NOVEL METHOD FOR CULTURE OF MICROALGAE

Abstract

Disclosed is a method for culture of microalgae, advantageously of the genus Crypthecodinium having a gene coding for the small 18s ribosomal RNA subunit having a genetic similarity of at least 96% with the same gene of the strain of the species Crypthecodinium cohnii deposited in the CCAP (Culture Collection of Algae and Protozoa) under number CCAP 1104/3 in a defined chemical medium.

Claims

1-19. (canceled)

20. Method for producing a biomass of microalgae comprising the steps of a) culturing the microalgae in a culture medium and b) harvesting the biomass from the culture medium, wherein step a) of culturing the microalgae comprises at least a first step a1) wherein said microalgae are cultivated in a defined medium, comprising macroelements comprising a mixture of sodium chloride (NaCl), magnesium chloride, sodium bicarbonate (N.sub.aHCO.sub.3), potassium hydrogen phosphate (K.sub.2HPO.sub.4), calcium chloride, iron (III) chloride (FeCl.sub.3 6H.sub.2O), boric acid (H.sub.3BO.sub.3) and a nitrogen source; microelements comprising a mixture of disodium ethylenediaminetetraacetic acid (Na.sub.2EDTA), manganese (II) chloride tetrahydrate (MnCl.sub.2 4H.sub.2O), zinc chloride (ZnCl.sub.2), cobalt chloride hexahydrate (CoCl.sub.2 6H.sub.2O), copper sulfate pentahydrate (CuSO.sub.4 5H.sub.2O), and nickel sulfate hexahydrate (NiSO.sub.4 6H.sub.2O); vitamins comprising a mixture of biotin, thiamin, cobalamin or vitamin B12 and pantothenate or vitamin B5; and a carbon source; for a sufficient time to reach a strain concentration between 5×10.sup.6 and 5×10.sup.8 cells/mL, advantageously between 10.sup.7 and 10.sup.8 cells/mL, and at least a second step a2) wherein one or more carbon source enrichment solution(s) are added, simultaneously or successively, to the culture medium and wherein the microalgae is a microalgae strain having a gene encoding the small subunit 18s ribosomal RNA having a genetic similarity of at least 96% with the same gene of the strain of the species Crypthecodinium cohnii deposited in the CCAP (Culture Collection of Algae and Protozoa) under number CCAP 1104/3.

21. Method according to claim 20, wherein in the defined medium of step a1), sodium chloride is at a concentration between 2 and 8 g/L, magnesium chloride is at a concentration between 0.6 and 1.4 g/L, sodium bicarbonate is at a concentration between 0.14 and 0.26 g/L, the nitrogen source is at a concentration between 1.7 and 3.1 g/L, potassium hydrogen phosphate is at a concentration between 0.3 and 0.7 g/L, calcium chloride is at a concentration between 1.4 and 2.6 g/L, iron (III) chloride is at a concentration between 0.016 and 0.024 g/L, boric acid is at a concentration between 0.046 and 0.74 g/L, disodium ethylenediaminetetraacetic acid is at a concentration between 0 and 0.08 g/L, manganese (II) chloride tetrahydrate is at a concentration between 0.001 and 0.02 g/L, zinc chloride is at a concentration between 0.0001 and 0.0008 g/L, cobalt chloride hexahydrate is at a concentration between 0.00005 and 0.0005 g/L, copper sulfate pentahydrate is at a concentration between 0.00007 and 0.004 g/L, nickel sulfate hexahydrate is at a concentration between 0.0005 and 0.003 g/L, biotin is at a concentration between 0.5×16.sup.−6 and 1.5×10.sup.−4 g/L, thiamin is at a concentration between 0.5×16.sup.−6 and 1.5×10.sup.−4 g/L, cobalamin is at a concentration between 0.5×16.sup.−6 and 1.5×10.sup.−4 g/L and pantothenate is at a concentration between 0.5×16.sup.−6 and 1.5×10.sup.−4 g/L.

22. Method according to claim 20, wherein in the defined medium of step a1), sodium chloride is at a concentration between 4 and 6 g/L, magnesium chloride is at a concentration between 0.8 and 1.2 g/L, sodium bicarbonate is at a concentration between 0.16 and 0.24 g/L, the nitrogen source is at a concentration between 1.9 and 2.9 g/L, potassium hydrogen phosphate is at a concentration between 0.4 and 0.6 g/L, calcium chloride is at a concentration between 1.6 and 2.4 g/L, iron (III) chloride is at a concentration between 0.018 and 0.022 g/L, boric acid is at a concentration between 0.048 and 0.72 g/L, disodium ethylenediaminetetraacetic acid is at a concentration between 0.015 and 0.06 g/L, manganese (II) chloride tetrahydrate is at a concentration between 0.002 and 0.01 g/L, zinc chloride is at a concentration between 0.00015 and 0.0006 g/L, cobalt chloride hexahydrate is at a concentration between 0.00007 and 0.0003 g/L, copper sulfate pentahydrate is at a concentration between 0.0009 and 0.0036 g/L, nickel sulfate hexahydrate is at a concentration between 0.0007 and 0.0028 g/L, biotin is at a concentration between 1×10.sup.−6 and 1×10.sup.−4 g/L, thiamin is at a concentration between 1×10.sup.−6 and 1×10.sup.−4 g/L, cobalamin is at a concentration between 1×10.sup.−6 and 1×10.sup.−4 g/L and pantothenate is at a concentration between 1×10.sup.−6 and 1×10.sup.−4 g/L.

23. Method according to claim 20, wherein the nitrogen source is selected among the group consisting in a nitrate salt, a glutamate salt, an ammonium salt and urea.

24. Method according to claim 20, wherein the carbon source is selected among the group consisting in one (or more) carbohydrate(s), one (or more) acetate(s), one or more alcohols, one or more complex molecules and mixtures thereof.

25. Method according to claim 20, wherein in the first step a1) the carbon source is at a concentration between 10 mM and 500 mM.

26. Method according to claim 20, wherein culture is in heterotrophic or mixotrophic conditions.

27. Method according to claim 20, wherein step a) of culturing the microalgae comprises a third step a3) wherein one or more nitrogen source and/or phosphorus source and/or macroelement source, and/or microelement source, and/or vitamin source, advantageously nitrogen and phosphorus source, enrichment solution(s) are added, simultaneously or successively, to the culture medium.

28. Method according to claim 20, wherein in the second step a2) the enrichment solution is a solution comprising at the same time the carbon source, the nitrogen source and the phosphorus source (a solution also called “C/N/P enrichment solution”).

29. Method according to claim 28, wherein the C/N/P enrichment solution has for a fixed quantity of carbon up to two times more or two times less nitrogen, and/or up to two times more or two times less phosphorus in relation to the ratio 891:26:1.

30. Method according to claim 29, wherein the C/N/P enrichment solution has the molar ratios selected from the group consisting in: 891:52:1, 891:26:1 and 891:13:1, 445:26:1, 445:13:1 and 445:6.5:1, 1782:104:1, 1782:52:1, and 1782:26:1.

31. Method according to claim 20, wherein in the defined medium of step a1), sodium chloride is at a concentration of 5 g/L, magnesium chloride is at a concentration of 1 g/L, sodium bicarbonate is at a concentration of 0.19 g/L, the nitrogen source (KNO.sub.3) is at a concentration of 2.4 g/L, potassium hydrogen phosphate is at a concentration of 0.5 g/L, calcium chloride is at a concentration of 1.9 g/L, iron (III) chloride is at a concentration of 0.02 g/L, boric acid is at a concentration of 0.06 g/L, disodium ethylenediaminetetraacetic acid is at a concentration of 0.03 g/L, manganese (II) chloride tetrahydrate is at a concentration of 0.0045 g/L, zinc chloride is at a concentration of 0.0003 g/L, cobalt chloride hexahydrate is at a concentration of 0.00015 g/L, copper sulfate pentahydrate is at a concentration of 0.0018 g/L, nickel sulfate hexahydrate is at a concentration of 0.0014 g/L, biotin is at a concentration of 0.000015 g/L, thiamin is at a concentration of 0.008 g/L, cobalamin is at a concentration of 0.00013 g/L, pantothenate is at a concentration of 0.0027 g/L and the carbon source is at a concentration of 30.00 g/L.

32. Method according to claim 20, wherein it further comprises downstream steps of, of lipid extraction form the harvested biomass, and optionally of DHEA extraction from the lipids recovered.

33. Method according to claim 32, wherein it further comprises a step of purifying the DHEA produced in the form of DHEA esters.

34. Method according to claim 20, wherein the biomass is cultured at a density at least greater than 50 g/L dry matter.

35. Method according to claim 20, wherein the biomass is cultured at a density at least greater than 100 g/L dry matter.

36. Method according to claim 20, wherein the biomass is cultured at a density at least greater than 150 g/L dry matter.

37. Method according to claim 20, wherein the biomass is cultured at a density at least greater than 200 g/L dry matter.

38. Method according to claim 20, wherein the biomass is cultured at a density at least greater than 250 g/L dry matter.

39. Method according to claim 20, wherein the biomass is cultured at a density at least greater than 270 g/L dry matter.

40. Method according to claim 20, wherein the microalgae are cultured for an enrichment in their docosahexaenoic acid (DHEA) content.

41. Method according to claim 26, wherein the cultured microalgae have a DHEA level at least greater than 20 g/L.

42. Method according to claim 26, wherein the cultured microalgae have a DHEA level at least greater than 30 g/L.

43. Method according to claim 27, wherein the cultured microalgae have a level of PUFAs other than DHEA lower than 1% of the total fatty acid content.

44. Method according to claim 27, wherein the cultured microalgae have a level of PUFAs other than DHEA lower than 0.8% of the total fatty acid content.

Description

[0137] Other advantages and features of the invention will become clear upon reading the examples described below as well as the appended drawings wherein:

[0138] FIG. 1 presents the results obtained during culture of a strain of the species Crypthecodinium cohnii (CCAP strain 1104/3) according to the method of the invention in chemically defined medium according to the invention with the addition of a carbon source enrichment solution, compared to a culture in rich medium (Example 1). FIG. 1A presents the evolution of the quantity of dry matter produced during the test. FIG. 1B presents the lipid profile of the oil contained in CCAP strain 1104/3 at the end of culture.

[0139] FIG. 2 presents the results obtained during culture of a strain of the species Crypthecodinium cohnii (CCAP strain 1104/3) according to the method of the invention in chemically defined medium according to the invention with the addition of a carbon source enrichment solution and a nitrogen and phosphorus source enrichment solution compared to culture in rich medium (Example 2). FIG. 2A presents the evolution of the quantity of dry matter produced during the test. FIG. 2B presents the lipid profile of the oil contained in CCAP strain 1104/3 at the end of culture.

[0140] FIG. 3 presents the results obtained during culture of a strain of the species Crypthecodinium cohnii (CCAP strain 1104/3) according to the method of the invention in chemically defined medium according to the invention with the addition of a carbon source enrichment solution and a macroelement, in particular containing nitrogen and phosphorus, microelement and vitamin enrichment solution, which makes it possible to supply the equivalent of twice the quantity of elements present in the defined medium used initially for a total mineral and vitamin supply corresponding to three times the initial quantity (“3×” total) after the additions, compared to culture in rich medium (Example 3). FIG. 3A presents the evolution of the quantity of dry matter produced during the test. FIG. 3B presents the lipid profile of the oil contained in CCAP strain 1104/3 at the end of culture.

[0141] FIG. 4 presents the results obtained during culture of a strain of the species Crypthecodinium cohnii (CCAP strain 1104/3) according to the method of the invention in chemically defined medium according to the invention with the addition of a C/N/P-type enrichment solution, compared to culture in rich medium (Example 4). FIG. 4A presents the evolution of the quantity of dry matter produced during the test. FIG. 4B presents the lipid profile of the oil contained in CCAP strain 1104/3 at the end of culture.

[0142] FIG. 5 presents the results obtained during culture of a strain of the species Crypthecodinium cohnii (CCAP strain 1104/3) according to the method of the invention in defined medium according to the invention with the addition of a macroelement, in particular containing nitrogen and phosphorus, microelement and vitamin enrichment solution and the addition of a C/N/P-type enrichment solution (Example 5). FIG. 5A presents the evolution of the quantity of dry matter produced during the test. FIG. 5B presents the lipid profile of the oil contained in CCAP strain 1104/3 at the end of culture.

EXAMPLES

Example 1: Culture of a Strain of the Species Crypthecodinium cohnii (CCAP Strain 1104/3) According to the Method of the Invention in Chemically Defined Medium According to the Invention with the Addition of a Carbon Source Enrichment Solution

[0143] Crypthecodinium cohnii (CCAP strain 1104/3) cultures were prepared in 1 to 5 L fermentors (bioreactors) used with dedicated computer-controlled automated systems. The system was adjusted in pH by adding acid (H.sub.2SO.sub.4). The culture temperature was set to 28° C. Agitation was provided by 2 agitator impellers, Rushton and HTPG4. The dissolved oxygen pressure was regulated in the medium throughout the culture, by agitation speed (200-800 rpm), air flow rate (0.6-1.6 vvm) and oxygen flow rate (0-1.6 vvm). The regulation parameters, integrated in the automated control system, made it possible to maintain a pO.sub.2 between 25% and 35%.

[0144] The total biomass concentration was monitored by measuring the dry mass (filtration on a Whatman GF/F filter then drying in a 105° C. oven for a minimum of 24 h before weighing).

[0145] The analyses of total lipids and FAME contents were carried out according to the methods classically described in the literature [Folch J. et al., A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem. 1957 May; 226(1):497-509].

[0146] The culture is prepared in the defined medium according to the invention, the composition of which is given in Table 2 below.

TABLE-US-00002 TABLE 2 Ingredient Concentration in g/L Macroelements NaCl 5 MgCl.sub.2 6H.sub.2O 1 N.sub.aHCO.sub.3 0.19 K.sub.2HPO.sub.4 0.5 CaCl.sub.2 2H.sub.2O 1.9 FeCl.sub.3 6H.sub.2O 0.02 H.sub.3BO.sub.3 0.06 KNO.sub.3 2.4 Microelements Na.sub.2EDTA 0.03 MnCl.sub.2 4H.sub.2O 0.0045 ZnCl.sub.2 0.0003 CoCl.sub.2 6H.sub.2O 0.00015 CuSO.sub.4 5H.sub.2O 0.0018 NiSO.sub.4 6H.sub.2O 0.0014 Vitamins Biotin 0.000015 Thiamin 0.008 Cobalamin 0.00013 Pantothenate 0.0027 Carbon source Glycerol 30

[0147] This chemically defined medium establishes a “1×” reference concentration for each of its components.

[0148] An organic carbon source enrichment solution, consisting of 712.3 g/L glycerol, makes it possible to feed the medium when the glycerol concentration measured in the fermentor drops below the 10 g/L threshold. This makes it possible to maintain the glycerol concentration between 10 g/L and 60 g/L, more preferentially between 20 g/L and 50 g/L.

[0149] A control culture is prepared in rich medium composed of 8.7 g/L yeast extract, 0.75 g/L KNO.sub.3, 5 g/L sea salts and 30 g/L glycerol. A first enrichment solution for the control culture is composed of concentrated KNO.sub.3 and yeast extract. These additions make it possible to increase the initially provided 1× quantity of KNO.sub.3 and yeast extract to a 3× quantity. An organic carbon source enrichment solution, consisting of 712.3 g/L glycerol, makes it possible to feed the medium when the glycerol concentration measured in the fermentor drops below the 10 g/L threshold. This makes it possible to maintain the glycerol concentration between 10 g/L and 60 g/L, more preferentially between 20 g/L and 50 g/L.

[0150] FIG. 1 presents the results obtained during this test.

[0151] FIG. 1A presents the evolution of the quantity of dry matter produced during the test as well as the quantity of fats produced based on the quantity of dry matter produced (% Fats/DM) as a function of time in the medium according to the invention (1× inorganic medium) compared to culture in rich medium.

[0152] In terms of biomass, 67.4 g/L is obtained with the medium according to the invention (compared to 174.3 g/L with rich medium) after 236 h of culture, with about 15.9% fat content (compared to 13.7% with rich medium).

[0153] FIG. 1B presents the lipid profile of the oil contained in CCAP strain 1104/3 at the end of culture. It is noted in particular that PUFAs other than DHEA are at trace levels (<1%, preferentially <0.8%).

[0154] Moreover, the DHEA content in the fats rises to 30.4%, compared to 39.7% in the rich medium.

[0155] Thus, the growth of the strain on the medium of the invention makes it possible to reach high cell densities, greater than 50 g/L biomass, with the addition of a carbon source enrichment solution, but does not make it possible to equal the performance obtained on the control rich medium.

Example 2: Culture of a Strain of the Species Crypthecodinium cohnii (CCAP Strain 1104/3) According to the Method of the Invention in Defined Medium According to the Invention with the Addition of a Carbon Source Enrichment Solution and a Nitrogen and Phosphorus Source Enrichment Solution

[0156] The results of this example are illustrated in FIG. 2.

[0157] Crypthecodinium (CCAP strain 1104/3) cultures were prepared in fermentors, in a manner similar to that described in Example 1, but with the additional supply of a mineral enrichment solution consisting of KNO.sub.3 at a concentration of 64.0 g/L and K.sub.2HPO.sub.4 at a concentration of 4.3 g/L, which makes it possible to increase the initial quantities of nitrogen and phosphorus and makes it possible to maintain productivity and thus to continue growth. The additions of this mineral enrichment solution are carried out once the culture reaches a cell density of 3×10.sup.7 cells/mL, or after 92 h of culture.

[0158] A control culture is prepared in complex medium, as described in Example 1.

[0159] Under the conditions of this example, the cell density and the lipid content of the cells are close to those obtained in complex medium, after 330 h of culture (FIG. 2A).

[0160] In terms of biomass, 168.6 g/L is obtained with the medium according to the invention (compared to 210 g/L with rich medium), with about 10% fats.

[0161] FIG. 2B presents the lipid profile of the oil contained in CCAP strain 1104/3 at the end of culture. It is noted in particular that PUFAs other than DHEA are at trace levels (<1%, preferentially <0.8%). Moreover, the quantity of DHEA is equivalent to the profile obtained in complex medium, with 40.4% compared to 39.7% DHEA in rich medium.

[0162] Thus, the growth of the strain on the medium of the invention, with the addition of a carbon source enrichment solution coupled with the addition of a nitrogen and phosphorus source enrichment solution, makes it possible to reach high cell densities close to those obtained on the control rich medium, and makes it possible to obtain a DHEA content in the fats identical to that of the control rich medium.

Example 3: Culture of a Strain of the Species Crypthecodinium cohnii (CCAP Strain 1104/3) According to the Method of the Invention in Defined Medium According to the Invention with the Addition of a Carbon Source Enrichment Solution and a Macroelement, in Particular Containing Nitrogen and Phosphorus, Microelement and Vitamin Enrichment Solution

[0163] The results of this example are illustrated in FIG. 3.

[0164] Crypthecodinium (CCAP strain 1104/3) cultures were prepared in fermentors, in a manner similar to that described in Example 2, but with a modification of the mineral enrichment solution.

[0165] In this example, the mineral enrichment solution is composed of all the minerals and vitamins present in the initial culture medium. The addition makes it possible to supply the equivalent of twice the quantity of minerals and vitamins present in the initial culture medium, for a total mineral and vitamin supply corresponding to three times the initial quantity (“3×” total) after the additions.

[0166] A control culture is prepared in complex medium, as described in Example 1.

[0167] The progressive supply of minerals and vitamins makes it possible to reach up to 274.6 g/L dry matter consisting of 18% fats with a DHEA content of 32.6% (see FIGS. 3A and B).

[0168] Thus, the conditions of Example 3 make it possible to exceed the performance obtained on the rich medium and the inorganic medium of Example 2.

[0169] FIG. 3B shows the lipid profile of the oil contained in CCAP strain 1104/3 at the end of culture. It is noted in particular that PUFAs other than DHEA are at trace levels (<1%, preferentially <0.8%).

[0170] Moreover, the DHEA content in the fats rises to 32.8% compared to 39.7% in the rich medium.

[0171] Thus, the growth of the strain on the medium of the invention, with the addition of a carbon source enrichment solution coupled with the addition of a macroelement, in particular containing nitrogen and phosphorus, microelement and vitamin enrichment solution, makes it possible to reach high cell densities greater than those obtained on the control rich medium, and makes it possible to obtain a DHEA content in the fats close to that of the control rich medium.

Example 4: Culture of a Strain of the Species Crypthecodinium cohnii (CCAP Strain 1104/3) According to the Method of the Invention in Defined Medium According to the Invention with the Addition of a C/N/P-Type Enrichment Solution

[0172] The results of this example are illustrated in FIG. 4.

[0173] Crypthecodinium (CCAP strain 1104/3) cultures were prepared in fermentors, in a manner similar to that described in Example 2.

[0174] The procedure was modified in terms of the nature of the enrichment solutions. In this example, the additional substrate is added in the form of additions of a solution called “C/N/P nutrient solution”. The nutrient solution consists of a carbon substrate (glycerol), an inorganic nitrogen source (KNO.sub.3) and an inorganic phosphorus source (K.sub.2HPO.sub.4). This nutrient solution has a defined molar ratio of carbon, nitrogen and phosphorus atoms: 891:26:1. This ratio was adjusted by the inventors to create a defined limitation of nitrogen and phosphorus, and thus to preferentially direct the metabolism toward DHEA production.

[0175] A control culture is prepared in rich medium as described in Example 1.

[0176] The addition of a C/N/P solution in a molar ratio of 891:26:1 makes it possible to reach 251.2 g/L dry matter consisting of 8% fats with a 59.3% DHEA content (FIGS. 4A and B).

[0177] Thus, according to this embodiment of the invention, the supply of carbon, nitrogen and phosphorus sources during culture in a chemically defined medium makes it possible to increase the DHEA content in the fats up to a value of 59.3%. This performance exceeds all those described in the prior art, whether in inorganic medium or in rich medium. This performance also makes it possible to improve the methods described in Examples 1, 2 and 3.

[0178] FIG. 4B shows the lipid profile of the oil contained in CCAP strain 1104/3 at the end of culture. It is noted in particular that PUFAs other than DHEA are at trace levels (<1%, preferentially <0.8%).

[0179] Moreover, the DHEA content in the fats rises to 59.3%, compared to 39.7% DHEA in rich medium.

[0180] Thus, compared to Examples 1, 2 and 3, the controlled supply of carbon, nitrogen and phosphorus source (C/N/P-type enrichment solution in a molar ratio of 891:26:1) makes it possible to significantly enrich the oil in DHEA, while maintaining high densities and productivities.

[0181] Conclusions Upon Reading the Results of the Examples

[0182] Microalgae strains concerned by the invention cultivated according to the invention make it possible to produce significant quantities of biomass, containing lipids that are rich in DHEA and low in DPA.

[0183] Indeed, the method of the invention in heterotrophic or mixotrophic conditions makes it possible to obtain a biomass concentration at least equal to if not greater than 50 g/L matter dry and may reach up to more than 250 g/L dry matter. This dry matter can consist of more than 10% fats, this content potentially going up to 60% lipids (FIG. 3A) with a DHEA content of up to more than 59% (FIG. 4B).

[0184] Generally, the quantity of DHEA obtained with the method of the invention is at least as great as that obtained using rich culture media as described in the prior art. For example, the results of Example 2 show that 40.4% DHEA is obtained with culture in defined medium according to the invention (Table 1) in a bioreactor and that 39.7% DHEA is obtained for the control culture (complex medium).

[0185] Other unwanted PUFAs, particularly DPA, generally represent less than 3%, and up to less than 0.01% of the fatty acids.

[0186] Generally, according to the method of the invention, PUFAs other than DHEA are at trace levels (<1%, preferentially <0.8%).

Example 5: Culture of a Strain of the Species Crypthecodinium cohnii (CCAP Strain 1104/3) According to the Method of the Invention in Defined Medium According to the Invention with the Addition of a Macroelement, in Particular Containing Nitrogen and Phosphorus, Microelement and Vitamin Enrichment Solution and the Addition of a C/N/P-Type Enrichment Solution

[0187] The culture was prepared in conditions similar to that of Example 1.

[0188] The culture is prepared in the defined medium according to the invention, the composition of which is given in Table 3 below.

TABLE-US-00003 TABLE 3 Compound Concentration (g/L) Macroelement solution NaCl 4 MgCl.sub.2 6H.sub.2O 0.97 NaHCO.sub.3 0.19 KNO.sub.3 3.69 K.sub.2HPO.sub.4 0.5 CaCl.sub.2 2H.sub.2O 1.9 FeCl.sub.3 6H.sub.2O 0.02 H.sub.3BO.sub.3 0.06 Microelement solution Na.sub.2EDTA 0 MnCl.sub.2 4H.sub.2O 0.009 ZnCl.sub.2 0.0006 CoCl.sub.2 6H.sub.2O 0.0003 CuSO.sub.4 5H.sub.2O 0.000034 NiSO.sub.4 6H.sub.2O 0.00033 Vitamin solution Biotin 0.0000075 Thiamin 0.004 Vitamin B12 0.00006 Pantothenate 0.0014 Carbon source Glycerol 20

[0189] In this example, a mineral enrichment solution consisting of all the minerals and vitamins present in the initial culture medium other than KNO.sub.3 and KH.sub.2PO.sub.4 is used to enrich the medium for a total mineral and vitamin supply corresponding to three times the initial quantity (“3×” total) after the additions. Moreover, the additional substrate is added in the form of additions of a solution called “C/N/P nutrient solution”. The nutrient solution consists of a carbon substrate (glycerol), an inorganic nitrogen source (KNO3) and an inorganic phosphorus source (K2HPO4). This nutrient solution has a defined molar ratio of carbon, nitrogen and phosphorus atoms: 445:13:1. This ratio was adjusted to create a defined limitation of nitrogen and phosphorus, and thus to preferentially direct the metabolism toward DHEA production while maintaining the biomass in a state allowing sufficient catalytic activity to maintain good productivity.

[0190] A control culture is prepared in rich medium as described in Example 1.

[0191] The addition of the mineral and vitamin enrichment solution and the C/N/P solution in a molar ratio of 445:13:1 makes it possible to reach 275 g/L dry matter consisting of 18% fats with a 37% DHEA content (FIGS. 5A and B).

[0192] Thus, according to this embodiment of the invention, supplying minerals and vitamins on the one hand, and carbon, nitrogen and phosphorus sources on the other, during culture in a chemically defined medium, makes it possible to obtain satisfactory performance in terms of fatty acid accumulation and DHEA content while obtaining good productivity.

[0193] FIG. 5B shows the lipid profile of the oil contained in CCAP strain 1104/3 at the end of culture. It is noted in particular that PUFAs other than DHEA are at trace levels (<1%).