METHOD FOR THE PROTEIN ENRICHMENT OF MICROALGAL BIOMASS

Abstract

The invention relates to a method for the protein enrichment of a microalga of the genus Chlorella, cultivated under heterotrophic conditions. The method is characterized in that the heterotrophic cultivation comprises a step intended to slow down the growth of the microalga, with the fermentation medium being deficient in a nitrogen-free nutritional source.

Claims

1.-15. (canceled)

16. A process for the protein enrichment of a microalga cultured heterotrophically in a fermentation medium, the microalga being of the genus Chlorella, wherein the heterotrophic culturing comprises a step of limiting a non-nitrogen nutrient in the fermentation medium, and wherein the protein content of the microalga is more than 50% by weight.

17. The process according to claim 16, wherein the non-nitrogen nutrient is a nutrient selected from the group consisting of glucose and phosphate.

18. The process according to claim 16, wherein the microalga pf the genus Chlorella is chosen from Chlorella sorokiniana or Chlorella protothecoides.

19. The process according to claim 16, wherein the heterotrophic culturing of the microalgae of the species comprises: a) a first step of growth of the microalgae, in which the glucose supply is not limiting; and b) a second step wherein the glucose supply is limited, wherein the limited glucose supply reduces the additional accumulation of storage substances.

20. The process according to claim 16, wherein the heterotrophic culturing of the microalgae of the species Chlorella protothecoides comprises a step of culturing with limiting a phosphate nutrient, wherein the growth rate is reduced and an increase in the protein content is obtained.

21. The process according to claim 20, wherein the heterotrophic culturing of the microalgae of the species Chlorella protothecoides comprises a step of culturing with limiting a phosphate nutrient, wherein the growth rate is reduced and an increase in the protein content is obtained, and further wherein the combined amount of glutamic acid and arginine together in the protein is more than 40%.

22. The process according to claim 16, wherein the fermentation temperature is modified to reduce the growth rate of the microalga.

23. The process according to claim 22, wherein the fermentation temperature is increased by about 1-5 C. relative to the optimum fermentation temperature.

24. The process according to claim 23, wherein the fermentation temperature is increased by about 3 C. relative to the optimum fermentation temperature.

25. The process according to claim 23, wherein the fermentation temperature increase leads to increasing the cooling capacity of the heat exchanger of the fermenter.

26. The process according to claim 16, wherein the fermentation temperature is about 31 C.

27. A process for the protein enrichment of a microalga cultured heterotrophically in a fermentation medium, wherein the heterotrophic culturing comprises a step directed towards slowing down the growth of said microalga by increasing the fermentation temperature relative to the optimum fermentation temperature, and limiting a non-nitrogen nutrient in the fermentation medium, and wherein the protein content of the microalga is more than 50% by weight.

28. The process according to claim 27, wherein the increased fermentation temperature is from 28 C. to 31 C.

29. The process according to claim 27, wherein the microalga is of the species Chlorella protothecoides.

30. A process for enriching the glutamic acid and/or arginine content of a heterotrophically cultivated microalga, the process comprising heterotrophic cultivation of the microalga in a fermentation medium, the fermentation medium comprising a limiting amount of a phosphate nutrient, and wherein the combined glutamic acid and arginine content of the protein content of the microalga is more than 40%.

31. The process a according to claim 30, wherein the microalga are of the species Chlorella protothecoides.

32. A microalgal biomass obtained via the process of claim 30.

Description

EXAMPLES

Example 1: Production of Chlorella sorokiniana in Fermentation of Sequential Batch Type without Limitation of the Supply of Nutrient Medium

[0116] The strain used is a Chlorella sorokiniana (strain UTEX 1663The Culture Collection of Algae at the University of Texas at Austin-USA).

[0117] Preculture: [0118] 600 ml of medium in a 2 L conical flask; [0119] Composition of the medium (table 1 below).

TABLE-US-00001 TABLE 1 Macro Glucose 20 elements K.sub.2HPO.sub.43H.sub.2O 0.7 (g/l) MgSO.sub.47H.sub.2O 0.34 Citric acid 1.0 Urea 1.08 Na.sub.2SO.sub.4 0.2 Na.sub.2CO.sub.3 0.1 clerol FBA 3107 (antifoam) 0.5 Micro Na.sub.2EDTA 10 elements CaCl.sub.22H.sub.2O 80 (mg/l) FeSO.sub.47H.sub.2O 40 MnSO.sub.44H.sub.2O 0.41 CoSO.sub.47H.sub.2O 0.24 CuSO.sub.45H.sub.2O 0.24 ZnSO.sub.47H.sub.2O 0.5 H.sub.3BO.sub.3 0.11 (NH.sub.4).sub.6Mo.sub.7O.sub.274H.sub.2O 0.04

[0120] The pH is adjusted to 7 before sterilization by addition of 8N NaOH.

[0121] Incubation is performed under the following conditions: [0122] duration: 72 h; [0123] temperature: 28 C.; [0124] shaking: 110 rpm (Infors Multitron incubator).

[0125] The preculture is then transferred to a 30 L Sartorius type fermenter.

[0126] Culture for Biomass Production:

[0127] The medium is identical to that of the preculture, but the urea is replaced with NH.sub.4Cl.

TABLE-US-00002 TABLE 2 Macro Glucose 20 elements K.sub.2HPO.sub.43H.sub.2O 0.7 (g/l) MgSO.sub.47H.sub.2O 0.34 Citric acid 1.0 NH.sub.4Cl 1.88 Na.sub.2SO.sub.4 0.2 clerol FBA 3107 (antifoam) 0.5 Micro Na.sub.2EDTA 10 elements CaCl.sub.2 80 (mg/l) FeSO.sub.47H.sub.2O 40 MnSO.sub.44H.sub.2O 0.41 CoSO.sub.47H.sub.2O 0.24 CuSO.sub.45H.sub.2O 0.24 ZnSO.sub.47H.sub.2O 0.5 H.sub.3BO.sub.3 0.11 (NH.sub.4).sub.6Mo.sub.7O.sub.274H.sub.2O 0.04

[0128] The initial volume (Vi) of the fermenter is adjusted to 13.5 L after inoculation.

[0129] It is finally brought to 16-20 L.

[0130] The parameters for performing the fermentation are as follows:

TABLE-US-00003 TABLE 3 Temperature 28 C. pH 5.0-5.2 by 28% w/w NH.sub.3 pO.sub.2 >20% (maintained by shaking) Shaking Minimum 300 rpm Air flow rate 15 l/min

[0131] When the glucose initially supplied is consumed, medium identical to the initial medium, without the antifoam, is supplied in the form of a concentrated solution containing 500 g/L of glucose and the other elements in the same proportions relative to the glucose as in the initial medium, so as to obtain a glucose content of 20 g/L in the fermenter.

[0132] Two other identical additions are performed in the same manner each time that the residual glucose concentration becomes zero.

[0133] Clerol FBA 3107 antifoam is added as required to avoid excessive foaming.

[0134] Results:

[0135] After 46 hours of culturing, 38 g/L of biomass with a protein content (evaluated by the N 6.25) of 36.2% are obtained.

Example 2: Production of C. sorokiniana in Fermentation of Fed-Batch Type with Limiting Glucose Supply

[0136] In this example, a supply of whole medium (fed-batch mode) is started after consumption of the glucose initially supplied. The other parameters for performing the fermentation are unchanged.

[0137] Glucose is supplied continuously using a 500 g/L concentrated solution. The supply rate is less than the consumption rate that the strain might achieve, such that the residual glucose content in the medium is kept at zero, i.e. the growth of the strain is limited by the glucose availability (glucose-limiting condition).

[0138] This rate is increased exponentially over time. The formula for calculating the supply rate is characterized by a factor which corresponds to the growth rate that the strain can adopt if it consumes all of the glucose supplied:

S=Soexp (.t)

[0139] S=glucose supply rate (in g/h)

[0140] So=initial glucose supply rate, determined as a function of the biomass present at the end of the batch. It is 12 g/h under our conditions.

[0141] =rate acceleration factor. It should be less than 0.11 h.sup.1, which is the growth rate of the strain in the absence of nutritional limitation.

[0142] t=fed-batch time (in h)

[0143] The salts are supplied if possible continuously, separately or mixed with the glucose. However, they may also be supplied sequentially in several portions.

[0144] Table 4 below gives the salt needs per 100 g of glucose.

TABLE-US-00004 TABLE 4 Macro Glucose 100 elements K.sub.2HPO.sub.43H.sub.2O 6.75 (g) MgSO.sub.47H.sub.2O 1.7 Citric acid 5.0 Na.sub.2SO.sub.4 1.0 Micro Na.sub.2EDTA 50 elements CaCl.sub.22H.sub.2O 400 (mg) FeSO.sub.47H.sub.2O 200 MnSO.sub.44H.sub.2O 2.1 CoSO.sub.47H.sub.2O 1.2 CuSO.sub.45H.sub.2O 1.2 ZnSO.sub.47H.sub.2O 2.5 H.sub.3BO.sub.3 0.6 (NH.sub.4).sub.6Mo.sub.7O.sub.274H.sub.2O 0.2

[0145] The concentrations of the elements other than the glucose were determined so that they were in excess relative to the nutritional requirements of the strain.

[0146] Clerol FBA 3107 antifoam is added as required to avoid excessive foaming.

[0147] Results: Effect of the Glucose Supply Rate in the Fed-Batch Mode

[0148] Tests were performed at various glucose supply rates in fed-batch mode. They are characterized by the applied. The protein content of the biomass obtained is evaluated by measuring the total nitrogen expressed by N 6.25.

TABLE-US-00005 TABLE 5 fed Duration Biomass Productivity % N Test (h.sup.1) (h) (g/l) (g/L/h) 6.25 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

[0149] These results show that working under glucose-limiting conditions makes it possible to increase the protein content.

[0150] Specifically, it is observed that, even with a high of 0.09, a protein content higher than that obtained without limitation as in Example 1 (39.3% instead of 36.2%) is obtained.

[0151] Increasing the limitation of the metabolism with glucose results in an additional improvement in the protein content.

[0152] Under the conditions of these tests, it is necessary to impose on the strain a of less than 0.06 h.sup.1 to obtain a protein content of greater than 50%.

[0153] It should be noted that this condition goes hand in hand with a reduction of the productivity: 0.56 g/L/h 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

[0154] In this example, the fermenter used is a 2L Sartorius Biostat B fermenter.

[0155] The fermentation is performed as in Example 2, but with ten times smaller volumes: the inoculum is 60 ml and the initial volume is 1.35 L.

[0156] Continuous supply of the medium is started according to the same principle as in Example 2, the salts in this case being mixed with the glucose in the feed manifold. The supply rate is accelerated according to the same exponential formula as in Example 2 by applying a p of 0.06 h.sup.1.

[0157] Chemostat

[0158] When a volume of 1.6 L is reached, i.e. a biomasses concentration of about 50 g/L, continuous functioning of chemostat type is implemented: [0159] 1. The fermenter is fed continuously at a rate of 96 ml/h with a nutrient medium solution containing 100 g/L of glucose, having the following composition:

TABLE-US-00006 TABLE 6 Macro Glucose 100 elements K.sub.2HPO.sub.43H.sub.2O 6.75 (g/l) MgSO.sub.47H.sub.2O 1.7 Citric acid 5.0 Na.sub.2SO.sub.4 1.0 Micro Na.sub.2EDTA 50 elements CaCl.sub.22H.sub.2O 400 (mg/l) FeSO.sub.47H.sub.2O 200 MnSO.sub.44H.sub.2O 2.1 CoSO.sub.47H.sub.2O 1.2 CuSO.sub.45H.sub.2O 1.2 ZnSO.sub.47H.sub.2O 2.5 H.sub.3BO.sub.3 0.6 (NH.sub.4).sub.6Mo.sub.7O.sub.274H.sub.2O 0.2 [0160] The concentrations of the elements other than the glucose were determined so that they were in excess relative to the nutritional requirements of the strain. [0161] 2. Medium is withdrawn continuously from the fermenter via a dip tube connected to a pump so as to keep the volume of the culture at 1.6 L.

[0162] Thus, the medium is renewed with a 0.06 (6%) fraction per hour. This renewal rate is referred to as the dilution rate (D).

[0163] In accordance with the principle of chemostat culturing, the growth rate of the strain () becomes established at the same value since the growth of the strain is limited by the glucose supply:

D==0.06 h.sup.1

[0164] Results

[0165] After 97 hours of chemostat functioning, the biomasses concentration becomes established at 48 g/L2 g/L and the protein content at 532%.

[0166] This mode of functioning makes it possible to obtain biomass with a high protein content by means of glucose limitation and the low growth rate imposed, while at the same time ensuring very good productivity, of the order of 2.9 g/L/h, by means of the high concentration of biomass.

Example 4: Production of Chlorella protothecoides in Fermentation of Batch Type with or without Limitation of the Phosphate Supply

[0167] The strain used is a Chlorella protothecoides (strain CCAP211/8DThe Culture Collection of Algae and Protozoa, Scotland, UK).

[0168] Preculture: [0169] 150 ml of medium in a 500 L conical flask; [0170] Composition of the medium: 40 g/L of glucose+10 g/l of yeast extract.

[0171] Incubation is carried out under the following conditions: duration: 72 h; temperature: 28 C.; stirring: 110 rpm (Infors Multitron incubator).

[0172] The preculture is then transferred into a 2 L Sartorius Biostat B fermenter.

[0173] Culture for Biomass Production:

[0174] The composition of the culture medium is as follows (in g/L):

TABLE-US-00007 TABLE 7 Glucose 80 Citric acid 4 NH.sub.4Cl 2 KH.sub.2PO.sub.4 3 (test 1)/2 (test 2)/1 (test 3) Na.sub.2HPO.sub.4 3 (test 1)/2 (test 2)/1 (test 3) MgSO.sub.47H.sub.2O 1.5 NaCl 0.5 Yeast extract 5

[0175] The phosphate supply is calculated so that it is in excess in test 1 and limiting for tests 2 and 3.

[0176] Clerol FBA 3107 antifoam is added as required to avoid excessive foaming.

[0177] The initial volume (Vi) of the fermenter is adjusted to 1 L after inoculation.

[0178] The parameters for performing the fermentation are as follows:

TABLE-US-00008 TABLE 8 Temperature 28 C. pH 6.5 by NH.sub.3 28 ww % pO.sub.2 >20% (maintained by shaking) Shaking Minimum 200 rpm Air flow rate 1 L/min

[0179] Results:

TABLE-US-00009 TABLE 9 Residual Duration Biomass Cumulative PO.sub.4 % N Test (h) (g/l) (h.sup.1) (mg/L) 6.25 1 36 38.1 0.09 800 48.1 2 45 36.5 0.07 0 56.1 3 54 36 0.05 0 61.2

[0180] The cumulative value corresponds to the growth rate of the biomass from inoculation.

[0181] The protein content is estimated by measuring the nitrogen content N6.25

[0182] These results show that a limitation of the phosphate supply, confirmed by the absence of residual phosphate at the end of fermentation, limits the growth speed (measured by the growth rate) and, as for the glucose limitation in the preceding examples, results in an increase in the protein content to reach values markedly greater than 50%.

Example 5: Production of Chlorella protothecoides at 28 C. in Fermentation of Fed-Batch Type Using a Phosphate-Limited Synthetic Medium

[0183] To obtain a high biomasses concentration, glucose is supplied during culturing (fed-batch) to avoid growth inhibition by glucose.

[0184] The supplies of salts, in particular phosphate, are conventionally performed at the start of fermentation (batch mode).

[0185] The culture medium is free of yeast extract.

[0186] As in Example 4, the strain used is a Chlorella protothecoides (strain CCAP211/8DThe Culture Collection of Algae and Protozoa, Scotland, UK).

[0187] Preculture: [0188] 500 ml of medium in a 2 L conical flask; [0189] Composition of the medium:

TABLE-US-00010 TABLE 10 Macro Glucose 40 elements (g/l) K.sub.2HPO.sub.4 3 Na.sub.2HPO.sub.4 3 MgSO.sub.47H.sub.2O 0.25 (NH.sub.4).sub.2SO.sub.4 1 Citric acid 1 clerol FBA 3107 (antifoam) 0.1 Microelements CaCl.sub.22H.sub.2O 30 and Vitamins FeSO.sub.47H.sub.2O 1 (mg/l) MnSO.sub.41H.sub.2O 8 CoSO.sub.47H.sub.2O 0.1 CuSO.sub.45H.sub.2O 0.2 ZnSO.sub.47H.sub.2O 0.5 H.sub.3BO.sub.3 0.1 Na.sub.2MoO.sub.42H.sub.2O 0.4 Thiamine HCl 1 Biotin 0.015 B12 0.01 Calcium pantothenate 0.03 p-Aminobenzoic acid 0.06

[0190] Incubation is performed under the following conditions: [0191] duration: 72 h; [0192] temperature: 28 C.; [0193] shaking: 110 rpm (Infors Multitron incubator).

[0194] The preculture is then transferred to a 30 L Sartorius type fermenter.

[0195] Culture for Biomass Production:

[0196] The medium is as follows:

TABLE-US-00011 TABLE 11 Macro Glucose 40 elements (g/l) KH.sub.2PO.sub.4 1.8 NaH.sub.2PO.sub.4 1.4 MgSO.sub.47H.sub.2O 3.4 (NH.sub.4).sub.2SO.sub.4 0.2 clerol FBA 3107 (antifoam) 0.3 Microelements CaCl.sub.22H.sub.2O 40 and Vitamins FeSO.sub.47H.sub.2O 12 (mg/l) MnSO.sub.41H.sub.2O 40 CoSO.sub.47H.sub.2O 0.1 CuSO.sub.45H.sub.2O 0.5 ZnSO.sub.47H.sub.2O 50 H.sub.3BO.sub.3 15 Na.sub.2MoO.sub.42H.sub.2O 2 Thiamine HCl 6 Biotin 0.1 B12 0.06 Calcium pantothenate 0.2 p-Aminobenzoic acid 0.2

[0197] The initial volume (Vi) of the fermenter is adjusted to 17 L after inoculation.

[0198] It is brought to a final volume of about 20-25 L.

[0199] The parameters for performing the fermentation are as follows:

TABLE-US-00012 TABLE 12 Temperature 28 C. pH 5.0-5.2 by 28% w/w NH.sub.3 pO.sub.2 20% 5% (maintained by shaking) Shaking Minimum 300 rpm Air flow rate 15 L/min

[0200] When the residual glucose concentration falls below 10 g/l, glucose in the form of a concentrated solution at approximately 800 g/l is introduced so as to maintain the glucose content between 0 and 20 g/l in the fermenter.

[0201] Results

[0202] The results are given as a function of time and of the C/P ratio which represents the amount of carbon consumed (originating from glucose) relative to the amount of phosphorus supplied (via phosphate).

TABLE-US-00013 TABLE 13 Duration C/P Residual Biomass Cumulative % N (h) (g/g) PO.sub.4 (g/L) (g/l) (h.sup.1) 6.25 0.0 2.25 3.0 13.0 13.0 1.43 15.5 0.126 20.0 25.0 0.65 28.5 0.113 35.9 28.0 70.0 0.00 54.3 0.103 38.4 36.0 120.0 0.00 80.0 0.091 56.5 40.0 145.0 0.00 89.5 0.085 68.5

[0203] These results show that the protein content increases markedly at the end of fermentation from the moment when all the phosphate supplied is consumed and the C/P exceeds a value of 60.

[0204] As regards the energy, the cooling capacity of the exchanger of this fermenter, fed with water at 25 C., is at 1.7 kW/m.sup.2 of exchange.

Example 6: Production of Chlorella protothecoides at 31 C. in Fermentation of Fed-Batch Type Using a Phosphate-Limited Synthetic Medium

[0205] This test is performed under the same conditions as in the preceding example, except for the temperature, which is raised to 31 C. (instead of 28 C.).

[0206] Results

[0207] The results are given as a function of time and of the C/P ratio which represents the amount of carbon consumed (originating from glucose) relative to the amount of phosphorus supplied (via phosphate).

[0208] The cumulative corresponds to the growth rate of the biomass from inoculation.

[0209] The protein content is estimated by measuring the nitrogen content N6.25.

TABLE-US-00014 TABLE 14 Duration C/P Residual Biomass Cumulative % N (h) (g/g) PO.sub.4 (g/L) (g/L) (h.sup.1) 6.25 0.0 2.25 3 13.0 11.2 1.54 13.5 0.102 42.9 20.0 23.7 0.83 26.7 0.098 36.6 28.0 60.1 0.00 51.7 0.101 35.1 36.0 99.3 0.00 70.2 0.09 51.2 40.0 121.6 0.00 79.8 0.085 58.9 44.0 143.9 0.00 88.8 0.079 71.9

[0210] These results first confirm those obtained at 28 C. Furthermore, increasing the temperature to 31 C. makes it possible to further reduce the growth rate and to increase the protein content (by about 5%).

[0211] Moreover, increasing the temperature to 31 C. instead of 28 C. makes it possible to very markedly improve the efficiency of the exchanger of the fermenter since this increases the temperature difference between the water feeding the exchanger and the fermentation must: the cooling capacity of the exchanger is raised to 3.5 kW/m.sup.2 instead of 1.7 kW/m.sup.2.

Example 7: Amino Acid Composition of the Biomass of Chlorella protothecoides Produced Under Phosphate-Deficient Conditions

[0212] The total amino acid composition of the microalgal biomasses produced according to the method detailed in standard ISO 13903: 2005 is determined.

[0213] The following biomasses are analyzed:

[0214] Batch (A): biomass of Chlorella protothecoides produced according to the conditions of Example 6, and having a protein content of between 60% and 70% (expressed as N625).

[0215] Batch (B): biomass of Chlorella protothecoides produced according to the conditions of Example 5, and having a protein content of between 45% and 60% (expressed as N625).

[0216] Batch (C): biomass of Chlorella protothecoides prepared according to test 1 of Example 4, and having a protein content of between 45% and 50% (expressed as N625).

[0217] Batch (D): biomass of Chlorella sorokiniana produced according to the conditions of Example 3, and having a protein content of between 50% and 60% (expressed as N625).

[0218] Table 15 below has the total amino acid composition of the biomass, expressed in relative percentages.

TABLE-US-00015 TABLE 15 Batch A Batch B Batch C Batch D relative % relative % relative % relative % Aspartic acid 3.7 6.4 7.9 9.0 Threonine 1.9 4.0 4.8 4.7 Serine 1.9 3.5 4.5 4.0 Glutamic acid 36.3 22.3 15.3 11.5 Glycine 2.2 4.0 5.2 5.9 Alanine 4.1 6.4 8.0 8.6 Valine 2.4 4.7 6.0 5.9 Isoleucine 1.4 2.8 3.6 3.8 Leucine 3.4 6.6 8.2 8.7 Tyrosine 1.7 2.6 3.3 3.9 Phenylalanine 1.8 3.3 4.0 4.7 Lysine 2.5 4.4 5.3 8.8 Histidine 0.9 1.7 2.1 2.3 Arginine 31.4 20.1 12.8 6.8 Proline 1.8 3.4 4.7 5.2 Cystine 0.8 0.9 1.0 1.6 Methionine 0.9 1.5 1.7 2.2 Tryptophan 0.9 1.3 1.5 2.3 TOTAL 100.0 100.0 100.0 100.0

[0219] The results obtained clearly show that, on the total amino acid composition of the Chlorella protothecoides biomasses, more than 40% (in relative terms) of the amino acids are glutamic acid and arginine if Chlorella protothecoides is cultured under conditions that enrich its protein content (not more than 30% under standard culture conditionscf. Batch C).

[0220] Moreover, this result is obtained only for Chlorella protothecoides, which tends to show the particular metabolic features of this microalga, with regard to Chlorella sorokiniana. Specifically, although having a high protein content, C. sorokinianaBatch Ddoes not produce more than 20% of glutamic acid and arginine.