KR101577820B1 - Novel culture process for a heterotrophic microalga - Google Patents

Abstract

The subject of the present invention is a novel heterotrophic microalgae comprising the steps of culturing a heterotrophic microalgae under heterotrophic culture conditions to produce an inoculum, inoculating the culture medium in a photobioreactor and culturing under autotrophic culture conditions, Cultivation methods, and methods for obtaining biomass from heterotrophic microalgae.

Heterotrophic culture conditions, heterotrophic microalgae, photobioreactors, biomass

Description

TECHNICAL FIELD [0001] The present invention relates to a novel culture method for heterotrophic microalgae,

The subject of the present invention is a new method of cultivation of heterotrophic microalgae and a method for obtaining biomass from heterotrophic microalgae.

Birds are an autotrophic organism, an organism that can synthesize organic matter by photosynthesis from carbon dioxide and light contained in the air. The two main groups of birds are distinguished as follows:

o Phytobenthos or macroalgae: Species fixed on the sea floor by fronds

Phytoplankton or microalgae: species floating or drifting in open water.

The major groups of microalgae are as follows:

- Diatoms,

- Wagyu Mojo River,

- Cladophylla algae (halftone algae),

- Nam Jo,

- Flushing River,

- Green algae (or green algae).

Thus, microalgae represent a broad group with the potential to supply compounds with biological activity that can be used in cosmetics, the pharmaceutical industry, and even agriculture-food industry.

In particular, certain algae, such as Chlorella (Chlorella) from the green alga is also dependent organisms and nutrient, that synthesize organic substances from the organic material that has already been produced by other organisms. Such microalgae are not only capable of synthesizing organic materials from inorganic materials such as carbon dioxide but also are called mixed nutrition organisms that can live like heterotrophic organisms in the absence of light or chemical energy.

It is known to cultivate some of these algae, in particular chlorella, in a fully fermenting tank in the presence of a source of organic carbon and a source of inorganic or organic nitrogen, in a fully heterotrophic manner, i.e. in the presence of oxygen. In this case, cultures grown in a heterotrophic manner in a dark room in the presence of organic carbon (e.g. glucose), oxygen (air) and under such conditions are not required for the growth of algae, Continue seeding in the fermenter.

This type of method makes it possible to achieve rapid growth of algae, especially at very high cell concentrations of more than 10 g of dry matter per liter, and even more than 50 g per liter. A disadvantage of this method is that it requires significant expenditure on energy production for the aeration and agitation of expensive organic carbon sources and fermentation tanks. In addition, the biochemical quality of the microalgae cultured under heterotrophic culture conditions can be enhanced by the addition of molecules with a high added value, especially those containing them (such as pigments such as chlorophyll, phycoerythrin, phycocyanin, lutein and beta-carotene, lipids such as linolenic acid , Eicosapentaenoic acid, phospho- and sulfolipids, amino acids such as proline, etc.).

It is also known to cultivate algae, in particular chlorella, in a completely autotrophic manner in the presence of light and carbonates (carbon dioxide) as well as nitrogen (nitrate, ammonium) and other minerals. These products are often obtained in shallow circular tanks which are slowly agitated. Again, the culture grown in an autotrophic manner in the presence of CO ₂ , bicarbonate and carbonate, and under these conditions is the result of photosynthesis, because the growth of algae is the result of photosynthesis. The disadvantage of this method is that rapid growth of algae can not be achieved due to the inability to set optimal values for parameters such as self-shielding phenomena, alternating day and night, temperature or pH of the culture medium of the culture. Thus, the growth of algae under these conditions is slow, so it is contaminated by other more competitive microbes, even protozoans.

It is known to cultivate chlorella and other algae or microalgae in a generally tubular closed biochemical bioreactor where the carbon dioxide can be injected at a high concentration while protecting the microalgae from contaminants, So that the performance of the tank can be improved. In addition, in an autotrophic culture method of algae in such confined media, seeding of the final culture is the result of continuous cultivation in an irradiated reactor which gradually grows. In these reactors, cell concentration can reach up to 5 g / l at the end of each subculture in inverse proportion to tube diameter.

The main advantage of microalgae cultivation is on the one hand the production of large quantities of biomass which is later used in agro-food industry, cosmetics or agriculture and on the other hand the production of molecules with high added value it contains. In these two cases, a challenge now to be solved is to develop a method for culturing microalgae that allows the biomass to be obtained in large quantities, i.e. above 1 g / l, while preserving the biochemical quality of the cultured microalgae.

It is known in the prior art that certain heterotrophic microalgae strains are capable of altering metabolism, and can be carried out in a heterotrophic manner and in an independent manner, and vice versa (Ogbonna et al: JC Ogbonna, H. Masui and H. Tanaka, in Journal of Applied Phycology, 1997, 9, pp 359-366).

Thus, the above document by Ogbonna et al. Describes the sequential cultivation of heterotrophic microalgae, in particular heterotrophic / autotrophic, of chlorella. This reference describes the cultivation of chlorella under the conditions of heterotrophic cultivation, wherein when the concentration of glucose is reduced to zero, the culture medium containing chlorella is transferred to a lighted flask for light stimulation under autotrophic culture conditions . Thus, transition from a stage under heterotrophic culture conditions to a stage under autotrophic culture conditions is carried out at a constant volume.

However, for those skilled in the art of microalgae cultivation, the two completely different culture conditions, i.e., the transition between independent nutrient and heterotrophic conditions, can only occur in a considerable re-adaptation phase to the new conditions of the bird, Is a transition from an oxygen-saturated medium to a carbon dioxide-saturated medium, from a medium in which carbon is an organic (glucose) medium to a medium in which carbon is a mineral (CO ₂ ), and from a darkroom to a brightroom. This re-adaptation step is particularly indicated by the concentration drop of microalgae. This was observed in the first column, left column, page 365 of the paper by Ogbona et al.: The concentration of microalgae is low at the end of 24 hours under independent nutrient conditions. In addition, a small increase in protein concentration is observed.

The Applicant has surprisingly found that, despite this technical bias, it is possible to increase the concentration of chlorophyll and protein in the microalgae, with excellent growth even in the independent nutrition stage, without reducing the re-adaptation step or the concentration of the microalgae during the change of the culture conditions, It has been found that cultures of microalgae can be obtained under heterotrophic conditions and cultures can be obtained under independent nutrient conditions. These novel methods include the production of inoculum by incubation of heterotrophic microalgae under heterotrophic culture conditions, inoculation of the culture medium with the inoculum obtained in a photobioreactor, and culture inoculated in a photobioreactor under autotrophic culture conditions Based on the culture of the medium.

More surprisingly, this improvement is most pronounced when the autotrophic culture step is performed in a closed-type photobioreactor. In addition, much higher cell concentrations of the inoculum obtained by incubation under heterotrophic culture conditions (as compared to the autotrophic culture conditions) allow the subculture to be initiated much more rapidly with the same volume of inoculum, It is possible to save the generation of generations and days of cultivation.

Accordingly, a first aspect of the present invention is

(a) preparing an inoculum by incubating one or more heterotrophic microalgae strains under heterotrophic culture conditions,

(b) inoculating the culture medium with the inoculum in a photobioreactor,

(c) culturing said microalgae in a photobioreactor under independent or mixed nutrition, preferably autotrophic culture conditions,

To a method for culturing heterotrophic microalgae.

According to another aspect of the present invention,

(a) culturing one or more heterotrophic microalgae strains under heterotrophic culture conditions to advantageously yield a concentration of heterotrophic microalgae of greater than 5 g / l to produce a pre-culture of said strain,

(b) advantageously an autotrophic or mixed nutrition, preferably an autotrophic culture, in order to obtain a chlorophyll concentration in excess of 0.01 g / l, a protein concentration in excess of 0.5 g / l and / or a microalgae concentration in excess of 1 g / Culturing the pre-culture obtained in step a) in a photobioreactor under the conditions

Wherein the volume of the preliminary culture in step b) is less than 1/5 of the volume of the culture.

According to the present invention there is provided a novel method comprising culturing a heterotrophic microalgae under heterotrophic culture conditions to produce an inoculum, inoculating the culture medium in a photobioreactor, and culturing under autotrophic culture conditions, Algae can be cultured and biomass can be obtained from heterotrophic microalgae.

According to the present invention, a heterotrophic microalgae means a microalgae capable of synthesizing an organic material in the presence of an organic carbon source and / or an inorganic or organic nitrogen source under the presence and absence of oxygen.

In the present invention, the inoculum is advantageously cultured under heterotrophic culture conditions with a significant concentration of heterotrophic microalgae advantageously obtained by culturing the heterotrophic microalgae in an amount above 5 g / l, more advantageously above 10 g / l Means a pure or substantially pure culture (i. E., The culture is not contaminated) of the heterotrophic microalgae that is less than one-fifth the volume of the desired final culture of the heterotrophic microalgae. The purpose of the inoculum is to become a pre-culture under heterotrophic culture conditions for rapid onset of culture under autotrophic conditions so that the desired concentration of heterotrophic microalgae is obtained within a minimum of time.

According to the present invention, inoculation refers to dilution of the inoculum in a culture medium suitable for culturing under autotrophic conditions, and the volume of the inoculum is less than one-fifth of the volume of the culture medium.

According to the present invention, the heterotrophic culture conditions are

A culture medium comprising at least one organic carbon source and / or an inorganic or organic nitrogen source,

- oxygen supply and

- optical member

≪ / RTI >

The optimal temperature for culturing microalgae under heterotrophic culture conditions is generally comprised between 20 and 40 ° C.

Optimal pH for culturing microalgae under heterotrophic culture conditions is generally included in 5-8.

Under heterotrophic culture conditions, the culture medium is agitated by the introduction of air and supplementally by a stirrer.

A culture medium for heterotrophic culture conditions means a medium containing the chemical elements necessary for the growth of microalgae in the absence of light and in the presence of oxygen.

Such media generally include the following:

At least one organic carbon source and / or an inorganic or organic nitrogen source,

- potassium and phosphorus sources (e.g., K ₂ HPO ₄ ),

- nitrogen and sulfur sources (e. _{G., (NH 4) 2 SO 4} ),

A magnesium source (e.g., MgCl ₂ ),

A calcium source (e.g., CaCl ₂ ),

An iron source (e.g., iron citrate),

- sources of trace elements (eg, salts of Cu, Zn, Co, Ni, B, Ti)

- water (for example, sterile water),

pH buffer: maintains an accurate, even optimal pH (eg KH ₂ PO ₄ ).

Examples of organic carbon sources for microalgae include glucose, corn steep, acetic acid, ethanol, urea, and the like.

Certain products such as Konstep have the advantage of being both a source of organic carbon, as well as a source of organic nitrogen, trace elements and iron.

Acetic acid and ethanol can not be used as a carbon source by many microorganisms and thus have the advantage of limiting microbial contamination of the microalgae culture. However, not all heterotrophic microalgae can use acetic acid or ethanol as a carbon source. It can be used for the cultivation of chlorella or other microalgae, but glucose can be used more generally.

As examples of inorganic nitrogen sources for microalgae, mention may be made of nitrates, nitrites and ammonium salts.

Examples of organic nitrogen sources for microalgae include algae and yeast extracts, konstep, urea, and the like.

One example of a culture medium composition for heterotrophic culture conditions is as follows:

Example of culture medium composition:

KNO ₃ 2 g

KH ₂ PO ₄ 2 g

MgSO ₄ .H ₂ O 2 g

Konstert 25 g

Glucose 25 g

5 g of urea

Water qsf 1000 mL

In accordance with the present invention, a photobioreactor or PBR refers to any type of device that has been demonstrated to be suitable for the regular propagation of photosynthetic microorganisms on a commercial scale in the presence of light and carbon dioxide. According to the present invention, the PBR may be open or closed. The PBR according to the present invention is preferably of a closed type.

The open PBR has an open top, light is coming in from the sun, and the gas dissolved in the open PBR is in equilibrium with the atmospheric gas above it.

An open PBR can be of various types, for example:

- lakes and ponds (inorganic salts are derived from soil and carbon dioxide is derived from air)

- Artificial tanks

- round tank with rotating stirrer

- Waterway

- Slope system.

In order to maximally prevent the self-shielding problem resulting in low growth of microalgae, the depth of the open PBR is about 10 to 30 cm.

All open PBR systems can be contaminated by external contaminants (all kinds of dust, insects and small animal carcasses, or new secretions). In order to exchange with the outside and to limit these contaminants to the maximum extent, closed PBRs have also been developed that strictly restrict the exchange of culture fungi with the outside.

In closed PBR, unlike in open PBR, light must pass through walls that act simultaneously to restrict gases and liquids. In this closed PBR, the concentration of carbon dioxide in the culture medium is much higher than in open PBR due to limitations.

In addition, the closed-type PBR provides the advantage of virtually eliminating the evaporation phenomenon or the dilution by the culture medium of the culture medium, and allows easy control of culture parameters such as temperature, pH, dissolved gas and nutrient concentration. It also provides the possibility of operation under a thin incubation thickness and thus the possibility of limiting the self-shielding phenomenon, resulting in an increase in the concentration of algae, thereby limiting the centrifugation energy during agitation and pumping energy and biomass collection during incubation.

A closed PBR is divided into various types according to its shape and its operating principle. Since there are so many combinations between different possibilities, only a few will be mentioned:

- tubular photobioreactor

■ Flat zigzag horizontal tubular reactor

Zigzag horizontal tubular reactors in flexible form

■ Overlapping zigzag horizontal tubular reactors

■ Partitioned Horizontal Tubular Reactor

■ Horizontal tubular reactors with dividers

■ Spiral wound tubular reactor

■ Triangle - Circulating Tubular Reactor

- Thin layer reactor

■ Flat alveolar panels

■ Have a window

■ Has rigid frame and flexible sheet

■ Has a meander

■ Have floating seats

■ Cylindrical expansion

■ Has a spherical spreader

- Vertical cylinders and sleeves

■ Vertical cylindrical reactor

■ Vertical sleeve

■ Horizontal sleeve.

According to the present invention,

- supply of carbon dioxide (e.g., natural air or CO ₂ enriched air, pure CO ₂ , carbonate or bicarbonate supplied), and

- The presence of natural light or artificial light

≪ / RTI >

In particular, cultures of microalgae should be irradiated with sufficient light intensity, but not lethal to microscopic algae, and the physical parameters of the culture (pH, CO ₂ , salinity, temperature, absorbance, etc.) should be controlled.

The optimum temperature for culturing microalgae under autotrophic culture conditions is generally comprised between 20 and 40 < 0 > C.

Optimal pH for culturing microalgae under autotrophic culture conditions is generally included in 5-8.

Also, agitation of the culture medium under autotrophic culture conditions is recommended.

The culture medium for autotrophic culturing conditions includes chemical elements necessary for the growth of microalgae in the presence of light and carbon dioxide.

Such media can include:

- potassium and phosphorus sources (e.g., K ₂ HPO ₄ ),

- nitrogen and sulfur sources (e. _{G., (NH 4) 2 SO 4} ),

A magnesium source (e.g., MgCl ₂ ),

A calcium source (e.g., CaCl ₂ ),

An iron source (e.g., iron citrate),

- sources of trace elements (eg, salts of Cu, Zn, Co, Ni, B, Ti)

- water (for example, sterile water),

- pH buffer: maintains an accurate, even optimal pH, or at least reduces its change (eg, KH ₂ PO ₄ ).

An example of a culture medium composition for autotrophic culture conditions is provided below:

Examples of the composition of the culture medium:

KNO ₃ 2 g

KH ₂ PO ₄ 2 g

MgSO ₄ -7H ₂ O 2 g

Yeast extract 5 g

5 g of urea

FeSO ₄ -7H ₂ O 100 mg

Solution of Trace Elements # 5 ml

1000 mL of water.

Composition of solution # of trace element:

H ₃ BO ₃ 2.86 g

MnSO ₄ -7H ₂ O 2.5 g

ZnSO ₄ -7H ₂ O 0.222 g

CuSO _{4 -} 5H ₂ O 79.0 mg

Na ₂ MO ₄ 21.0 mg

Distilled water 1000 ml.

Under autotrophic culture conditions, as in heterotrophic culture conditions, microalgae show considerably longer doubling times than bacteria (about 24 hours instead of 20 minutes to 1 hour).

According to the present invention, the mixed nutrient culture conditions are

- oxygen supply,

- Carbon dioxide supply,

- presence of light and

A culture medium comprising at least one organic carbon source and / or an inorganic or organic nitrogen source

≪ / RTI >

When the carbon source is acetic acid, the oxidative decomposition of acetic acid forms carbon dioxide in the culture medium, so that the supply of carbon dioxide may be omitted.

The optimum temperature for culturing microalgae under mixed nutrient culture conditions is generally comprised between 20 and 40 ° C.

Optimal pH for culturing microalgae under mixed nutrient culture conditions is generally included in 5-8.

The culture medium for the mixed nutrient culture conditions is the same as the culture medium under the heterotrophic culture conditions described above, but the concentration of all components may be slightly increased, and there is added 1 l per 1 l of the microalgae A few grams of carbon may be added.

Also, gentle agitation of the culture medium under mixed nutrition culture conditions is recommended.

Step a) of the process according to the invention advantageously comprises several successive incubation sub-steps under heterotrophic culture conditions.

Step a) of the process according to the invention preferably comprises 2 to 4 consecutive incubation sub-steps under heterotrophic culture conditions.

The continuous culture sub-steps are advantageously carried out with an increase in the culture volume.

The dilution factor between each successive incubation step is advantageously comprised between 1/5 and 1/50, preferably between 1/10 and 1/30.

The starting pre-culture of the microalgae required for carrying out step a) of the process advantageously amounts to about 1 l.

Thus, for example, if step a) of the method according to the invention comprises three consecutive incubation sub-steps and the dilution factor between each successive incubation step is 1/10, step a)

- a preliminary culture of about 1 l of microalgae

Dilution of about 1 l of the pre-culture of about 9 l of the culture medium

- Cultivation under the conditions of about 10 l of heterotrophic culture conditions thus obtained

- dilution of about 10 l of medium in about 90 l of culture medium

- Culturing under about 100 l of heterotrophic culture conditions thus obtained

. ≪ / RTI >

It should be noted that the dilution factor between consecutive incubation sub-steps need not necessarily be the same. Thus, after having a dilution factor of 1/10 (a dilution of about 1 l of the pre-culture of microalgae in about 9 l of the culture medium), a dilution factor of 1/20 in the subsequent culture sub-step (obtained at about 190 l of culture medium Diluted to about 10 l).

Step a) of the process according to the invention preferably corresponds to culturing the heterotrophic microalgae until a concentration of greater than 1 g / l is obtained.

The carbon source of the heterotrophic culture medium during step a) of the process according to the invention is advantageously introduced semi-discontinuously during said subculturing sub-steps. The semi-discontinuous mode of culture referred to as "feed-batch" is carried out by adding the carbon source of the heterotrophic culture medium in a linear or controlled manner. Thus, the initiation culture medium will not contain all of the carbon source initially provided in the culture medium composition, but will be gradually added to the culture medium. This is effective in blocking contamination, especially bacteria.

The inoculum prepared in step a) of the process according to the invention is preferably sterile.

According to the present invention, the aseptic inoculum means a pure culture of microalgae strains free of all detectable foreign or parasitic microorganisms, due to culture in a sterile bioreactor.

Step A ') may be advantageously provided before step a), and step A') is a preliminary incubation step of the heterotrophic microalgae under autotrophic culture conditions. Step a) The purpose of this step A ') is to promote the appearance of photosynthetic systems (chloroplasts, etc.) in microalgae to facilitate the transition from the heterotrophic culture conditions of step a) to the autotrophic culture conditions of step c) will be.

In step b) of the method according to the invention, the volume of the inoculum advantageously corresponds to 1/5 to 1/50, preferably 1/10 to 1/30 of the volume of the culture medium.

Step c) of the process according to the invention is preferably carried out until the chlorophyll concentration above 0.01 g / l and / or the protein concentration above 0.5 g / l and / or the microalgae concentration above 1 g / l are attained It corresponds to culturing nutrient microalgae.

Heterotrophic microalgae according to the present invention are preferably algae, particularly Chlorella (Chlorella), three or four des mousse (Scenedesmus) and bunch El rope sheath (Muriellopsis), and more preferably, Chlorella vulgaris (Chlorella vulgaris), or diatoms, For example, from Odontella aurita .

The selected strain is advantageously rich in chlorophyll.

The heterotrophic microalgae strains are preferably strains obtained by adapting to heterotrophic growth.

According to the present invention, adaptation to heterotrophic growth is meant to selectively isolate strains that can best grow under heterotrophic culture conditions. This adaptation method comprises an optional first step of exposing the microalgae of interest to UV radiation or a mutagen, e. G., Acriflamine, and then introducing one or more organic carbon sources and / or inorganic or organic Culturing the microalgae in a petri dish in the absence of light on a gelled medium containing a nitrogen source, selecting colonies of microalgae that exhibit the best growth, and optionally culturing the colonies thus selected in heterogeneous nutrient culture conditions individually in a liquid medium . Methods without an optional step of exposing to UV radiation are described, for example, in page 2 of patent document GB 1,109,659. A method having an optional step of exposing to UV radiation or a mutagen is described, for example, in patent application WO 00/36084.

Another aspect of the present invention is a method for producing biomass from heterotrophic microalgae comprising the steps of: d) collecting biomass from the heterotrophic microalgae obtained at the end of the culture according to step c) To a method according to the invention.

According to the present invention, the collection of biomass from heterotrophic microalgae means the separation of heterotrophic microalgae from its culture medium and the collection of thus obtained heterotrophic microalgae, so-called "algal paste ". The so-obtained " seaweed paste "generally contains 15 to 30% dry matter.

The step d) of collecting the biomass from the microalgae preferably comprises a centrifugation step or is carried out by filtration of the microalgae culture, and more preferably by centrifugation.

If filtration is used, tangent type filtration will preferably be used.

The process for obtaining a biomass according to the invention further comprises the step of drying the biomass collected from the heterotrophic microalgae obtained at the end of step d). Step e) of drying the biomass collected from the microalgae is preferably carried out by atomization or lyophilization.

The method according to the invention advantageously further comprises the step of freezing f) the biomass collected from the heterotrophic microalgae obtained at the end of step d), said freezing step being carried out under vacuum or under a controlled atmosphere .

The following examples illustrate preferred embodiments of the process according to the invention, but do not limit the scope of the invention.

<Examples>

Example 1 : Culture of Chlorella vulnaris according to the method of the present invention

The culture of Chlorella vulnaris according to example 1

- from 1 l of the pre-culture to three incubation sub-steps (under 1/4 dilution factor between the first sub-step and the second sub-step and 1/25 dilution between the second sub-step and the third sub- 24 dilution factor) and incubated for 5 days in each sub-step to obtain an inoculum,

- Inoculate a culture medium for autotrophic culture conditions with an inoculum having a dilution factor of 1/50,

- incubation for 2 days under autotrophic culture conditions

.

Thus, 1 l of a pre-culture of chlorella bulgur microalgae was cultured under aeration and stirred in culture medium A for 5 days at 35 ° C:

1 l &lt; tb &gt; Composition of culture medium A:

KNO ₃ 2 g

KH ₂ PO ₄ 2 g

MgSO ₄ .H ₂ O 2 g

Konstert 25 g

Glucose 25 g

5 g of urea

Water qsf 1000 mL

pH 6.5.

One liter of a pre-culture of chlorella bulgaris microalgae was diluted in 24 liters of culture medium A.

25 l of the culture medium containing the thus obtained chlorella Bulgarian microalgae was cultivated under heterotrophic culture conditions, i.e. in a standard 25 l fermenter such as used for bacterial fermentation, in the absence of light and in the presence of air for 5 days. As glucose was consumed by chlorella bulgur microalgae in the culture, glucose was added discontinuously and in an automated manner.

25 l of the culture of the thus obtained chlorella Bulgarian microalgae was diluted in 575 l of the culture medium A.

600 l of the culture medium containing the thus obtained chlorella digestive microalgae was cultured in a standard 600 l fermenter such as that used for bacterial fermentation in the absence of light and in the presence of air for 5 days. As glucose was consumed by chlorella bulgur microalgae in the culture, glucose was added discontinuously and in an automated manner.

600 l of the thus obtained culture of the chlorella bulgaris microalgae contained an inoculum of microalgae of chlorella bulgaris with a concentration of chlorella bulgaris microalgae of 15 g / l.

Inoculating the culture medium B with the inoculum in a closed tubular photobioreactor was carried out by diluting 600 l of the obtained chlorella bulgaris microalgae with 29,400 l of culture medium B.

1 l Composition of culture medium B:

Urea 0.3 g / l

Ammonium nitrate 0.4 g / l

KH ₂ PO ₄ 0.35 g / l

Magnesium sulfate 0.5 g / l

0.5 g / l of iron (I) sulfate

Solution of trace element of 0.01 ml / l of the following composition:

Zinc sulfate 75 g / l

Borax 6 g / l

Cobalt sulfate 24 g / l

Copper sulfate 24 g / l

410 g / l manganese sulfate

And 0.01 ml / l ammonium molybdate solution 9.2 g / l

Was added to the solution of the macro-nutrients.

The pH of the culture medium will be adjusted to 6.5 to 7 by the injection of carbon dioxide.

30,000 l of the culture medium containing the thus obtained chlorella digestive microalgae was cultured in a 30,000 l (or 30 m ³ ) tubular photobioreactor for 2 days in the presence of light and carbon dioxide.

After 2 days of culture, a concentration of 3 g / l chlorella bulgaris microalgae, a chlorophyll concentration of 0.12 g / l and a protein concentration of 1.5 g / l were obtained.

Example 2: Culture of Chlorella vulgaris under stand-alone nutrient culture conditions alone

Example 2 is a comparative example of Example 1 and cultivation of chlorella bulgaris was carried out under stand-alone nutrient culture conditions alone.

The culture of Chlorella vulnaris according to example 2

- 5 l culturing stages from 1 l of the pre-culture under autotrophic culture conditions (having a 1/10 dilution factor between the first 4 steps and a 1/30 dilution factor between the 4th and 5th incubation steps) , Incubating for 7 days for the first 4 steps and 9 days for the 5th step

.

Thus, 1 l of a pre-culture of chlorella bulgaris microalgae was cultured in a closed 1 l tubular photobioreactor for 7 days.

These preliminary cultures were diluted in 9 l of culture medium B.

10 L of the culture medium containing the thus obtained chlorella Bulgarian microalgae was cultured under autotrophic culture conditions in a closed 10 L tubular photobioreactor, that is, in the presence of light and carbon dioxide for 7 days.

10 l of the thus obtained culture of chlorella bulgaris microalgae was diluted in 90 l of culture medium B.

100 L of the culture medium containing the thus obtained chlorella Bulgarian microalgae was cultivated under autotrophic culture conditions in a closed 100 L tubular photobioreactor, that is, in the presence of light and carbon dioxide for 7 days.

100 L of the thus-obtained culture of the chlorella Bulgarian microalgae was diluted in 900 L of culture medium B.

1000 L of the culture medium containing the thus obtained chlorella Bulgarian microalgae was cultivated under autotrophic culture conditions in a closed 1000 l tubular photobioreactor, that is, in the presence of light and carbon dioxide for 7 days.

The concentration of chlorella Bulgaris microalgae was 3 g / l in 1000 l of the cultured microalgae thus obtained.

1000 L of the thus-obtained culture of the chlorella Bulgarian microalgae was diluted in 29,000 l of culture medium B.

30,000 l of the culture medium containing the thus obtained chlorella digestive microalgae was cultivated in a closed 30,000 l (or 30 m ³ ) tubular photobioreactor under autotrophic culture conditions, that is, for 9 days in the presence of light and carbon dioxide.

After a total of 30 days of culture, a concentration of 3 g / l chlorella bulgaris microalgae, a chlorophyll concentration of 0.12 g / l and a protein concentration of 1.5 g / l were obtained.

Comparison of Example 1 / Example 2:

In order to obtain the same concentration of chlorella bulgaris having the same biochemical quality (amount of chlorophyll, amount of protein), culture for 30 days (Example 2) is required under the condition of autotrophic culture alone, It is clear that using the method according to the invention requires 17 days of culture (i.e., 13 days of time (44%)) (Example 1).

Claims (22)

(a) producing an inoculum by culturing a strain of at least one heterotrophic microalga under heterotrophic culture conditions, (b) inoculating said inoculum into a culture medium in a photobioreactor, and (c) culturing said microalgae in a photobioreactor under independent nutrient or mixed nutrition culture conditions Lt; RTI ID = 0.0 &gt; heterotrophic microalgae. &Lt; / RTI &gt; The method according to claim 1, wherein step a) comprises several successive incubation sub-steps under heterotrophic culture conditions. 3. The method of claim 2, wherein said continuous culture sub-steps are performed with increasing culture volume. 4. The method according to claim 3, characterized in that the dilution factor between each successive incubation step is comprised between 1/5 and 1/50. 5. Process according to any one of claims 1 to 4, characterized in that step a) corresponds to culturing the heterotrophic microalgae until a concentration of greater than 5 g / l is obtained. Method according to any one of claims 2 to 4, characterized in that the carbon source of the heterotrophic culture medium during step a) is introduced during the continuous culture sub-step in a semi-discontinuous manner . 5. A method according to any one of claims 1 to 4, characterized in that the inoculum is sterile. 5. Process according to any one of claims 1 to 4, characterized in that the volume of the inoculum in step b) corresponds to 1/5 to 1/50 of the culture volume. The method according to any one of claims 1 to 4, wherein the photobioreactor is selected from a closed-type photobioreactor and an open-type photobioreactor. 5. Method according to any one of the preceding claims, wherein step c) comprises obtaining at least one of a chlorophyll concentration greater than 0.01 g / l, a protein concentration greater than 0.5 g / l and a microbial concentration greater than 1 g / l Lt; RTI ID = 0.0 &gt; heterotrophic microalgae &lt; / RTI &gt; The method according to any one of claims 1 to 4, wherein the heterotrophic microalgae are selected from chlorophytes or diatoms. The method according to any one of claims 1 to 4, wherein the heterotrophic microalgae strain is obtained by adapting to heterotrophic growth. 5. A method according to any one of claims 1 to 4, comprising the step (d) of collecting the biomass from the heterotrophic microalgae obtained at the end of the culture according to step c) &Lt; RTI ID = 0.0 &gt; 5. &Lt; / RTI &gt; 14. The method according to claim 13, wherein step (d) of collecting biomass from the microalgae comprises centrifuging the culture of the microalgae. The method according to claim 13, further comprising the step (e) of drying the biomass collected from the heterotrophic microalgae obtained at the end of step d). 14. The method according to claim 13, further comprising the step (f) of freezing the biomass collected from the heterotrophic microalgae obtained at the end of step d). The method according to claim 1, wherein the culture condition in step c) is an autotrophic culture condition. 5. A method according to claim 4, characterized in that the dilution factor between each successive incubation step is comprised between 1/10 and 1/30. Method according to claim 5, characterized in that step a) corresponds to incubating the heterotrophic microalgae until a concentration of greater than 10 g / l is obtained. The method according to claim 8, characterized in that the volume of the inoculum in step b) corresponds to 1/10 to 1/30 of the culture volume. 10. The method according to claim 9, wherein the photobioreactor is a sealed photobioreactor. 12. The method according to claim 11, characterized in that the heterotrophic microalgae are green algae selected from Chlorella , Scenedesmus and Muriellopsis , or diatoms of Odontella aurita How to.