KR20150139509A - Microalgal biomass protein enrichment method - Google Patents

Abstract

The present invention relates to a method for increasing the protein content of microalgae of the genus Chlorella grown under heterotrophic conditions which aims at limiting the growth of the microalgae by deficient fermentation medium in terms of non-nitrogen source of nutrients The method comprising the steps of:

Description

MICROALGAL BIOMASS PROTEIN ENRICHMENT METHOD [0002]

The invention microalgae, more specifically, Chlorella (Chlorella) in the microalgae, more specifically, the chlorella Thoreau Kearney Ana (Chlorella sorokiniana) Chlorella species or prototype Te Koi Death (Chlorella protothecoides species microalgae.

Algae (large algae and microalgae) are characteristic but not yet sufficiently studied. Algae is rarely used in food, chemicals and bio-energy production. However, birds hide significant value components, and the abundance and richness of these components can only be recognized by marine animals that live on these birds.

In fact, microalgae are a source of vitamins, lipids, proteins, sugars, pigments and antioxidants.

Therefore, algae and microalgae are of interest in the industry or in agriculture where they are used to produce dietary supplements, functional foods, cosmetics or pharmaceuticals.

In particular, all microalgae are photosynthetic microorganisms that live in large quantities in all biological habitats exposed to light.

Monoclonal culturing of microalgae is performed on an industrial scale in a photobioreactor (independent nutrient conditions; light and CO ₂ present) or (in some cases) fermentors (heterotrophic conditions; cancerous environment and presence of carbon sources) .

In fact, a few species of microalgae, such as Chlorella , Nitzschia , Cyclotella , Tetraselmis , Crypthecodinium , Schizochytrium , Lt; / RTI > can be grown under non-existent conditions.

In addition, heterotrophic conditions may be established by those skilled in the art:

- using a fermenter identical to that used for bacteria and yeast, with all of the culture parameters being controllable; And

- to produce biomass in a much larger amount than the amount of biomass obtained by light-based cultivation

, It is estimated that the cost of culturing under heterotrophic conditions is one tenth of the cost of culturing under photoperiod conditions.

For the beneficial use of microalgae, generally the components of interest, for example,

- Chlorophyll a, b and c, β-carotene, astaxanthin, lutein, phycocyanin, xanthophyll, picoeles, Trin, etc.);

- proteins to optimize nutritional quality; or

- Especially

· Not only to be used for biofuel,

To be used as food or feed for humans or animals when the selected microalgae produce polyunsaturated fatty acids, ie PUFAs, which are so-called "essential" (ie, provided as diets because they are not produced naturally by humans or animals)

To optimize the fatty acid content (to make less than 60%, even less than 80%, based on the building weight of microalgae)

It is necessary to control the fermentation conditions so that the accumulation of the fermentation conditions can be achieved.

Therefore, in order to achieve these results, first, a high cell density (HCD) fermentation method was thoroughly studied to obtain maximum yield and production of protein or lipid.

The purpose of this HCD incubation was to obtain the desired product at the highest concentration possible in the shortest time possible.

These teachings may be, for example Chlorella pinji crude N-Sys (Chlorella zofingiensis ), that is, the growth of the microalgae has been shown to be directly correlated with the production of astaxanthin (Wang and Peng, 2008, World J Microbiol . Biotechnol . , 24 (9), 1915-1922).

However, the fact that the growth is kept at the maximum rate (μ / h) is not always correlated with the high productivity of the desired product.

In fact, it became clear to the experts in the field that microalgae require nutrient stress to limit the growth of microalgae, for example, to make them store large amounts of lipid.

At present, growth and production are carried out separately in fermentation methods.

For example, in order to promote the accumulation of polyunsaturated fatty acids (here, docosahexaenoic acid, i.e. DHEA), patent application WO 01/54510 advises to separate cell growth and production of polyunsaturated fatty acids.

Thus, in the case of the microalgae Schizochytrium sp. Strain ATCC 20888, it is performed to promote high cell density (greater than 100 g / l) in the first growth stage proceeding without oxygen limitation; Then, in the second step, the oxygen supply is progressively reduced so that the microalgae are stressed and the growth of the microalgae is delayed to start production of the fatty acids of interest.

Microalgae Crypthecodinium cohnii) In, it is a low glucose concentration (about 5 g / ℓ), and hence at a low growth rate in accordance with, the highest docosahexaenoic acid (DHEA, polyunsaturated fatty acids) content obtained (Jiang and Chen, 2000, Process Biochem ., 35 (10), 1205-1209).

These results clearly demonstrate that the rate of product formation may be positively or negatively associated with microalgae growth, and that even speed and microalgae growth can be expressed in combination.

As a result, it is wise to control the cell growth rate if product formation is not correlated with high cell growth.

Generally, one of ordinary skill in the art will choose a method to control microbial growth by controlling fermentation conditions (Tp, pH, etc.) or by controlling the feeding of nutrients to the fermentation medium (semi-continuous conditions, so- .

If one of ordinary skill in the art would choose to control microalgae growth under heterotrophic conditions by the supply of carbon sources, those skilled in the art will generally recognize that microalgae (e. G., DHEA type polyunsaturated fatty acids) (Mr. Connie, Euglena gracilis , etc.) is adapted to a carbon source (pure glucose, acetate, ethanol, etc.).

Temperature can also be an important parameter,

- for example, the synthesis of polyunsaturated fatty acids by specific microalgae species, for example Chlorella minutissima ) is promoted at a temperature lower than the temperature required for optimal growth of the microalgae;

On the contrary, the yield of lutein by chlorella protocheids, grown under heterotrophic conditions, is higher when the production temperature is raised from 24 ° C to 35 ° C.

Chlorella protocheids is legitimately recognized as one of the finest oil producing microalgae.

Under heterotrophic conditions, microalgae rapidly convert carbohydrates to triglycerides (greater than 50% of microalgae dry matter).

To optimize such triglyceride production, one skilled in the art will optimize the carbon stream in the direction of oil production by acting on the nutrient environment of the fermentation medium.

Therefore, oil is known to accumulate when a sufficient amount of carbon is supplied, but under nitrogen-deficient conditions.

In such cases, the C / N ratio plays a decisive role, so that the best results are obtained by acting directly on the nitrogen content without limiting the glucose content.

It is not surprising that this nitrogen deficiency affects cell growth and thus the growth rate is 30% lower than that of normal microalgae (Xiong et al. , Plant Physiology , 2010, 154, pp. 1001-1011) .

To illustrate these results, Xiong et al. In the above article show that if chlorella biomass is divided into its five major components, carbohydrate, lipid, protein, DNA and RNA (which account for 85% of biomass dry matter) If the C / N ratio does not affect the DNA, RNA and carbohydrate content, then in fact the C / N ratio indicates that it is significant for protein and lipid content.

Therefore, chlorella cells grown to lower the C / N ratio contain 25.8% protein and 25.23% lipid, while the chlorella cells grown to have a high C / N ratio have 53.8% lipid and 10.5% Can be synthesized.

Therefore, in order to optimize oil production, a person skilled in the art must control the carbon flow by turning the flow of carbon in the direction of oil production in case of loss of protein production; This redistributes the carbon flow and accumulates lipid storage materials when microalgae are present in the nitrogen deficient medium.

Therefore, when these teachings are considered, one of ordinary skill in the art will recognize that the opposite task of metabolic control to produce protein-

Feeding a large amount of nitrogen source to the fermentation medium while maintaining a constant supply of carbon source to be converted to protein;

- The growth of microalgae should be stimulated

To perform fermentation conditions that promote the achievement of a low C / N ratio.

It is important to change the carbon flow in the direction of protein production (and thus biomass production) in case of production loss of storage lipids.

In the context of the present invention, the Applicants have chosen to study the novel pathway by disclosing an alternative solution to the method traditionally addressed by those skilled in the art.

Therefore, the present invention relates to a method for increasing the protein content of microalgae grown under heterotrophic conditions, that is, microalgae of the genus Chlorella, more particularly microalgae of chlorella norota or chlorella protothecidis, Involves targeting a non-nitrogen nutrient source to the fermentation medium to defeat the growth of the micro-algae.

This step is a heterotrophic culture step in which the non-nitrogen nutrient factor is fed to the medium in an amount not sufficient for the microalgae to grow. It should be noted that " insufficient amount " does not mean that these nutrients are not supplied. This nutritional deficiency stage is a slowing phase (limiting) of cell metabolism without completely inhibiting cell metabolism.

In the meaning of the present invention, " increased amount " means increasing the protein content in the biomass by at least 15 wt%, preferably by at least 20 wt%, so that the protein content in the biomass reaches 50 wt% or more.

The present invention more specifically includes a method for heterotrophic culture of microalgae, comprising the step of targeting the growth of microalgae by deficient in a non-nitrogen source of nitrogen in the fermentation medium.

The present invention therefore relates to a method of increasing the protein content of microalgae grown under heterotrophic conditions, said microalgae being microalgae of the genus Chlorella, more particularly chlorella norota or chlorella protothecidis, And targeting the nutrient source to the fermentation medium to limit the growth of the microalgae so that the protein content in the biomass can reach over 50% by weight.

By "deficient in non-nitrogen nutrient sources in the fermentation medium" is meant that one or more of the non-nitrogen nutrients in the nutrient is supplied to the microalgae in an amount not sufficient for the microalgae to grow.

Specifically, the nutrient source is deficient to achieve a growth rate of 10% to 60% lower than the growth rate when the nutrient source is not restricted. In particular, the growth rate can be increased by 10% to 60%, in particular by 10%, 15%, 20%, 25%, or 20%, compared to the growth rate when the glucose is not restricted, 30%, 35%, 40%, 45%, 50%, or 55%. Preferably, the growth rate is reduced by 15% to 55%.

The duration of the culturing step, which includes deprivation of nutrients, particularly glucose, is at least 1 hour, preferably at least 10 hours, more preferably at least 20 hours, especially from 30 to 60 hours.

As a result, the remaining non-nitrogen nutrients in the culture medium are not present, and microalgae consume nutritive factors almost simultaneously with these nutrients. However, the absence of residual non-nitrogen nutrients in the culture medium is distinguished from the situation when nutrients are not fully supplied to microalgae.

An essential measure in the context of the present invention is stress, a limitation of cell growth by cell stress caused by deficiency of non-nitrogen nutrients in the fermentation medium.

Therefore, this strategy breaks the technological bias that increasing biomass and increasing cell growth are necessary to increase protein content in biomass.

By " non-nitrogen nutrient source " is meant a nutrient selected from the group consisting of, for example, glucose and phosphate.

As will be exemplified below, advantageously

Limiting the growth of chlorella norovirus by deficiency of glucose in the fermentation medium; And

Limiting the growth of chlorella protothecoides by deficiency of phosphate in the fermentation medium

Can be selected.

Tukhi, according to the above strain, nutrients are supplied so as to obtain a growth rate of 0.06 h ^-1 to 0.09 h ^-1.

In a very specific embodiment, the Chlorella thalliokina strain is strain UTEX 1663 (University of Texas Algae Culture Collection, Austin, USA). In a very specific embodiment, the chlorella protocheids strain is the strain CCAP211 / 8D (Bird and protozoa culture collection, Scotland, UK).

Optionally, the growth of the microalgae can be limited by adding a substance that inhibits cell growth, such as a sulfate, in the culture medium.

In addition, when not limited to any one theory, the Applicant believes that the glucose flux in microalgae of the genus Chlorella genus is normally

1. Basic Metabolism

2. Growth, ie formation of protein-enhanced biomass

3. Storage materials (lipids and carbohydrates, such as starch)

As shown in FIG.

This principle explains the natural changes in protein content as microalgae grow, despite the steady supply of nitrogen.

Therefore, the Applicant has found that, in order to increase the protein in the microalgae biomass, the growth of microalgae should be restricted, and the consumption of nutrients other than nitrogen of microalgae, such as glucose,

- make all of the glucose consumed for protein production pathways,

- to prevent accumulation of storage materials, for example, lipids

.

In fact, blocking nitrogen deficiency can prevent metabolic flows from going toward lipid production.

It may be advantageous to prevent the metabolic flows from progressing in the lipid production direction as described above, as long as the use of specific inhibitors selectively blocks any synthesis of the storage material.

Indeed, for any number of inhibitors used in lipid, or even starch (storage carbohydrates typical of microalgae) synthesis, is known as:

In the case of lipids, cerulenin is used as an inhibitor of fatty acid synthesis, and as an inhibitor of Streptomyces tricycles toxytricini ) have been described as inhibitors of lipases and the like;

In the case of starch, it is known that imino sugars (obtained by simple substitution of the nitrogen atom of oxygen atoms in the ring of the sugars) are potent inhibitors of glycosidase, glycosyltransferase, glycogen phosphorylase and UDP-Galp mutase have.

The method of the present invention therefore comprises fermenting microalgae biomass under heterotrophic conditions, comprising a first step of growing the biomass and a second step of removing the non-nitrogen nutrient source from the fermentation medium.

The second step can increase the protein in the biomass. Particularly, the second step can make the protein content of the biomass reach to more than 50% by weight (based on the weight of the building).

In a first preferred embodiment according to the present invention, a method for heterotrophic culture of microalgae, in particular chlorella noroquiniana,

A first stage in which microalgae are grown in accordance with the consuming capacity of microalgae in order to obtain a large amount of biomass rapidly; And

The second step in which the microalgae produce a protein, by setting the glucose feed rate to a value that is clearly below the glucose consumability of the microalgae in order to prevent further accumulation of storage materials or to promote their consumption;

.

As illustrated below, the first step in the growth of microalgae is performed in a discontinuous or " ash " manner in which the initial glucose feed is consumed entirely by microalgae to produce a base biomass.

The second step, in which the protein is produced,

- after the initially supplied glucose has been consumed, the complete medium is fed in a semi-continuous manner, using the so-called "feed-in" system; Conditions in which other operational parameters of the fermentation remain unchanged.

Glucose is supplied continuously and the feed rate is slower than the maximum consumption rate of the strain, so the remaining glucose content in the medium is maintained at zero.

Therefore, the growth of the strain is limited by the availability of glucose (glucose restriction conditions); or

- Conditions under which a continuous spinner incubator is used, in which the growth rate (μ) of the strain is kept at the minimum value and the growth of the strain is limited by the glucose supply

Lt; / RTI >

In this way of operation, biomass with high protein content can be obtained due to the limitation of glucose and the low growth rate imposed (both of which guarantee very good productivity).

In a second preferred embodiment according to the invention, the heterotrophic culture method of microalgae, in particular chlorella protothecidis, comprises the step of growing microalgae, in which the growth rate is limited if the phosphate feed is restricted, Thereby increasing the protein content. Thus, a method for heterotrophic culture of microalgae of the species Chlorella prototypedes includes heterotrophic culturing of microalgae in the absence of phosphate to reduce the rate of growth and thereby increase the protein content.

The invention will be better understood with the aid of the following examples, which are meant to be illustrative and non-limiting.

Example

Example  1: Chlorella using continuous-ash fermentation without limiting supply of nutrient media Sorokiniana  production

The strain used was Chlorella norovirus (strain UTEX 1663; University of Texas Algae Culture Collection, Austin, USA).

Pre-culture:

- 600 ml of medium in a 2 L Erlenmeyer flask;

- Composition of the medium (see Table 1 below)

Massive element (g / l) Glucose 20 K ₂ HPO ₄ .3H ₂ O 0.7 MgSO ₄ .7H ₂ O 0.34 Citric acid 1.0 Urea 1.08 Na ₂ SO ₄ 0.2 Na ₂ CO ₃ 0.1 Clerol FBA 3107 (antifoaming agent) 0.5 Trace element
(Mg / l) Na ₂ EDTA 10 CaCl ₂ .2H ₂ O 80 FeSO ₄ .7H ₂ O 40 MnSO ₄ · 4H ₂ O 0.41 CoSO ₄ .7H ₂ O 0.24 CuSO ₄ · 5H ₂ O 0.24 ZnSO ₄ .7H ₂ O 0.5 H ₃ BO ₃ 0.11 _{(NH 4) 6 Mo 7 O} 27 · 4H 2 O 0.04

Before sterilization, the pH was adjusted to 7 by the addition of 8 N NaOH.

The incubation was carried out under the following conditions:

- Duration: 72 hours;

- Temperature: 28 캜;

- Shaking speed: 110 rpm (Infors Multitron thermostat)

The pre-culture was then transferred to a 30 L Sartorius fermenter.

Biomass  Production culture:

The medium was the same as the medium used for the preliminary culture except that NH ₄ Cl was used instead of urea.

Massive element (g / l) Glucose 20 K ₂ HPO ₄ .3H ₂ O 0.7 MgSO ₄ .7H ₂ O 0.34 Citric acid 1.0 NH ₄ Cl 1.88 Na ₂ SO ₄ 0.2 Clorol FBA 3107 (antifoaming agent) 0.5 Trace element
(Mg / l) Na ₂ EDTA 10 CaCl ₂ 80 FeSO ₄ .7H ₂ O 40 MnSO ₄ · 4H ₂ O 0.41 CoSO ₄ .7H ₂ O 0.24 CuSO ₄ · 5H ₂ O 0.24 ZnSO ₄ .7H ₂ O 0.5 H ₃ BO ₃ 0.11 _{(NH 4) 6 Mo 7 O} 27 · 4H 2 O 0.04

After inoculation, it was adjusted to the initial volume (V _i) of the fermenter to 13.5 ℓ.

The volume of the fermenter was increased to a final volume of 16 to 20 liters.

The operating parameters of the fermentation were as follows:

Temperature 28 ℃ pH Using _{NH 3 28% (w / w} ) alignment to 5.0 to 5.2 pO ₂ > 20% (maintained by shaking) Shaking Speed 300 rpm minimum Air flow rate 15 L / min

When the initially supplied glucose was consumed, the same medium as that of the first medium containing no defoaming agent was added at a concentration of 500 g / l of glucose (the same ratio as the glucose in the initial medium) to the other medium to obtain a glucose content in the fermenter of 20 g / Was supplied in the form of a concentrated solution containing the other components.

Two other identical additional media were fed in the same manner, with each remaining glucose concentration becoming zero.

Clorol FBA 3107 defoamer was added as needed to prevent excessive bubble formation.

result:

After 46 hours of incubation, 38 g / l of biomass with a protein content of 36.2% (as assessed by N 6.25) was obtained.

Example  2: Of glucose  While limiting supply Oil price formula  Seeds used for fermentation. Sorokiniana  production

In this example, the supply of the complete medium (fed-batch) was started after the initially supplied glucose was consumed. Other operating parameters of the fermentation were maintained unchanged.

Glucose was continuously fed using a 500 g / l concentrated solution. Since the feeding rate was slower than the maximum consumption rate of the strain, the residual glucose content in the medium was maintained at 0, i.e. the growth of the strain was limited by the availability of glucose (glucose restriction conditions).

This consumption rate increased exponentially with time. The formula used to estimate the added flux is characterized by the factor (μ) corresponding to the growth rate that the strain can adopt if the strain consumes all of the glucose supplied:

S = So x exp (μ.t)

S = glucose feed flow rate (g / h)

So = initial glucose feed rate, determined by the amount of biomass present after ash culture. And 12 g / h under the conditions of the present invention.

μ = flow rate acceleration factor. It should be less than 0.11 h ^-1 , which is the growth rate of the strain when nutritional restrictions are not established.

t = duration of oil price operation (h).

Salts were supplied continuously, if possible, provided separately or mixed with glucose. However, the salt may also be divided into several portions and continuously supplied.

Table 4 below shows the amount of salt required for 100 g of glucose.

Massive element (g) Glucose 100 K ₂ HPO ₄ .3H ₂ O 6.75 MgSO ₄ .7H ₂ O 1.7 Citric acid 5.0 Na ₂ SO ₄ 1.0 Trace element
(Mg) Na ₂ EDTA 50 CaCl ₂ .2H ₂ O 400 FeSO ₄ .7H ₂ O 200 MnSO ₄ · 4H ₂ O 2.1 CoSO ₄ .7H ₂ O 1.2 CuSO ₄ · 5H ₂ O 1.2 ZnSO ₄ .7H ₂ O 2.5 H ₃ BO ₃ 0.6 _{(NH 4) 6 Mo 7 O} 27 · 4H 2 O 0.2

The concentration of components other than glucose was determined to be excessive compared to the nutrient requirement of the strain.

Clorol FBA 3107 defoamer was added as needed to prevent excessive bubble formation.

result: Oil price formula  During operation Glucose  Influence of supply rate

Tests were performed according to various glucose feed rates in a fed-batch mode. The glucose feed rates were characterized by the applied [mu]. The protein content in the obtained biomass was evaluated by measuring the total amount of nitrogen expressed as N 6.25.

Test Supply μ
(h ^-1 ) duration
(h) Biomass
(g / l) productivity
(g / l / h) N 6.25% One 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 indicate that glucose restriction can increase protein content.

The actual protein content was even higher when the μ was as high as 0.09 (39.3% versus 36.2%), as in Example 1, when the glucose limit was not achieved.

A more stringent restriction of glucose metabolism has resulted in an additional increase in protein content.

In order to obtain a protein content of more than 50% under these test conditions, it was necessary to add μ to the strain at less than 0.06 h ^-1 .

It should be noted that this condition is closely related to the decrease in productivity: 0.56 g / ℓ / h to 1.35 g / ℓ / h (test 3).

Example  3: Glucose  Continuously limiting supply Chemical agent  Fermentation is used Seed. Sorokiniana  production

In this example, a 2 L Sartorius Biostat B fermenter was used.

Fermentation was carried out as in Example 2 except that the volume was one tenth of the original volume, the volume of inoculation was 60 ml, and the initial volume was 1.35 l.

Continuous feeding of the medium was started according to the same principle as in Example 2, in which case the salt was mixed with glucose in the feed tank. The feed rate was accelerated according to the same exponential formula applied to the exponential formula of Example 2, applying .mu. To 0.06 h.sup. ^{- 1} .

Chemical agent

When the volume reached 1.6 liters (i.e., when the biomass concentration reached approximately 50 g / l), the operation of the continuous fractional crystallization system was initiated:

1. In a fermenter, a nutrient broth solution containing 100 g / l of glucose and having the following composition was continuously fed at a flow rate of 96 ml / h:

Massive element (g / l) Glucose 100 K ₂ HPO ₄ .3H ₂ O 6.75 MgSO ₄ .7H ₂ O 1.7 Citric acid 5.0 Na ₂ SO ₄ 1.0 Trace element
(mg / l) Na ₂ EDTA 50 CaCl ₂ .2H ₂ O 400 FeSO ₄ .7H ₂ O 200 MnSO ₄ · 4H ₂ O 2.1 CoSO ₄ .7H ₂ O 1.2 CuSO ₄ · 5H ₂ O 1.2 ZnSO ₄ .7H ₂ O 2.5 H ₃ BO ₃ 0.6 _{(NH 4) 6 Mo 7 O} 27 · 4H 2 O 0.2

The concentrations of the components other than glucose were determined to be excessive compared to the nutrient requirement of the strain.

2. The medium was continuously withdrawn from the fermenter by means of a siphon tube connected to the pump so that the volume of the culture medium was maintained at 1.6 L.

This replaced the 0.06 g / l (6%) fraction of the medium per hour. The substitution rate at this time is referred to as the dilution rate (D).

Since the growth of the strain was restricted by the glucose supply, the growth rate () of the strain was established to be the same value according to the principle of the method of culture of the fractional medium:

D = μ = 0.06 h ^-1

result

After 97 hours of operation by the chemical method, the biomass concentration was stabilized at 48 g / ℓ ± 2 g / ℓ and the protein content was stabilized at 53 ± 2%.

This mode of operation has the advantage of limiting biomass with high protein content due to the limitations of glucose and the low growth rate imposed (both of which guarantee very good productivity of about 2.9 g / l / h) and high concentrations of biomass Can be obtained.

Example  4: Chlorella in which ash fermentation is used, with or without limited phosphate supply Protocheids  production

The strain used was Chlorella protocheidis (strain CCAP21 1 / 8D; bird and protist culture collection, Scotland, UK).

Pre-culture:

150 ml of medium in a 500 ml Erlenmeyer flask;

- Composition of medium: glucose 40 g / ℓ + yeast extract 10 g / ℓ

The incubation was carried out under the following conditions: Duration: 72 hours; Temperature: 28 占 폚; Shaking speed: 110 rpm (Infors multitron thermostat).

Then, the preliminary culture liquid was transferred into a 2 L Sartorius biostat B fermenter.

Biomass  Production culture:

The composition of the culture medium was (g / l) as follows:

Glucose 80 Citric acid 4 NH ₄ CL 2 KH ₂ PO ₄ 2 (Test 1)
Or 3 (Test 2) Na ₂ HPO ₄ 2 (Test 1)
Or 3 (Test 2) MgSO _4, 7H ₂ O 1.5 NaCl 0.5 Yeast extract 5

The amount of phosphate supplied was estimated to be limited in Test 1 and to be excessive in Test 2. Clorol FBA 3107 defoamer was added as needed to prevent excessive bubble formation. After inoculation, it was adjusted to the initial volume (V _i) of the fermenter 1 to ℓ.

The operating parameters of the fermentation were as follows:

Temperature 28 ℃ pH Fit to 6.5 using NH ₃ 28% (w / w) pO ₂ > 20% (maintained by shaking) Shaking Speed At least 200 rpm Air flow rate 1 l / min

result:

Test Duration (h) Biomass
(g / l) Cumulative μ
(h ^-1 ) Residual PO ₄
(Mg / l) N 6.25
% One 45 36.5 0.07 0 56.1 2 36 38.1 0.09 800 48.1

These results demonstrate that the feed restriction of phosphate, identified by the absence of residual phosphate after fermentation, (as in the previous examples, increased the protein content to a value of greater than 50% Lt; RTI ID = 0.0 > (as measured by < / RTI >

Claims (8)

A method of increasing the protein content of microalgae grown under heterotrophic conditions, wherein the microalgae is of the genus Chlorella and the heterotrophic culture is characterized in that the protein content in the biomass is less than or equal to 50% by limiting the growth of the microalgae by deficient in non- % ≪ / RTI > by weight of the composition. 2. The method of claim 1, wherein the deficient non-nitrogen nutrient source is a nutrient selected from the group consisting of glucose and phosphate. 3. The method according to claim 1 or 2, wherein the nutrient source is deficient to obtain a growth rate 10% to 60% lower than the growth rate when the nutrient source is not restricted. 4. The method according to any one of claims 1 to 3, wherein the microalgae in the genus Chlorella are selected from the group consisting of chlorella thallokina and chlorella protocheodis. The method of claim 4 wherein the nutrient material is characterized in that the supply to achieve a growth rate of 0.06 h ^-1 to 0.09 h ^-1. 6. The method according to any one of claims 1 to 5, characterized in that the duration of the deficient culturing step is at least 1 hour, preferably at least 10 hours, more preferably at least 20 hours, especially from 30 to 60 hours Lt; / RTI > The method according to any one of claims 1 to 6, wherein the heterotrophic culture of chlorella norovirus kinase microalgae comprises the steps of:
a. A first step of growing a microalgae while adjusting a feeding rate of glucose to the consumability of the microalgae in order to rapidly obtain a large amount of biomass; And
b. A second step in which the microalgae produce a protein by setting the glucose supply rate to a value that is clearly below the glucose consumability of the microalgae in order to prevent further accumulation of storage materials or promote their consumption
≪ / RTI > The method according to any one of claims 1 to 6, wherein the heterotrophic culture of chlorella protochedes species microalgae is carried out by culturing in the absence of phosphate to limit the growth rate and thereby increase the protein content ≪ / RTI >