FR3003873A1 - PROCESS FOR ENRICHING PROTEIN FROM BIOMASS OF MICROALGUES - Google Patents

Abstract

The invention relates to a protein enrichment method of a microalga grown in heterotrophy, microalgae of the genus Chlorella, characterized in that the heterotrophic culture comprises a step for limiting the growth of said microalgae by a deficiency of the medium of fermentation in a non-nitrogenous nutritional source.

Description

The present invention relates to a process for enriching proteins in the biomass of microalgae, more particularly of the genus Chlorella, more particularly of the species Chlorella sorokiniana or Chlorella protothecoides. The algae, macro and micro, have a richness largely unexplored. Their exploitation for food, chemical or bioenergetic purposes is still very marginal. They contain, however, valuable components, which only the marine fauna, which feed on them, are truly capable of appreciating their richness and abundance. Microalgae are indeed sources of vitamins, lipids, proteins, sugars, pigments and antioxidants. Algae and microalgae are thus of interest to the industrial sector which uses them for the manufacture of food supplements, functional foods, cosmetics, medicines or for aquaculture. Microalgae are primarily photosynthetic microorganisms that colonize all biotopes exposed to light. On an industrial scale, their monoclonal culture is carried out in photobioreactors (autotrophic conditions: in the light with CO2) or for some of them, also in fermenters (heterotrophic conditions: in the dark in the presence of a carbon source). Some microalgae species are indeed able to grow in the absence of light: Chlorella, Nitzschia, Cyclotella, Tetraselmis, Crypthecodinium, Schizochytrium. It is moreover estimated that culture in heterotrophic conditions costs 10 times less expensive than in phototrophic conditions because, for those skilled in the art, these heterotrophic conditions allow: the use of fermentors identical to those used for bacteria and yeasts and allow control of all culture parameters. - the production of biomasses in a quantity much higher than what is obtained by culture based on the light. The profitable exploitation of the microalgae generally requires the control of the fermentation conditions making it possible to accumulate its components of interest, such as: the pigments (chlorophyll a, b and c, fl-carotene, astaxanthin, lutein, phycocyanin, xanthophylls , phycoerythrin ...) whose demand is growing, both for their remarkable antioxidant properties, as for their contribution of natural colors in the diet, - the proteins, in order to optimize their nutritional qualities; or - lipids, in order to optimize their fatty acid content (up to 60%, see 80% by weight of their dry matter), in particular for: - biofuel applications, but also - applications in human or animal nutrition , when the selected microalgae produce polyunsaturated fatty acids or PUFAs called "essential" (ie brought by the food because they are not naturally produced by the man or the animal). To achieve this result, first fermentation processes to obtain high cell densities (HCD for High-Cell-Density) were thus much worked, so as to obtain maximum yields and productivities in proteins or lipids.

The objective of these HCD cultures was to obtain the highest possible concentration of the desired product in the shortest time. This precept applies, for example, to the biosynthesis of astaxanthin by Chlorelle zofingiensis, where the growth of the microalgae has been directly correlated with the production of this compound (Wang and Peng, 2008, World J. Microbiol., Biotechnol., 24 ( 9), 1915-1922). However, maintaining growth at its peak rate (p, in h-1) is not always correlated with the high production of the desired product. It quickly became apparent to specialists in the field that, for example, microalgae must be subjected to a nutritional stress which limits their growth when they wish to produce large lipid reserves. It is therefore now decoupling growth / production in fermentative processes. For example, to promote the accumulation of polyunsaturated fatty acids (here docosahexaenoic acid or DHA), patent application VVO 01/54510 recommends dissociating cell growth and the production of polyunsaturated fatty acids. In the microalga Schizochytrium sp strain ATCC 20888, a first phase of growth without limitation in oxygen is thus carried out, so as to favor obtaining a high cell density (more than 100 g / l) and then, in a second phase, gradually slow down the oxygen supply, in order to stress the microalgae, slow down its growth and trigger the production of the fatty acids of interest. In the microalgae Crypthecodinium cohnii, the highest content of docosahexaenoic acid (DHA, polyunsaturated fatty acid) is obtained at low glucose concentration (of the order of 5 g / 1), and thus at a low growth rate (Jiang and Chen, 2000, Process Biochem., 35 (10), 1205-1209). These results illustrate that product formation kinetics can be positively and negatively associated with the growth of microalgae, or even a combination of both. Therefore, in cases where the formation of products is not correlated with high cell growth, it is advisable to control the cell growth rate. In general, a person skilled in the art chooses to control the growth of microalgae by controlling the fermentation conditions (Tp, pH, etc.), or by the regulated supply of nutritional components of the fermentation medium (semi-continuous conditions called " fed-batch "). If he chooses to control the growth of microalgae in heterotrophy by the supply of carbon sources, the skilled person generally chooses to adapt the carbon source (pure glucose, acetate, ethanol ...) to the microalga (C. cohnii, Euglena gracilis ...) depending on the metabolite produced (for example a polyunsaturated fatty acid type DHA). Temperature may also be a key parameter: - For example, it has been reported that the synthesis of polyunsaturated fatty acids in certain species of microalgae, such as EPA by Chlorella minutissima, is favored at a lower temperature than required for optimal growth of said mi croalg ue; - In contrast, the yield of lutein is higher in Chlorella protothecoides grown in heterotrophy, when the production temperature is increased from 24 to 35 ° C.

Chlorella protothecoides is justly recognized as one of the best oil-producing microalgae. In heterotrophic conditions, it rapidly converts carbohydrates into triglycerides (more than 50% of its dry matter). To optimize this production of triglycerides, those skilled in the art are led to optimize the carbon flow towards the production of oil, by acting on the nutritional environment of the fermentation medium. It is thus known that the accumulation of oil occurs during a sufficient carbon supply, but under conditions of nitrogen deficiency. The C / N ratio is here decisive, and it is accepted that the best results are obtained by acting directly on the nitrogen content, the glucose content not being limiting.

Not surprisingly, this nitrogen deficiency affects cell growth, resulting in a 30% lower growth rate than the normal growth rate of the microalgae (Xiong et al., Plant Physiology, 2010, 154, pp1001 - 1011).

To explain this result, Xiong et al, in the article cited above, demonstrate indeed that if one divides the biomass of Chlorella into its main components, ie carbohydrates, lipids, proteins, DNA and RNA (representing 85 % of its dry matter), if the C / N ratio has no impact on the DNA, RNA and carbohydrate content, it becomes preeminent for the protein and lipid content.

Chlorella cells cultured with a low C / N ratio contain 25.8% protein and 25.23% lipid, while a high C / N ratio allows the synthesis of 53.8% of the protein. lipids and 10.5% protein. To optimize its oil production, it is therefore essential for the skilled person to control the carbon flow by deflecting it to the production of oil, to the detriment of the production of proteins; the carbon flux is redistributed and accumulates in lipid reserve substances when the microalgae are placed in nitrogen-deficient medium. With this teaching, for the production of protein-rich biomasses, the skilled person is led to take the opposite of this metabolic control, ie work the fermentation conditions by favoring rather a low C / N ratio, and so : - make a significant contribution of nitrogen source to the fermentation medium, while keeping the carbon source load constant, which will be converted into proteins and - stimulate the growth of the microalgae. It is a question of modifying the carbon flow towards the production of proteins (and thus of biomasses), to the detriment of the production of the reserve lipids. In the context of the invention, the applicant company has chosen to explore an original way by proposing an alternative solution to that conventionally envisaged by those skilled in the art. The invention thus relates to a process for enriching the proteins of a microalga grown in heterotrophy, a microalgae of the genus Chlorella, more particularly Chlorella sorokiniana or Chlorella protothecoides, a heterotrophic culture method which comprises a step intended to limit the growth of said microorganism. microalgae by a deficiency of the fermentation medium in a nutritional source non-nitrogenous. This step is a heterotrophic culture step where a non-nitrogenous nutrient is provided in insufficient quantity in the medium to allow the growth of the microalgae.

For the purposes of the invention, "enrichment" means an increase in the protein content of the biomass of at least 15%, preferably at least 20% by weight, of to achieve a protein content of the biomass of more than 50% by weight.

The invention more specifically covers a process for the heterotrophic cultivation of said microalgae comprising a step aimed at limiting the growth of said microalgae by a deficiency of the fermentation medium in a non-nitrogen nutritional source. Thus, the present invention relates to a protein enrichment process of a microalga grown in heterotrophy, microalgae of the genus Chlorella, more particularly Chlorella sorokiniana or Chlorella protothecoides, the process comprising the heterotrophic culture which comprises a step intended to limit the growth of said microalgae by a deficiency of the fermentation medium in a non-nitrogenous nutritional source, thus making it possible to achieve a protein content of the biomass of more than 50% by weight. By "deficiency of the fermentation medium into a non-nitrogenous nutritional source" is meant a culture in which at least one of the non-nitrogenous nutritive factors is provided to the microalgae in an amount insufficient to allow its growth. This results in an absence of non-nitrogenous residual nutrient factor in the culture medium, the microalgae consuming this nutritive factor as and when it is added. Within the meaning of the invention, the essential criterion is therefore the limitation of stress-induced cell growth, a cellular stress caused by the lack of a non-nitrogenous nutrient in the fermentation medium.

This strategy goes against the technical prejudice that to increase the protein content of biomass, it is inevitable to increase this biomass and therefore cell growth. The term "non-nitrogenous nutritional source" means a nutrient chosen for example from the group consisting of glucose and phosphates.

As will be exemplified below, it is advantageous to choose to limit the growth of: - Chlorella sorokiniana by a glucose deficiency of the fermentation medium, - Chlorella protothecoides by a phosphate deficiency of the fermentation medium.

Optionally, the restriction of the growth of said microalgae can be obtained by adding in the culture medium substances that inhibit cell growth, such as sulphates.

Moreover, without being bound by any theory, the Applicant Company has found that the flow of glucose is normally used in microalgae of the genus Chlorella sorokiniana in a precise order of priority: 1. basal metabolism, 2. growth, ie formation of a protein-rich biomass; 3. reserve substances (lipids and carbohydrates such as starch). This principle makes it possible to explain natural variations in protein content during the growth of the microalgae, despite the constant supply of nitrogen. The applicant company has thus found that, in order to enrich the biomass of microalgae with proteins, it is necessary to limit the growth of the microalga and to control its consumption in a nutritive source other than nitrogen, for example in glucose so as to: - dedicate the consumption in whole glucose to the protein production pathways, - avoid the accumulation of reserve substance such as lipids. In fact, avoiding a nitrogen deficiency makes it possible to prevent the deviation of metabolic fluxes towards the production of lipids. Optionally, it may be advantageous to go as far as completely blocking any synthesis of spare material, by acting through specific inhibitors. Indeed, a certain number of inhibitors of the lipid synthesis pathways are known, even starch (reserve carbohydrate par excellence of green microalgae): - for lipids, cerulenin is described as an inhibitor of synthesis fatty acids, or lipstatin, a natural substance produced by Streptomyces toxytricini, as a lipase inhibitor ... - for starch, iminosugars (obtained by simple substitution of the endocyclic oxygen atom of sugars by an atom of Nitrogen) are historically known as potent inhibitors of glycosidases, glycosyltransferases, glycogen phosphorylases, or UDP-Galp mutase. Thus, the method comprises the fermentation of a biomass of microalgae under heterotrophic conditions with a first stage of growth of the biomass and with a second stage of deficiency of the fermentation medium into a non-nitrogenous nutritional source.

This second step makes it possible to enrich the biomass with protein. In particular, it makes it possible to achieve a protein content of the biomass of more than 50% by weight (by weight of dry matter).

In a first preferred embodiment according to the invention, the process for the heterotrophic culture of said microalgae, in particular Chlorella sorokiniana, comprises: a first stage of growth of microalgae, where the rate of glucose supply is adjusted to their capacity to consumption, so as to rapidly obtain a large biomass, o a second stage of protein production by microalgae, where the glucose supply rate is set at a value well below their capacity to consume glucose, so as to to prevent the additional accumulation of reserve substances, or even to favor their consumption. As will be exemplified below, the first microalgae growth step is carried out batchwise or "batch" mode, in which the glucose initially supplied is entirely consumed by the microalgae, leading to the production of a basic biomass. The second protein production step is carried out under conditions in which: - a complete medium feed is introduced in a semi-continuous mode, called "fed-batch" mode, after the consumption of the glucose initially supplied; the other driving parameters of the fermentation are unchanged. The glucose is fed continuously and the feed rate is then less than the consumption rate that the strain could achieve so that the residual glucose content in the medium is maintained zero. The growth of the strain is then limited by the availability of glucose (glucose-limiting condition). a continuous operation of the chemostat type, in which the growth rate of the strain (p) is maintained at its minimum value, the growth of the strain being limited by the glucose supply. This mode of operation makes it possible to obtain a biomass with a high protein content thanks to the limitation in glucose and the low rate of growth imposed while ensuring a very good productivity. In a second preferred embodiment according to the invention, the method of heterotrophic culture of said microalgae, in particular Chlorella protothecoides, comprises a step of growth of microalgae in which a limitation of the phosphate supply limits the speed of growth and causes a increase in protein content. Thus, the heterotrophic culture of microalgae of the Chlorella protothecoides species comprises a heterotrophic culture step with a phosphate deficiency, the growth rate thus being reduced and resulting in an increase in the protein content. The invention will be better understood with the aid of the examples which follow, which are intended to be illustrative and not limiting.

EXAMPLES Example 1 Production of Sorokiniana Chlorella in "Sequential Batch" Type Fermentation Without Limiting Nutrient Intake The strain used is a Chlorella sorokiniana (strain UTEX 1663 - The Culture Collection of Algae at the University of Texas at Austin - USA). Preculture: - 600 mL of medium in a 2 L Erlenmeyer flask; - Composition of the medium (Table 1 below) Table 1. Macro Glucose 20 elements (g / L) K2HPO4 3H20 0.7 MgSO4 7H20 0.34 Citric acid 1.0 Urea 1.08 Na2SO4 0.2 Na2CO3 0.1 clerol FBA 3107 (antifoam) 0.5 Micro Na2EDTA 10 elements (mg / L) CaCl2.2H2O 80 FeSO4.7H2O 40 MnSO4.4H2O 0.41 C0SO4.7H2O 0.24 CuSO4.5H2O 0.24 ZnSO4.7H2O 0.5 H3B03 0.11 (NI-14) 6Mo7027.4F120 0.04 The pH is adjusted to 7 before sterilization by addition of 8N NaOH.

The incubation takes place under the following conditions: duration: 72 h; temperature: 28 ° C; agitation: 110 rpm (lnfors Multitron Incubator).

The preculture is then transferred to a 30L fermentor of the Sartorius type. Culture for biomass production: The medium is identical to that of preculture, but the urea is replaced by NH4Cl. Table 2. Macro Glucose 20 elements (g / L) K2HPO4 3H20 0.7 MgSO4 7H20 0.34 Ac Citric 1.0 NH4Cl 1.88 Na2SO4 0.2 clerol FBA 3107 (antifoam) 0.5 Micro Na2EDTA 10 elements (mg / L) CaCl2 80 FeSO4.7H2O 40 MnSO4.4H2O 0.41C0SO4.7H2O 0.24 CuSO4.5H2O 0.24 ZnSO4.7H2O 0.5 H3B03 0.11 (NI-14) 6Mo7027.4F120 0.04 Volume initial (Vi) of the fermenter is adjusted to 13.5 L after seeding. It is brought to 16 - 20 L in final. The driving parameters of the fermentation are as follows: Table 3. Temperature 28 ° C pH 5.0 - 5.2 by NH3 28% w / w P02> 20% (stirred) Stirring 300 RPM mini Air flow 15 L / min When the glucose initially supplied is consumed, a mixture of medium identical to the initial medium, without the antifoam, is produced in the form of a concentrated solution containing 500 g / l of glucose and the other elements in the same o proportions relative to glucose than in the initial medium, so as to obtain a glucose content of 20 g / L in the fermenter. Two other identical additions are made in the same way each time the residual concentration of glucose becomes zero.

Clerol FBA 3107 antifoam is added on demand to prevent excessive foaming. Results: After 46 h of culture, 38 g / L of biomass is obtained at a protein content (evaluated by N 6.25) of 36.2%. EXAMPLE 2 Production of C. sorokiniana during fermenting type "fed batch With limiting glucose supply In this example, a complete medium input ("fed-batch" mode) is started after consumption of the glucose initially supplied. The other driving parameters of the fermentation are unchanged. Glucose is continuously fed with a concentrated solution of 500 g / L. The feed rate is less than the rate of consumption that the strain could achieve so that the residual glucose content in the medium is maintained at zero, that is, the growth of the strain is limited by the availability in glucose (glucose-limiting condition). This speed is increased over time exponentially. The formula used to calculate the rate of addition is characterized by a factor p which corresponds to the growth rate that the strain can adopt if it consumes all the glucose supplied: S = So x exp (p, t) S = flow rate glucose supply (in g / h) So = initial glucose supply rate, determined according to the biomass present at the end of the batch. It is 12 g / h in our conditions. p = flow acceleration factor. It must be less than 0.11 thread which is the growth rate of the strain in the absence of nutritional limitation. t = duration of the fed-batch (in h) The salts are brought if possible continuously, separately or in mixture with the glucose. But they can also be brought sequentially in several times. Table 4 below gives the salt requirements per 100 g of glucose.

Table 4. Macro Glucose 100 elements (g) K2HPO4 3H20 6.75 MgSO4 7H20 1.7 Ac Citric 5.0 Na2SO4 1.0 Micro Na2EDTA 50 elements (mg) CaCl2 2H20 400 FeSO4.7H2O 200 MnSO4.4H2O 2.1 C0SO4 .7H20 1.2 CuSO4.5H20 1.2 ZnSO4.7H20 2.5 H3B03 0.6 (N1-14) 6Mo7027.4F120 0.2 The concentrations of the elements other than glucose were determined to be in excess by relative to the nutritional needs of the strain.

Clerol FBA 3107 antifoam is added on demand to prevent excessive foaming. Results: effect of the rate of glucose supply during fed-batch Tests were carried out at different rates of glucose supply in "fed-batch". They are characterized by the applied p. The protein content of the biomass obtained is evaluated by measuring the total nitrogen expressed in N 6.25. Table 5.

Test p fed (h-1) Time (h) Biomass Productivity `) / 0 N 6.25 (g / L) (g / L / h) 1 0.06 78 43.6 0.56 49.2 2 0.07 54 35.1 0.65 43.1 3 0.09 48 64.9 1.35 39.3 These results show that working with limiting glucose increases the protein content. It is indeed observed that, even with a high p of 0.09, a higher protein content is obtained than that obtained without limitation as in Example 1 (39.3% instead of 36.2%). Increasing the limitation of glucose metabolism results in further improvement in protein content.

Under the conditions of these tests, it is necessary to impose on the strain a p less than 0.06 h -1 to obtain a protein content greater than 50%. It should be noted that this condition goes hand in hand with a reduction in productivity: 0.56 g / LJh instead of 1.35 g / L / h with test 3.

Example 3 Production of C. sorokiniana in continuous fermentation of chemostat type with limiting glucose supply In this example, the fermentor used is a 2L of Sartorius Biostat B type.

It is carried out as Example 2, but with volumes 10 times lower: the inoculum is 60 ml and the initial volume of 1.35L. The continuous supply of the medium is started according to the same principle as in Example 2, the salts being in this case mixed with the glucose in the feeding nanny. The feed rate is accelerated according to the same exponential formula as in Example 2 by applying a p of 0.06 h -1. Chemostat When a volume of 1.6 L is reached, ie a biomass concentration of approximately 50 g / L, continuous operation of the chemostat type is set up: 1. The fermenter is fed continuously at a flow rate of 96 ml / h with a solution of nutrient medium at 100 g / l of glucose of the following composition: Table 6. Macro Glucose 100 elements (g / L) K2HPO4 3H20 6.75 MgSO4 7H20 1.7 Ac Citric 5.0 Na2SO4 1 0 Micro Na2EDTA 50 elements (mg / L) CaCl2 2H20 400 FeSO4.7H2O 200 MnSO4.4H2O 2.1 C0SO4.7H2O 1.2 CuSO4.5H2O 1.2 ZnSO4.7H2O 2.5 H3B03 0.6 (NI-14 ) 6Mo7027.4F120 0.2 Concentrations of other elements than glucose were determined to be in excess of the nutritional requirements of the strain. 2. Medium is withdrawn continuously from the fermenter using a dip tube connected to a pump to maintain the volume of the culture at 1.6L. Thus, the medium is renewed with a fraction 0.06 (6%) per hour. This renewal rate is called the dilution ratio (D).

In accordance with the principle of chemostat culture, the growth rate of the strain (p) is established at the same value because the growth of the strain is limited by the supply of glucose: D = p = 0.06 h 1 Results After 97 hours of chemostat operation, the biomass concentration stabilizes at 48 g / L +/- 2 g / L and the protein content at 53 +/- 2%. This operating mode makes it possible to obtain a biomass with a high protein content thanks to the limitation in glucose and the low rate of growth imposed while ensuring a very good productivity, of the order of 2.9 g / L / h, thanks to the high concentration of biomass. EXAMPLE 4 Production of Chlorella Protothecoides in Batch Type Fermentation With or Without Limitation of Phosphate Intake The strain used is a Chlorella protothecoides (strain CCAP211 / 8D - The Culture Collection of Algae and Protozoa, Scotland, UK). Preculture: 150 mL of medium in a 500 mL Erlenmeyer flask; o Composition of the medium: 40 g / l of glucose + 10 g / l of yeast extract. The incubation takes place under the following conditions: duration: 72 h; Temperature: 28 ° C; agitation: 110 rpm (lnfors Multitron Incubator). The preculture is then transferred to a 2 L fermentor of the Sartorius Biostat B type.

Culture for biomass production: The composition of the culture medium is as follows (in g / L): Table 7. Glucose 80 Citric acid 4 NH4Cl 2 KH2PO4 2 (test 1) or 3 (test 2) Na2HPO4 2 (test 1 ) or 3 (test 2) MgSO4, 7H20 1.5 NaCl 0.5 Yeast extract 5 Phosphate intake is calculated to be limiting in test 1 and in excess in test 2. Clerol antifoam FBA 3107 is added on demand to prevent excessive foaming. The initial volume (Vi) of the fermenter is adjusted to 1 L after seeding. The fermentation control parameters are as follows: Table 8. Temperature 28 ° C pH 6.5 by NH3 28% w / w P02> 20% (stirred) Agitation 200 RPM mini Air flow 1 L / min Results: Table 9. Run Time (h) Biomass p cumulative residual PO4 (n) / 0 N 6.25 (g / L) (h-1) (mg / L) 1 45 36.5 0.07 0 56.1 2 36 38.1 0.09 800 48.1 These results show that a limitation of phosphate intake, confirmed by the absence of residual phosphate at the end of fermentation, limits the growth rate (measured by the cumulative p ) and, as the limitation by glucose in the previous examples, causes an increase in the protein content to reach values well above 50%. 25

Claims (5)

REVENDICATIONS1. Process for the protein enrichment of a microalga grown in heterotrophy, a microalgae of the genus Chlorella, characterized in that the heterotrophic culture comprises a step aimed at limiting the growth of said microalga by a deficiency of the fermentation medium in a non-nitrogen nutritional source , thereby achieving a protein content of the biomass of more than 50% by weight. 2. Method according to claim 1, characterized in that the non-nitrogen deficient nutritional source is a nutrient selected from the group consisting of glucose and phosphates. 3. Method according to either of claims 1 and 2, characterized in that the microalgae of the genus Chlorella is selected from the group consisting of Chlorella sorokiniana and Chlorella protothecoides. 4. Method according to claim 3, characterized in that the heterotrophic culture of microalgae of the Chlorella sorokiniana species comprises: a. a first stage of growth of microalgae, where the rate of glucose supply is adjusted to their consumption capacity, so as to rapidly obtain a large biomass, b. a second stage of protein production by microalgae, where the rate of glucose supply is set at a value well below their glucose consumption capacity, so as to prevent the additional accumulation of reserve substances, or even favor their consumption. 5. Process according to claim 3, characterized in that the heterotrophic culture of microalgae of the Chlorelle protothecoides species comprises a step of culture with a phosphate deficiency which limits the growth rate and causes an increase in the protein content.