Method for the protein enrichment of microalgal biomass

Abstract

The invention relates to a method for the protein enrichment of a heterotrophically cultured microalga, the microalga being of the genus Chlorella, even more particularly Chlorella protothecoides, characterized in that it comprises: a first step directed toward limiting the ammonium supply so as to obtain a microalgal biomass with a protein content of less than 50% expressed as N.6.25, preferably less than 30%, more preferentially between 20 and 25%; a second step in which the ammonium supply in the fermentation medium is increased so as to obtain a protein content of greater than 50%, preferably greater than 60%, more preferentially greater than 65%.

Claims

1. A method for protein enrichment of heterotrophically cultured microalga of the genus Chlorella, the method comprising: a first step comprising cultivation of Chlorella in a fermentation medium, the fermentation medium comprising a limited supply of ammonia, wherein the microalga is cultivated to possess a protein content of less than 50% expressed as N.6.25, and wherein cultivation in the limited supply of ammonia provides a specific rate of nitrogen consumption by the microalga of less than 0.005 g/g/h; and a second step, wherein the ammonia present in the fermentation medium is increased, and wherein the microalga is cultivated until a protein content of a biomass of the microalga is increased to greater than 50% expressed as N.6.25.

2. The method as claimed in claim 1, wherein in the first step, pH regulation of the fermentation medium is performed by addition of an NH.sub.3/KOH mixture, and wherein in the second step, pH regulation of the fermentation medium is performed with NH.sub.3.

3. The method of claim 2, wherein a mole percent of the NH.sub.3/KOH mixture is about 70-45% NH.sub.3 and 30-55% KOH.

4. The method of claim 2, wherein a mole percent of the NH.sub.3/KOH mixture is about 65-55% NH.sub.3 and 35-45% KOH.

5. The method of claim 1, wherein the ammonia supply in the second step is increased by about 1.5 to 2 fold over the ammonia supply in the first step.

6. The method of claim 1, wherein in the second step, a specific rate of nitrogen consumption by the microalga is greater than 0.01 g/g/h.

7. The method of claim 1, wherein a growth rate of the microalga is maintained substantially constant during both the first step and the second step.

8. The method of claim 7, wherein the growth rate is maintained at 0.07 h.sup.−1 to 0.09 h.sup.−1.

9. The method of claim 8, wherein, the growth rate is maintained at about 0.0810.

10. The method of claim 1, wherein the first step comprises a batch phase, wherein 20 g/l of glucose is supplied to a fermentation medium during cultivation of Chlorella, and; a first exponential fed-batch phase, wherein a growth rate is maintained at 0.08 h.sup.−1 until the glucose supplied in the batch phase is completely consumed; wherein during the first exponential fed-batch phase, a concentration of ammonia in the fermentation medium is limited by regulating a pH of the fermentation medium with a mixture of NH3 and KOH, wherein during the first exponential fed-batch phase, biomass comprising less than 25% protein expressed as N.6.25 is obtained, and wherein the limited concentration of ammonia provides a specific rate of nitrogen consumption by the microalga of less than 0.005 g/g/h; and the second step comprises a second exponential fed-batch phase, wherein the growth rate is maintained at 0.08 h.sup.−1, and wherein during the second exponential fed-batch phase, the concentration of ammonia in the fermentation medium is no longer limited and the pH of the fermentation medium is regulated by adding 100% aqueous ammonia solution to the fermentation medium, and wherein during the second exponential fed-batch phase, biomass comprising more than 50% protein expressed as N.6.25 is obtained.

11. The method of claim 1, wherein the microalgal biomass comprises 45% arginine relative to total amino acids of the biomass.

12. The method of claim 10, wherein the microalgal biomass comprises 45% arginine relative to total amino acids of the biomass.

13. The method of claim 1, wherein the microalga is Chlorella protothecoides.

14. The method of claim 1, wherein the microalgal biomass comprises more than 45% of glutamic acid and arginine relative to the total amino acids of the microalgal biomass.

15. The method of claim 10, wherein the microalgal biomass comprises more than 45% of glutamic acid and arginine relative to the total amino acids of the microalgal biomass.

16. The method of claim 1, wherein the first step comprises cultivating the microalga until a protein content from 20 to 25% expressed as N.6.25 is obtained and the second step comprises cultivating the microalga until a protein content of the microalgal biomass is increased to greater than 60% expressed as N.6.25.

17. The method of claim 1, wherein the first step comprises cultivating the microalga until a protein content from 20 to 25% expressed as N.6.25 is obtained.

18. The method of claim 1, wherein the second step comprises cultivating the microalga until a protein content of the microalgal biomass is increased to greater than 60% expressed as N.6.25.

19. The method of claim 10, wherein during the second exponential fed-batch phase, biomass comprising more than 60% protein expressed as N.6.25 is obtained.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 illustrates the change in the N.6.25 as a function of the glucose consumed.

(2) FIG. 2 illustrates the change in the specific rate of nitrogen consumption (qN) as a function of the glucose consumed.

(3) FIG. 3 illustrates the change in the N.6.25 as a function of time and the amount of each amino acid as a weight percentage of dry biomass as a function of time.

(4) FIG. 4 illustrates the change in the N.6.25 as a function of time and the amount of total or particular fatty acids as a weight percentage of dry biomass as a function of time.

(5) FIG. 5 illustrates the change in the N.6.25 as a function of time and the amount of total or particular sugars as a weight percentage of dry biomass as a function of time.

EXAMPLES

Example 1: Preparation of a Biomass of C. Protothecoides Rich in Protein with a High Content of Glutamic Acid and Arginine

(6) The strain used is a Chlorella protothecoides (strain CCAP211/8D—The Culture Collection of Algae and Protozoa, Scotland, UK).

(7) Preculture: 150 mL of medium in a 500 mL Erlenmeyer flask; Composition of the medium: 40 g/L of glucose+10 g/L of yeast extract.

(8) Incubation is performed under the following conditions: time: 72 h; temperature: 28° C.; shaking: 110 rpm (Infors Multitron Incubator).

(9) Culturing in batch and then fed-batch mode

(10) Preparation and Initial Batch Medium prepare and filter a mixture of KOH at 400 g/l (41%)/NH3 at 20% v/v (59%); sterilize 20 L fermenter at 121° C./20 min; inoculate with 2 conical flasks of 500 mL of preculture (OD.sub.600 nm of 15); regulation of the pH at 5.2 with the KOH/NH.sub.3 mixture; starting shaking speed of 300 rpm; aeration: 15 L/min of air; pO.sub.2 regulation at 30% by modifying the shaking; temperature: 28° C.

(11) Feeding glucose: 500 g/L ammonium sulfate: 25 g/L monobasic sodium phosphate: 17 g/L monobasic potassium phosphate: 23 g/L magnesium sulfate heptahydrate: 20 g/L iron sulfate: 120 mg/L calcium nitrate: 610 mg/L solution of trace elements: 45 mL/L solution of vitamins: 3.6 mL/L

(12) TABLE-US-00001 solution of trace elements (for 2 liters) Ingredients (g) CuSO.sub.4•5H.sub.2O 0.22 ZnSO.sub.4•7H.sub.2O 28 MnSO.sub.4•1H.sub.2O 16 FeSO.sub.4•7H.sub.2O 2.2 Citric acid 60 H.sub.2O qs 2

(13) TABLE-US-00002 Solution of vitamins Ingredients (g/l) Thiamine HCl 13.5 Biotin 0.7 Pyridoxine 6.75

(14) Fermentation Procedure provide the equivalent of 20 g/L of glucose before inoculation when the glucose concentration=0 g/L, start feeding in glucose in fed-batch mode; use a flow rate that makes it possible to set the growth rate at 0.08 h.sup.−1 regulate to pH 5.2 with the 41% KOH/59% NH.sub.3 mixture when 2 kg of glucose have been consumed by the microalga, switch the system to pH regulation with NH.sub.3 alone, when the biomass reaches 100 g/L by weight of dry matter, and about 3.5 kg of glucose have been fed in, the glucose feeding is stopped.

(15) Results:

(16) Two tests were performed under these same conditions and the results are given in table I and in the following graphs:

(17) TABLE-US-00003 TABLE I Test 1 Test 2 F2 140519 F5 140623 Final titer (%) Final titer (%) (for 3.6 kg of (for 3.4 kg of glucose consumed) glucose consumed) N.6.25 66.0 65.7 Total amino acids 44.7 43.2 Content of Arg and Glu 46 47 relative to the total amino acids Total fatty acids 10.2 10.1 Total sugars 20.3 21.7 Color of the biomass Yellow Yellow

(18) FIG. 1 illustrates the change in the N.6.25 as a function of the glucose consumed. These two tests reflect some noteworthy results: the production of a yellow biomass with an N.6.25 content of more than 65%.

(19) FIG. 2 illustrates the change in the specific rate of nitrogen consumption (qN) as a function of the glucose consumed.

(20) It is seen that the specific rate of nitrogen consumption is at its maximum after the nitrogen limitation has been lifted (at 2 kg of glucose consumed), and then decreases gradually. The similar rates between the two tests also reflect the good repeatability of the protocol.

(21) Full analysis of the amino acids present in the biomass was performed on a sample taken just before lifting the limitation, and on several samples after the pulse.

(22) The results are shown in FIG. 3.

(23) It is noted that just before lifting the nitrogen limitation, the sum of the amino acids is low (16.3%) and there is no predominance among the various amino acids.

(24) One hour after lifting the nitrogen limitation, it is noted that the amino acid which undergoes the greatest increase is glutamic acid, followed by arginine. The content of the other amino acids also increases, but to a much lower extent.

(25) The increase in the N.6.25 is thus above all correlated with the increase in glutamic acid and arginine.

(26) In addition to these analyses, full analysis of the fatty acids present in the biomass was performed on a sample taken just before lifting the nitrogen limitation and on several samples after the pulse.

(27) The results are shown in FIG. 4.

(28) The total content of fatty acids in the biomass, which is 19.2% before the pulse, falls to 10.2%. The predominant fatty acid which follows this curve is oleic acid.

(29) The fatty acids thus accumulate in the biomass when it is deficient in nitrogen.

(30) Full analysis of the sugars present in the biomass was also performed on a sample taken just before lifting the nitrogen limitation and on several samples after lifting the nitrogen limitation. The results are shown in FIG. 5.

(31) The total content of sugars in the biomass, which is 37.5% before lifting the nitrogen limitation, falls to 20% and then stagnates. The predominant sugar which follows this curve is glucose.

(32) The sugars are thus also stored in the biomass when it is deficient in nitrogen.

(33) The content of sugars then appears to stabilize, unlike the fatty acid content which continues to decrease.

(34) The salt content of the biomass was measured by measuring the calcination residue: it is 9%.