 Gly / 20 Glc 4.19 16.42 61.3 5 Gly / 25 Glc 3.76 17.47 51.64 30g / L Glc 1.10 18.80 14.17

[0087]

[0088]

[0089] Example 6: Variation of PC production as a function of the Gly / Glc proportion in the culture medium (Glycerol concentration less than 5gd). This example complements example 5, the culture conditions are similar, the only difference is in the glycerol concentrations tested which are less than or equal to 5g / l in order to quantify the limit glycerol concentration allowing the inhibition of phycocyanin synthesis by glucose in the culture medium to be lifted. Productivity (mg / g / h) substrate PC productivity (mg / gX / h) MS (gX / L) PC concentration (mg / gX) 30g Glc 0.68 11.45 14.31 Ig / L Gly / 29 Glc 0.66 11.41 14 2g / L Gly / 28 Glc 0.84 11.59 17.47 3g / L Gly / 27 Glc 0.86 11.7 17.56 4g / L Gly / 26 Glc 1.03 11.65 21.3 5g / L Gly / 25 Glc 1.01 11.62 20.77 30g / L Gly 1.29 12.84 24.19

[0090]

[0091]

[0092]

[0093] Example 7: Variation in PC production as a function of the proportion of Glycerol added to a medium containing 30g / l of milk permeate at the start of culture. Different amounts of glycerol are added to a medium composed of milk permeate in order to observe its effect on the synthesis of phycocyanin and the lifting of the inhibition of lactose (glucose + sucrose) on the production of this pigment. The basic medium contains 30g / l of milk permeate. The added glycerol is symbolized by the contraction "gly". 6 glycerol concentrations are tested (0, 1, 2, 3, 4 and 5g / l). Productivity (mg / L / h) glycerol Productivity in PC (mg / g X / h) MS (gX / 1) Concentration in PC (mg / gX) 0g / l 0.13 19.4 1.09 lg / 1 0.53 18.7 4.74 2g / l 1.05 21 8.42 3g / l 1.45 21.7 11.26 4g / l 1.90 19.7 16.22 5g / l 2.31 17.4 22.33 Similarly, as described by Stadnichuk et al., (1998), glucose has a limiting effect on the amount of phycocyanin produced by the Galdieria sulphuraria strain, with a concentration of less than 15 mg of phycocyanin per g of dry matter at the end of growth (15 mg PC / g of DM). In contrast, the strain grown in the presence of glycerol only accumulates approximately 40 mg PC / g of DM, for the same culture time (240 h). By extending, the culture grown in glycerol can have accumulated up to 70 mg of PC / g of DM, while the one grown in glucose will have a quantity of PC / g of DM which will remain more or less constant over time. In all other cases (glucose-glycerol mixture) the biomass produced as well as the quantity of PC present in the biomass remains constant and even higher than that of glycerol for the same culture time.These values ​​are between 50 and 60 mg PC / g DM, more than three times higher than what is found in the biomass of cells grown on glucose alone.

[0094] In conclusion, the addition of small quantities of glycerol to the medium makes it possible to lift the inhibition of PC synthesis linked to the presence of glucose in the medium, whether it is present in the form of simple sugar (glucose), or complex (lactose, sucrose, or other ...).

[0095] This method allows cells to be grown using glucose (or a glucose-containing carbon source) as the main carbon source, and glycerol to be used only as a PC synthesis inducer and not as the main substrate. Glucose or glucose-containing carbon sources are generally less expensive than glycerol, and generally allow higher growth rates to be obtained than on glycerol, thus saving on fermentation costs. REFERENCES

[0096] • Gross et al., 1995; • Oesterhelt et al., 1999 - 2007 • Stadnichuk et al., 1998 • Moon & al. (2014) • Jaouen & al. (1999) • FR 2 789 399 • FR 1752674 filed on March 30, 2017 • WO 2017 / 050917, • WO 2017 / 050918, • WO 2017 / 093345

Claims

Claims

1. Method for culturing unicellular red algae (URA) for the production of a biomass rich in phycocyanins, comprising the steps (i) of culturing said URA in mixotrophic or heterotrophic mode on a culture medium comprising a carbon source comprising glucose, and (ii) of recovering the biomass, characterized in that an amount of glycerol is added to the culture medium to increase the production of phycocyanin compared to the culture without glycerol and in that the weight ratio of glycerol / carbon source comprising glucose (Gly / Glu) is at least 1 / 15.

2. Method according to claim 1, characterized in that the ARUs are the genera Cyamdioschyzon, Cyanidium or Galdieria, in particular among the species Cyamdioschyzon merolae 10D, Cyamdioschyzon merolae DBV201, Cyanidium caldarium, Cyanidium daedalum, Cyanidium maximum, Cyanidium partitum, Cyanidium rumpens, Galdieria daedala, Galdieria maxima, Galdieria partita or Galdieria sulphuraria.

3. Method according to one of claims 1 or 2, characterized in that the carbon source comprising glucose is chosen from glucose and glucose in a complex form such as lactose, fructose or polysaccharides comprising glucose, and mixtures thereof.

4. A method according to claim 3, characterized in that the carbon source is milk permeate comprising lactose.

5. Method according to one of claims 1 to 4, characterized in that the culture medium comprises between 0.05 g / L and 200 g / L of carbon source comprising glucose.

6. Method according to one of claims 1 to 5, characterized in that the culture step is in mixotrophic mode with continuous or discontinuous lighting.

7. Method according to one of claims 1 to 6, characterized in that the addition of glycerol to the culture medium is carried out at the start of culture or in culture media once the biomass has reached a determined density.

8. Method according to one of claims 1 to 7, characterized in that the glycerol / carbon source weight ratio comprising glucose (Gly / Glu) is at least 1 / 14, more preferably at least 1 / 10, even more preferably at least 1 / 5.

9. Method according to claim 8, characterized in that the ratio Gly / Glu weight ratio ranges from 1 / 8 to 1 / 1, preferably from 1 / 7 to 1 / 2, more preferably from about 1 / 5 to about 1 / 3.

10. Process for the preparation of phycocyanins characterized in that that it comprises the production of a biomass according to one of claims 1 to 9 and a step (iii) of extracting phycocyanins from the previously recovered biomass.