PROCESS FOR ENRICHING THE BIOMASS OF MICROALGAE OF THE THRAUSTOCHYTRIUM GENUS WITH DHA AND WITH ARG AND GLU AMINO ACIDS

Abstract

The present invention relates to a process for enriching a biomass of microalgae of the Thraustochytrium genus with DHA and with arginine and glutamic acid amino acids, characterized in that it comprises a step aimed at limiting the rate of growth of the microalga while at the same time maintaining or continuously introducing a source of nitrogen in or into the fermentation medium.

Claims

1- A process for enriching a biomass of microalgae of the Thraustochytrium genus with docosahexaenoic acid (DHA) and with arginine and glutamic acid amino acids, comprising limiting the growth rate of the microalga in a fermentation medium, determining a ratio of growth rates μ/μmax of the microalgae, concurrently with said limiting maintaining or continuously introducing a nitrogen source in or into the fermentation medium as soon as the value of the ratio of the growth rates μ/μmax of the microalgae becomes less than 0.2, to produce a biomass.

2- The process according to claim 1, wherein the microalgae are of the genus Schizochytrium sp. or Schizochytrium mangrovei genus.

3- The process according to claim 1, wherein the microalgae are a strain selected from strain references CNCM I-4469 and CNCM I-4702 deposited with the Collection Nationale de Cultures de Microorganismes [French National Collection of Microorganism Cultures] of the Institut Pasteur on Apr. 14, 2011 and Nov. 22, 2012, respectively.

4- The process according to claim 1, wherein the limitation of the growth rate of the microalga is obtained by reducing or exhausting trace elements in the fermentation medium or by limiting the O.sub.2 transfer.

5- The process it according to claim 1, further comprising harvesting the biomass, optionally preparing a cell extract or lysate from this biomass, and then optionally extracting a DHA-rich crude oil.

6- The process according to claim 1, wherein the biomass obtained comprises at least 45% of DHA by weight of total fatty acids.

7- The process according to claim 1, wherein the biomass obtained comprises at least 40% of proteins by weight of biomass (g/g) expressed in N.6.25, including at least 10% of arginine and at least 25% of glutamic acid by weight relative to total amino acids.

8- A process for producing a biomass, comprising, culturing microalgae of genus Thraustochytrium in a fermentation medium to enrich docosahexaenoic acid (DHA), arginine and glutamic acid amino acid content, feeding a source of nitrogen to the medium, assessing μ/μmax during said culturing, wherein μ is the actual growth rate of the microalga and μmax is the is optimal growth rate of the microalgae, maintaining or adding said nitrogen source to the fermentation medium when μ/μmax of the microalgae becomes less than 0.2, optionally, controlling the supply oxygen to the fermentation medium such that μ drops to μ/μmax <0.2 rapidly, and recovering a biomass, including at least 10% of arginine and at least 25% of glutamic acid by weight relative to total amino acid content.

9- The process according to claim 8, wherein the microalgae is one of Schizochytrium sp. Strain CNCM I-4469, or Schizochytrium mangrovei strain CNCM I-4702.

10- The process according to claim 1, wherein the biomass includes at least 25% of DHA by weight of total fatty acids.

11- The process according to claim 9, wherein the biomass includes at least 45% of DHA by weight of total fatty acids.

12- The process according to claim 1, wherein the biomass includes at 15% of arginine and at least 40% of glutamic acid.

13- The process according to claim 9, wherein the biomass includes at 15% of arginine and at least 40% of glutamic acid.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0091] Within the context of the invention, the applicant company has chosen to explore an original route for optimizing the production of DHA and of arginie and glutamic acid amino acids by proposing a novel way of conducting fermentation.

[0092] The applicant company has thus found, which goes against the technical preconceptions on the subject, that it is possible to produce by fermentation microalgal biomasses: [0093] rich in lipids (more than 25% by dry weight of biomass, preferably at least 30%), the predominant fatty acid of which is docosahexaenoic acid (DHA), and [0094] rich in arginine and glutamic acid amino acids (more than 35% by weight of the total amino acids, preferably at least 55%),

[0095] without it being essential, as described in the prior art, to maximize the C/N ratio (consumed carbon to consumed nitrogen, mole/mole).

[0096] The applicant company has thus found that it is possible to modify the lipid and amino acid composition of the biomass produced by fermentation, through the maintaining, which is not conventional for a lipid production, of the nitrogen feed throughout the fermentation even when the growth rate μ/μmax is less than 0.2.

[0097] Indeed, the applicant company has understood that, when the μ/μmax ratio becomes less than 0.2, following a limitation of a nutritive substrate other than the nitrogenous or carbon-based substrates, it is possible to move the metabolic productions toward the production of arginine and glutamic acid amino acids, while at the same time retaining a considerable DHA production.

[0098] In one embodiment, the limitation which makes it possible to reduce the growth rate can be the limitation of the oxygen supply (OTR, oxygen transfer rate).

[0099] In particular, the OTR during the fermentation phase is preferably from 30 to 35 mmol/l/h.

[0100] The growth limitation can also be induced by exhausting trace elements or minerals, preferably chosen from phosphate, magnesium or potassium.

[0101] More particularly, the applicant company has found that it is necessary to supply nitrogen, preferentially in aqueous ammonia form (used for example in pH regulation), or that it is necessary to maintain the nitrogen supply, until the end of the culture, provided that μ is less than 20% of μmax.

[0102] In one preferred embodiment, the initial nitrogen supply is added to by the regulation of the pH, the nitrogen consumed thus being compensated for by that of the regulation of the pH. This makes it possible to obtain a C/N ratio (consumed carbon to consumed nitrogen, mole/mole) at the end of the culture of less than 20, for example of between 10 and 15, and preferably of approximately 15.

[0103] The strains to be used in the methods of the present invention are of the Thraustochytrium genus, more particularly Schizochytrium mangrovei or Schizochytrium sp. Such strains are known to those skilled in the art.

[0104] In the course of their research, the applicant company has identified several microalgal strains of great interest which produce DHA. The applicant company is especially quite particularly interested in two strains that it has identified.

[0105] The first strain is a strain of Schizochytrium sp., deposited in France on Apr. 14, 2011 with the Collection Nationale de Cultures de Microorganismes [French National Collection of Microorganism Cultures] (CNCM) of the Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France, under number I-4469 and also in China with the China Center for Type Culture Collection (CCTCC) of the University of Wuhan, Wuhan 430072, P.R. China under number M 209118. This strain mainly produces DHA and to a lesser extent palmitic acid and palmitoleic acid. It was characterized by partial sequencing of the gene encoding 188 RNA (SEQ ID No 1):

TABLE-US-00001   1 GAGGGTTTTA CATTGCTCTC aTTCCaATAG CAaGACGCGA AGCGCCCCGC ATTGATATTT  61 CTCGTCACTA CCTCGTGGAG TCCACATTGG GTAATTTACG CGCCTGCTGC CTTCCTTGGA 121  TGTGGTAGCC GTCTCTCAGG CTCCCTCTCC GGAGTCGAGC CCTAACTCCC CGTCACCCGT 181  TATAGTCACC GTAGGCCAAT ACCCTACCGT CGACAACTGA TGGGGCAGAA ACTCAAACGA 241  TTCATCGCTC CGAAAAGCGA TCTGCTCAAT TATCATGACT CACCAAGAGA GTTGGCTTAG 301  ACCTAATAAG TGCGGCCCTC CCCGAAAGTC GGGCCCGTAC AGCACGTATT AATTCCAGAA 361  TTACTGCAGG TATCCGTATA AAGGAACTAC CGAAGGGATT ATAACTGATA TAATGAGCCG 421  TTCGCAGTTT CACAGTATAA TTCGCTTATA CTTACACATG CATGGCTTAG TCTTTGAGA

[0106] which made it possible to identify it as being a strain of Schizochytrium sp. type. This strain will be subsequently denoted “CNCM I-4469” in the present application.

[0107] Moreover, the second strain is a strain of Schizochytrium mangrovei. It produces DHA and palmitic acid in relatively equal proportions. It was deposited by the applicant company in France on Nov. 22, 2012 with the Collection Nationale de Cultures de Microorganismes (CNCM) of the Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, under number CNCM I-4702. It was characterized by sequencing of the genes encoding 18 S rRNA (SEQ ID No 2):

TABLE-US-00002   1 GGTTTTACAT TGCTCTCATT CCGATAGCAA AACGCATACA CGCTTCGCAT CGATATTTCT  61 CGTCCTACCT CGTGGAGTCC ACAGTGGGTA ATTTACGCGC CTGCTGCTAT CCTTGGATAT 121 GGTAGCCGTC TCTCAGGCTC CCTCTCCGGA GTCGAGCCCT AACTCTCCGT CACCCGTTAT 181 AGTCACCGTA GTCCAATACA CTACCGTCGA CAACTGATGG GGCAGAAACT CAAACGATTC 241 ATCGACCAAA AWAGTCAATC TGCTCAATTA TCATGATTCA CCAATAAAAT CGGCTTCAAT 301 CTAATAAGTG CAGCCCCATA CAGGGCTCTT ACAGCATGTA TTATTTCCAG AATTACTGCA 361 GGTATCCATA TAAAAGAAAC TACCGAAGAA ATTATTACTG ATATAATGAG CCGTTCGCAG 421 TCTCACAGTA CAATCGCTTA TACTTACACA GCAG

[0108] which made it possible to identify it as being a strain of Schizochytrium mangrovei type. This strain will be subsequently denoted “CNCM I-4702” in the present application.

[0109] Moreover, the fermenting processes according to the present invention are carried out under heterotrophic culturing conditions. These conditions adapted to the microalgae under consideration and also the culture media are well known to those skilled in the art.

[0110] The carbon source necessary for the growth of the microalga is preferably glucose.

[0111] Preferably, the glucose supply is such that the glucose concentration during the fermentation is maintained at a concentration of 20 g/l or more. At the end of fermentation, the glucose concentration is at least 5 g/l.

[0112] The nitrogen source may be extracts of yeast, urea, sodium glutamate, ammonium sulfate, aqueous ammonia with pH regulation, used alone or in combination.

[0113] Generally, the culturing step comprises a preculturing step to revive the strain, then a step of culturing or fermentation proper. The latter step corresponds to the step of production of the lipids of interest, in particular of DHA.

[0114] Preferably, the pH is regulated during the fermentation at a pH of between 5 and 7, preferably approximately 6.

[0115] Preferably, the temperature during the fermentation is 26-30° C., preferably approximately 28° C.

[0116] The fermentation time is preferably at least 50 hours, preferably between 65 and 90 hours, even more preferably between 70 and 85 hours.

[0117] The fermentation process according to the present invention makes it possible to obtain (or is carried out in such a way as to obtain) a biomass comprising at least 45% of DHA by weight of total fatty acids. In addition, the process guarantees a lipid content by weight relative to the biomass of at least 25%. Thus, the biomass is indeed enriched with DHA.

[0118] Moreover, the fermentation process according to the present invention makes it possible to obtain (or is carried out in such a way as to obtain) a biomass comprising at least 40% of proteins by weight relative to the biomass. In addition, the proportion of glutamic acid relative to the total amino acids is at least 25%. The arginine proportion is at least 10%.

[0119] For the CNCM I-4702 strain, the results obtained with the fermentation process according to the invention are a biomass comprising approximately 47% of DHA by weight of total fatty acids, with a lipid content by weight relative to the biomass of approximately 35%, and approximately 53% of proteins with a proportion of glutamic acid of approximately 40% and of arginine of approximately 16%.

[0120] For the CNCM I-4469 strain, the results obtained with the fermentation process according to the invention are a biomass comprising approximately 52% of DHA by weight of total fatty acids, with a lipid content by weight relative to the biomass of approximately 26%, and approximately 43% of proteins with a proportion of glutamic acid of approximately 26% and of arginine of approximately 10%.

[0121] When reference is made to a percentage by weight, it is understood to be by dry weight.

[0122] Aside from the biomass, the present invention also relates to a cell extract or lysate prepared from this biomass. In particular, this extract or lysate is prepared from the biomass recovered after fermentation. This extract or lysate is rich in DHA and in arginine and glutamic acid amino acids.

[0123] The cells may be ruptured to extract the lipid content in various ways, including mechanical, chemical and enzymatic ways.

[0124] An oil can subsequently be extracted from the cell lysate.

[0125] Thus, the method for producing lipids of interest, preferably DHA, and arginine and glutamic acid amino acids, comprises the fermenting process according to the present invention, harvesting the biomass, preparing a cell extract or lysate and extracting a crude oil comprising the lipids of interest, preferably DHA and optionally arginine and glutamic acid amino acids.

[0126] The term “approximately” is intended to mean the value + or −10% of said value, preferably + or −5% of said value.

[0127] The invention will be understood more clearly from the following examples which are intended to be illustrative and nonlimiting.

EXAMPLES

Example 1: Conditions for Culturing the CNCN I-4702 Strain

[0128] The protocol comprises preculturing for inoculation of the fermenter at 0.1 g/l of biomass for the Schizochytrium mangrovei CNCM I-4702 strain.

[0129] Preculturing

[0130] The preculturing (100 ml of medium) in a 500 ml baffled Erlenmeyer flask lasts for 24 h at 28° C.

[0131] All of the components of the medium are sterilized by filtration.

TABLE-US-00003 TABLE I Preculture medium % (g/g) Anhydrous glucose 3 Yeast extract 0.4 Monosodium glutamate 6.42 NaCl 1.25 MgSO.sub.4•7(H.sub.2O) 0.4 KCl 0.05 CaCl.sub.2•2(H.sub.2O) 0.01 NaHCO.sub.3 0.05 KH.sub.2PO.sub.4 0.4 Stock solution vitamins B1, B6, B12 0.1 Stock solution trace elements 0.8

[0132] Culturing

[0133] The medium is sterilized in 3 parts.

[0134] The glucose is sterilized with the KH.sub.2PO.sub.4 for an addition just before T.sub.0.

[0135] The remainder of the salts are sterilized in the fermenter with 0.75 ml/l of Clearol FBA 3107. The trace elements and vitamins are sterilized by filtration.

[0136] The volume at T.sub.0 represents 75% of the final volume. The pH is adjusted at T.sub.0 using aqueous ammonia, then it is regulated at 6, still with aqueous ammonia.

TABLE-US-00004 TABLE II Culture medium % (W/W) KH.sub.2PO.sub.4 0.80 (NH.sub.4).sub.2SO.sub.4 0.33 Na.sub.2SO.sub.4 0.67 NaCl 0.27 CaCl.sub.2•2(H.sub.2O) 0.03 MgSO.sub.4•7(H.sub.2O) 1.00 Anhydrous glucose 6.00 Stock solution vitamins B1, B6, B12 0.20 Stock solution trace elements 0.27

[0137] A fed batch of glucose (concentration: 500 g/l) is supplied continuously starting from T.sub.0 at a constant rate (to be adjusted according to calculations) so as not to be at a concentration lower than 20 g/l. At the end, the glucose will be exhausted without descending below 5 g/l at the time fermentation is stopped.

[0138] The culturing is carried out at 28° C. and lasts from 70 to 85 hours with a fixed and constant OTR (oxygen uptake rate) of 20 to 30 mmol of O.sub.2/l/h.

[0139] Stock Solutions

TABLE-US-00005 vitamins g/l B1 45 B6 45 B12 0.25

TABLE-US-00006 Trace elements g/l MnCl.sub.2•2H.sub.2O 8.60 CoCl.sub.2•6H.sub.2O 0.2 NiSO.sub.4•6H.sub.2O 7.50 Na.sub.2MoO.sub.4•2H.sub.2O 0.15 ZnSO.sub.4•7H.sub.2O 5.70 CuSO.sub.4•5H.sub.2O 6.50 FeSO.sub.4•7H.sub.2O 32.00 Zinc acetate 0.01 EDTA Brought to pH >3

[0140] Two fermentation conditions are implemented: [0141] As a control: “standard” conditions, in which the C/N ratio (consumed carbon to consumed nitrogen) is maximized so as to produce essentially lipids by interrupting the nitrogen supply but not that of the carbon-based substrate, this being without limitation of O.sub.2. These conditions are thus nitrogen deficient. Suppression of the nitrogen supply takes place when one or more salts are exhausted. Actual growth is then impossible or very limited: the cell multiplication rate drops to the benefit of the lipid enrichment of the cells present. The overall mass of the cells increases but the number of cells changes little since the growth rate falls. [0142] According to the invention: Conditions which make it possible to produce lipids rich in DHA with amino acids rich in arginine and glutamic acid by limiting the growth rate by limiting O.sub.2 transfer such that μ drops to μ/μmax <0.2 rapidly, while at the same time maintaining the supply of glucose and nitrogen preferentially through regulation of the pH with aqueous ammonia.

[0143] FIG. 1 presents the change in the proportion of arginine and glutamic acid among the amino acids as a function of the C/N calculated at the end of culture.

[0144] It appears that the process promotes the production of arginine and glutamic acid amino acids provided that the C/N ratio is less than 15 (# μ/μmax<0.2).

[0145] Table III below reflects, for the CNCN I-4702 strain, the fatty acid and amino acid composition of the biomass produced according to the “conventional” operating conditions and the operating conditions in accordance with the invention.

TABLE-US-00007 TABLE III Use of the Conventional process of culture the invention Lipids relative to Biomass (g/g) 0.60 0.35 Proteins relative to Biomass according to N 0.12 0.53 6.25 (g/g) DHA/Fatty acids (g/g) 0.24 0.47 Aspartic Acid relative to Σ TAA (g/g) 0.12 0.05 Threonine relative to Σ TAA (g/g) 0.06 0.03 Serine relative to Σ TAA (g/g) 0.06 0.03 Glutamic Acid relative to Σ tAA (g/g) 0.11 0.40 Glycine relative to Σ TAA (g/g) 0.05 0.03 Alanine relative to Σ TAA (g/g) 0.07 0.04 Valine relative to Σ TAA (g/g) 0.06 0.03 Isoleucine relative to Σ TAA (g/g) 0.05 0.03 Leucine relative to Σ TAA (g/g) 0.08 0.04 Tyrosine relative to Σ TAA (g/g) 0.04 0.02 Phenylalanine relative to Σ TAA 0.04 0.03 (g/g) Lysine relative to Σ TAA (g/g) 0.07 0.04 Histidine relative to Σ TAA (g/g) 0.02 0.01 Arginine relative to Σ TAA (g/g) 0.06 0.16 Proline relative to Σ TAA (g/g) 0.05 0.03 Cystine relative to Σ TAA (g/g) 0.02 0.01 Methionine relative to Σ TAA (g/g) 0.03 0.02 Tryptophan relative to Σ TAA (g/g) 0.02 0.01

[0146] The glutamic acid proportion relative to the sum of the amino acids is multiplied by 3.75 and the arginine proportion relative to the sum of the amino acids is multiplied by 2.75.

[0147] The lipid composition is reduced, but the DHA content of the fatty acids is almost multiplied by two.

Example 2: Conditions for Culturing the CNCN I-4469 Strain

[0148] The conditions for culturing this microalga are the same as those of example 1 (with the exception of the level of inoculum chosen in preculture, of about 5 g/l for Schizochytrium sp.).

[0149] According to, two culture conditions implemented “conventionally” and according to the invention.

[0150] Table IV below reflects the fatty acid and amino acid composition of the biomass produced according to the “conventional” operating conditions and the operating conditions in accordance with the invention.

TABLE-US-00008 TABLE IV Use of the process Conventional of the culture invention Lipids relative to Biomass (g/g) 0.46 0.26 Proteins relative to Biomass according to N 0.21 0.43 6.25 (g/g) DHA/Fatty acids (g/g) 0.42 0.52 Aspartic Acid relative to Σ TAA (g/g) 0.12 0.08 Threonine relative to Σ TAA (g/g) 0.05 0.04 Serine relative to Σ TAA (g/g) 0.05 0.04 Glutamic Acid relative to Σ tAA (g/g) 0.15 0.26 Glycine relative to Σ TAA (g/g) 0.05 0.05 Alanine relative to Σ TAA (g/g) 0.08 0.06 Valine relative to Σ TAA (g/g) 0.06 0.05 Isoleucine relative to Σ TAA (g/g) 0.04 0.03 Leucine relative to Σ TAA (g/g) 0.08 0.06 Tyrosine relative to Σ TAA (g/g) 0.04 0.03 Phenylalanine relative to Σ TAA (g/g) 0.04 0.03 Lysine relative to Σ TAA (g/g) 0.06 0.05 Histidine relative to Σ TAA (g/g) 0.02 0.02 Arginine relative to Σ TAA (g/g) 0.06 0.10 Proline relative to Σ TAA (g/g) 0.04 0.07 Cystine relative to Σ TAA (g/g) 0.02 0.01 Methionine relative to Σ TAA (g/g) 0.02 0.02 Tryptophan relative to Σ TAA (g/g) 0.02 0.01

[0151] For the Schizochytrium sp. strain, the effects are identical but smaller. Moreover, an increase of 75% in the proportion of proline among the amino acids is also noted.

[0152] The arginine and glutamic acid amino acid contents increase respectively by 60% and 75%, while the protein content doubles.

[0153] The DHA content in the fatty acids increases by 23%.