PROCESS FOR THE PRODUCTION OF CYANOBACTERIA OF THE GENUS APHANIZOMENON

Abstract

The present invention relates to a method for producing cyanobacteria of the genus Aphanizomenon comprising the following steps: (a) inoculating, in an aqueous culture medium, cyanobacteria of the genus Aphanizomenon and microalgae of the genus Chlorella in quantities such that the initial dry mass ratio (microalgae of the genus Chlorella) / (microalgae of the genus Chlorella + cyanobacteria of the genus Aphanizomenon) is between 5% and 70%; (b) maintaining the co-culture under illumination whereby the cyanobacteria of the genus Aphanizomenon and the microalgae of the genus Chlorella grow in the aqueous culture medium; then (c) physically separating the cyanobacteria of the genus Aphanizomenon from the microalgae of the genus Chlorella and recovering the cyanobacteria of the genus Aphanizomenon.

Description

Title of the invention: METHOD FOR PRODUCING CYANOBACTERIA OF THE GENUS APHANIZOMENON Technical field

[0001] The present invention applies to the general field of microorganism production and, more particularly, to the production of cyanobacteria of the genus Aphanizomenon and in particular of cyanobacteria of the species Aphanizomenon flos-aquae.

[0002] Indeed, the present invention proposes a method for the mass production of cyanobacteria of the genus Aphanizomenon, and in particular cyanobacteria of the species Aphanizomenon flos-aquae, by implementing a co-culture of the latter with microalgae of the genus Chlorella, either autotrophically or cyclotrophically. PRIOR TECHNIQUE

[0003] Among the known cyanobacteria, the species Aphanizomenon flos-aquae (AFA) is the subject of increasing interest.

[0004] Indeed, this algae has a composition rich in vitamins, in particular vitamin B12 and vitamin C, in minerals, in essential fatty acids, in proteins containing all the essential amino acids, in pigments such as chlorophylls and phycocyanins, potential antioxidants.

[0005] Such a composition offers real industrial potential in the food, pharmaceutical, and cosmetic sectors. For this reason, AFA is often sold as a food supplement. AFA is also being studied for its potential bioactive properties, with preliminary studies suggesting immunomodulatory, antioxidant, and anti-inflammatory effects.

[0006] This freshwater cyanobacterium is often found, during algal blooms, in nutrient-rich lakes, especially those that are alkaline and shallow, such as Klamath Lake (Oregon, USA) where the harvesting of AFA is an important commercial activity.

[0007] However, the state of the art reports few attempts at controlled culture of this cyanobacterium outside its natural environment. Moreover, the data available in the literature only report culture in small volumes and with poor growth performance (maximum concentration = 170 mg dry matter / L) [1].

[0008] Furthermore, as illustrated in the experimental section below, the inventors' work has shown that, while autotrophic AFA culture is feasible when sterility conditions are maintained, the transition to open culture leads to a cessation of growth, or even biomass lysis, particularly due to contamination by exogenous microorganisms.

[0009] It therefore seems that the culture, in particular non-axenic AF A, is not easy and that achieving large volumes of implementation represents a real challenge.

[0010] Also, the inventors set themselves the goal of proposing a process for producing AFAs in mass without presenting the disadvantages of the processes currently used. Description of the invention

[0011] The stated goals and others are achieved by the invention which proposes a process for producing large quantities of cyanobacteria of the genus Aphanizomenon such as AFA, thanks to the positive effects of the presence of chlorella during the non-axenic culture of cyanobacteria of the genus Aphanizomenon such as AFA, whether in autotrophy or cyclotrophy.

[0012] Indeed, the inventors' work has shown that, when Chlorella is present, a stable consortium is established, allowing for prolonged AFA culture in culture systems that do not exhibit strict containment from the external environment (so-called "open culture systems"), without the appearance of significant microbial contamination. Thus, the presence of Chlorella stabilizes the culture. Furthermore, the presence of Chlorella also stabilizes AFA cultures during the cyclotrophy process.

[0013] Thus, the present invention is based on a stable consortium of two types of phototrophic microorganisms, i.e., which exploit photon capture processes (e.g., photosynthesis) in order to acquire energy and grow, the two types of phototrophic microorganisms being cyanobacteria of the genus Aphanizomenon and microalgae of the genus Chlorella.

[0014] More particularly, the present invention relates to a process for producing cyanobacteria of the genus Aphanizomenon comprising the following steps:

[0015] a) inoculate, in an aqueous culture medium, cyanobacteria of the genus Aphanizomenon and microalgae of the genus Chlorella in quantities such that the initial mass ratio in dry matter (microalgae of the genus Chlorella) / (microalgae of the genus Chlorella + cyanobacteria of the genus Aphanizomenon) is between 5% and 70% (inclusive);

[0016] b) maintain the co-culture under illumination, whereby cyanobacteria of the genus Aphanizomenon and microalgae of the genus Chlorella develop in the aqueous culture medium; then

[0017] c) physically separate cyanobacteria of the genus Aphanizomenon from microalgae of the genus Chlorella and recover cyanobacteria of the genus Aphanizomenon.

[0018] By "process for producing cyanobacteria of the genus Aphanizomenon", means a process for producing large quantities of cyanobacteria of the genus Aphanizomenon, i.e. volumes used greater than 100 liters of culture medium and concentrations of cyanobacteria of the genus Aphanizomenon greater than 170 mg dry matter / L of culture medium.

[0019] In the context of the process according to the present invention, any species of cyanobacteria of the genus Aphanizomenon can be used and produced. In a particular embodiment, the cyanobacteria of the genus Aphanizomenon produced by the process according to the invention are cyanobacteria of the species Aphanizomenon flos-aquae (AFA).

[0020] In the context of the process according to the present invention, any species of microalgae of the genus Chlorella may be used. In one particular embodiment, the microalgae of the genus Chlorella used in the process according to the invention are selected from the group consisting of microalgae of the species Chlorella luteoviridis, microalgae of the species Chlorella pyrenoidosa, microalgae of the species Chlorella vulgaris, and mixtures thereof. In a more particular embodiment, the microalgae of the genus Chlorella used in the process according to the invention are microalgae of the species Chlorella vulgaris.

[0021] To take advantage of the presence of microalgae of the species Chlorella in the aqueous culture medium containing cyanobacteria of the genus Aphanizomenon, it is necessary to have neither too little nor too much of the microalgae of the species Chlorella.

[0022] Indeed, by using an initial mass ratio in dry matter (microalgae of the genus Chlorella) / (microalgae of the genus Chlorella + cyanobacteria of the genus Aphanizomenon) of less than 5%, no effect is observed on the cyanobacteria of the genus Aphanizomenon in culture i.e. the culture of cyanobacteria of the genus Aphanizomenon with such a quantity of microalgae of the species Chlorella behaves like a culture without microalgae of the species Chlorella.

[0023] Similarly, using an initial mass ratio in dry matter (microalgae of the genus Chlorella) / (microalgae of the genus Chlorella + cyanobacteria of the genus Aphanizomenon) greater than 70%, we observe a prevalence of the microalga which will rapidly dominate the co-culture.

[0024] In a particular embodiment of step a) of the process according to the invention, the initial mass ratio of dry matter (microalgae of the genus Chlorella) / (microalgae of the genus Chlorella + cyanobacteria of the genus Aphanizomenon) is between 10% and 65%, in particular between 20% and 60%, and, in particular, between 27% and 55%.

[0025] Furthermore, the mass concentration of cyanobacteria of the genus Aphanizomenon inoculated during step a) is typically greater than or equal to 100 mg dry matter / L of culture medium, in particular greater than or equal to 125 mg dry matter / L of culture medium and, in particular, in the order of 150 mg dry matter / L of culture medium (i.e. 150 mg dry matter / L ± 10 mg dry matter / L of culture medium).

[0026] In step a) of the process according to the invention, cyanobacteria of the genus Aphanizomenon and microalgae of the genus Chlorella may be introduced into the culture medium one after the other or simultaneously. Advantageously, cyanobacteria of the genus Aphanizomenon and microalgae of the genus Chlorella are introduced into the culture medium simultaneously.

[0027] By "aqueous culture medium" is meant an aqueous medium comprising the nutrients enabling the growth of cyanobacteria of the genus Aphanizomenon and microalgae of the genus Chlorella. Typically, this medium comprises one or more elements selected from the group consisting of sodium nitrate (NaNO3), potassium monohydrogen phosphate (K2HPO4), magnesium sulfate (MgSO4), calcium chloride (CaCl2), citric acid, EDTA, sodium carbonate (Na2CO3), iron ammonium citrate, boric acid (H3BO3), manganese chloride (MnCl2), zinc sulfate (ZnSO4), sodium molybdate (Na2MoO4), copper sulfate (CuSO4) and cobalt nitrate (Co(NO3)2). As a specific example of an aqueous culture medium usable within the framework of the invention, we can cite the BG-11 culture medium also known as "modified ATCC medium 616".

[0028] In a first embodiment, the aqueous culture medium used is devoid of organic carbon. In this first embodiment, step b) of the process according to the invention is carried out by autotrophy corresponding to the mode of nutrition of living organisms that can feed solely on inorganic foods in the presence of an external energy source such as, in this case, light (photoautotrophy).

[0029] In a second embodiment, the aqueous culture medium used comprises organic carbon. Any source of organic carbon conventionally used in microorganism cultures is usable in the aqueous culture medium used in the invention. By way of illustrative and non-limiting examples of usable organic carbon sources in the invention, one can cite a lysate of microorganisms such as a bacterial lysate, beef juice, a wheat grain culture, a rice culture, a milk by-product such as whey and in particular powdered whey, or peptones such as soy peptone. In this second embodiment, step b) of the process according to the invention is carried out in Mixotrophy. This mode of nutrition of living organisms is characterized by the fact that they are able to feed themselves either by autotrophy, or by heterotrophy which corresponds to the need for a living organism to feed on pre-existing organic constituents, or by both trophic modes simultaneously.

[0030] In a third embodiment, the aqueous culture medium is initially devoid of organic carbon and the cyanobacteria of the genus Aphanizomenon and the microalgae of the species Chlorella are left in autotrophic culture, during step b), before an input of organic carbon as previously defined.

[0031] The culture conditions implemented during step b) of the process according to the invention are the conditions classically implemented for the culture of photosynthetic microorganisms and well known to those skilled in the art.

[0032] Typically, the pH of the aqueous culture medium, during step a) and / or during step b) of the process according to the invention is greater than 6, in particular between 6.5 and 10 and, in particular, between 8 and 9. As illustrated in the experimental part below, the pH of the aqueous culture medium during step a) is in particular greater than the pH of the aqueous culture medium during step b) of the process according to the invention.

[0033] Advantageously, steps a) and b) of the process according to the invention are carried out at a temperature between 16°C and 30°C and in particular at a temperature of around 25°C (i.e. 25°C ± 1°C).

[0034] In step b) of the process according to the invention, the co-culture of cyanobacteria of the genus Aphanizomenon and microalgae of the genus Chlorella, i.e., the aqueous culture medium containing the cyanobacteria of the genus Aphanizomenon and the microalgae of the genus Chlorella, is under natural or artificial, intermittent or continuous illumination, the cyanobacteria and microalgae using light as an energy source during photosynthesis. Advantageously, the illumination of the co-culture of cyanobacteria of the genus Aphanizomenon and microalgae of the genus Chlorella is constant artificial illumination. For this purpose, any artificial source of light energy, such as, for example, a lamp, a light bulb, a laser, light-emitting diodes (or LEDs), a fluorescent bulb, and / or a chemiluminescent source, can be used in the present invention.It is evident that, whatever the artificial light energy source used, it must emit light of an intensity and wavelength suitable for the photosynthesis of cyanobacteria of the genus Aphanizomenon and microalgae of the genus Chlorella. As a particular example, light-emitting diodes, and in particular light-emitting diodes having an irradiance, in the photosynthetically active radiation spectrum, measured at the liquid surface, equal to 100 pmol.s*.m2, can be used in step b) of the process according to the invention.

[0035] In step b) of the process according to the invention, the co-culture of cyanobacteria of the genus Aphanizomenon and microalgae of the genus Chlorella, i.e., the aqueous culture medium containing the cyanobacteria of the genus Aphanizomenon and the microalgae of the genus Chlorella, is typically subjected to agitation. This agitation results in a homogeneous co-culture, allowing for better access to light and nutrients. Any technique for agitating the culture medium is usable within the scope of the invention. By way of specific examples, agitation by pumping, agitation by blades or propellers, or agitation by a magnetic drive system such as a magnetic stirrer may be cited. As a more specific example, the agitation of the co-culture in step b) is by blade agitation. Typically, the agitation in step b) of the process according to the invention is carried out at a moderate speed.For the purposes of this invention, "moderate speed" means a tangential speed at the end of the agitator less than or equal to 4 m / s, in particular less than or equal to 3 m / s and, in particular, between 0.5 m / s and 2 m / s.

[0036] Typically, step b) of the process according to the invention lasts for a sufficient time to obtain at least a doubling of the cyanobacteria of the genus Aphanizomenon. Advantageously, step b) of the process according to the invention lasts for a time longer than the doubling time of the cyanobacteria of the genus Aphanizomenon. In particular, step b) of the process according to the invention can last at least 2 days, at least 10 days, at least 20 days, at least 30 days, at least 40 days, at least 50 days, or even at least 100 days.

[0037] Step c) of the process according to the present invention consists of a physical separation between cyanobacteria of the genus Aphanizomenon and microalgae of the genus Chlorella, whereby, on the one hand, cyanobacteria of the genus Aphanizomenon are recovered in the form of a phase rich in cyanobacteria of the genus Aphanizomenon and, on the other hand, a first culture must free of cyanobacteria of the genus Aphanizomenon and comprising microalgae of the genus Chlorella and possibly contaminating microorganisms.

[0038] Any technique enabling physical separation based on a morphological or phenotypic difference between cyanobacteria of the genus Aphanizomenon and microalgae of the genus Chlorella can be used in step c) of the process. For example, this could be a difference in size, shape, surface properties, density, propensity for aggregation or flocculation.

[0039] According to a particular embodiment, step c) of physical separation of the process according to the invention comprises a sieving step or a filtration or gravity separation step. An example of filtration usable in the invention is tangential flow filtration or frontal filtration. The filtration can be carried out using a membrane. Advantageously, step c) of the physical separation of the process according to the invention includes a sieving step. In particular, step c) of the physical separation of the process according to the invention includes a sieving step using a sieve, and in particular a vibrating sieve. Typically, the porosity of the sieve used in step c) is greater than 30 µm and in particular between 30 µm and 300 µm.

[0040] Once the cyanobacteria of the genus Aphanizomenon have been recovered, they can be used for any of the current applications in the food, pharmaceutical, and cosmetic sectors. Furthermore, some of the recovered Aphanizomenon cyanobacteria can be used to inoculate an aqueous culture medium as previously defined and repeat steps a) to c) of the process according to the invention.

[0041] Furthermore, the process according to the invention may include an additional step following step c) consisting of recovering the Chlorella microalgae. This additional step consists of physically separating the Chlorella microalgae from the first culture broth obtained at the end of step c).

[0042] Everything previously described for the physical separation in step c) applies mutatis mutandis to the recovery of Chlorella microalgae in this additional step. It should be noted, however, that the porosity of the sieve used in this step is greater than 1 µm and, in particular, between 1 µm and 30 µm. At the end of this additional step, a phase rich in Chlorella microalgae and a second culture broth are obtained, free of Aphanizomenon cyanobacteria and Chlorella microalgae, and possibly containing contaminating microorganisms.

[0043] Once the Chlorella microalgae have been recovered, they can be used for any of the current applications in the food, pharmaceutical, and cosmetic sectors. Furthermore, some of the recovered Chlorella microalgae can be used to inoculate an aqueous culture medium as previously defined and repeat steps a) to c) of the process according to the invention. Advantageously, when recovered Chlorella microalgae are used to inoculate an aqueous culture medium or to be reintroduced into an aqueous culture medium, this inoculation or reintroduction is carried out simultaneously with the inoculation or reintroduction of Aphanizomenon cyanobacteria, whether recovered or not.

[0044] When the second culture mash contains contaminating microorganisms, these can be physically separated from the mash to generate a phase rich in contaminating microorganisms. Therefore, the process according to the invention may include an additional step of recovering contaminating microorganisms following the recovery of microalgae of the genus Chlorella.

[0045] By "contaminating microorganisms" is meant microorganisms capable of growing in the aqueous culture medium as previously defined and affecting the growth of cyanobacteria of the genus Aphanizomenon and microalgae of the genus Chlorella. Examples of such microorganisms include bacteria or microalgae such as microalgae of the genus Nanochloropsis.

[0046] Everything previously described for the physical separation in step c) applies mutatis mutandis to the recovery of contaminating microorganisms in this additional step. It should be noted, however, that the porosity of the sieve used in this step is less than 1 µm, specifically less than 0.65 µm and, in particular, less than 0.2 µm. At the end of this additional step, a phase rich in contaminating microorganisms and a second culture mash free of microorganisms are obtained.

[0047] The contaminating microorganisms recovered at the end of this additional step can be lysed and thus provide a source of usable organic carbon, as previously mentioned, in the aqueous culture medium during the process according to the invention. This particular embodiment corresponds to a cyclotrophic process.

[0048] As a reminder, cyclotrophy is a process described in patent application FR 3 085 960 Al [2] in the name of Kyanos Biotechnologies and exploited by that company, consisting of producing AFA in mixed culture (comprising different microorganisms) through successive cycles of culture / separation. The culture is carried out in culture equipment that does not contain the surrounding environment and is mixotrophic (i.e., in the presence of both organic substrate and light), while the separation steps allow (i) the harvesting and concentration of the AFA and (ii) the specific separation of the other microorganisms in order to lyse them before the return of the nutrient stream thus produced to the culture basin.

[0049] Other features and advantages of the present invention will become apparent to the person skilled in the art upon reading the examples below given by way of illustration and not limitation, with reference to the attached figures. Brief description of the drawings

[0050] Figure 1 shows the evolution of the reconstituted biomass (X) over time for five different pure AFA cultures during autotrophic growth. Cultures A and B were grown to a maximum volume of 1 L before use. Cultures C, D, and E were grown to maximum volumes of 10 L, 25 L, and 3 L, respectively, before collapsing. The black arrows represent the cessation of culture due to the observation of lysis or the complete absence of AFA in the culture.

[0051] Figure 2 shows the evolution of the reconstituted biomass (X) over time for two different high-volume AFA cultures in the presence of autotrophic chlorella. Culture A was grown to more than 18 L and culture B to more than 30 L.

[0052] DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS Materials and Methods Microorganisms used

[0053] The Aphanizomenon flos-aquae strain used in this study is a strain isolated by KYANOS BIOTECHNOLOGIES. This strain is regularly subcultured on liquid medium to ensure its maintenance. A portion of the maintenance cultures is used as preculture inoculum in the event that culture trials are carried out.

[0054] The Chlorella vulgaris strain used in this study was obtained from the Algae Research Supply microalgal collection (ref ACCv-01000). The supplied inoculum was then regularly subcultured onto liquid medium to ensure its maintenance. A portion of the maintenance cultures is used as preculture inoculum in the event that culture trials are carried out. Growing environments

[0055] The culture medium used is a BG11 medium (detailed composition provided in the appendix, tables 1 to 4 below), native for autotrophic culture.

[0056] [Tables 1] Product Concentration g / L Na2MgEDTA 0.1 Ferric ammonium citrate 0.6 Citric acid, H2O 0.6 CaCl2, 2H2O 3.6 Osmosis water, sterilized 20 minutes at 121°C or filtered (store at 4°C)

[0057] Table 1: Composition of stock solution 1

[0058] [Tables2] Product Concentration ng / L MgSO4, 7H2O 7.5 Osmosis water, sterilized for 20 minutes at 121°C or filtered (store at 4°C)

[0059] Table 2: Composition of stock solution 2

[0060] [Tables3] Product Concentration g / L K2HPO4 0.6 Osmosis water, sterilized for 20 minutes at 1-21°C or filtered (store at 4°C)

[0061] Table 3: Composition of stock solution 3

[0062] [Tables4] Product Concentration g / L or mL / L Stock Solution 1 (mL) 10 Stock Solution 2 (mL) 10 Stock Solution 3 (mL) 10 Na2CO3 (g) 0.02 NaNO3 (g) 1.5 Osmosis water, pH adjusted to 9, sterilized for 20 minutes at 121°C

[0063] Table 4: Composition of the BG11 nutrient medium solution

[0064] This medium is used with added soy peptone (supplier: Cari Roth, reference 2365.4) at a final concentration of 1.0 gL', for cyclotrophic culture™.

[0065] For the sake of experimental simplification, the organic substrate consisting of the soybean peptone solution is added by scoops into the culture medium. Operating conditions of the cultures

[0066] The co-cultures of A'Aphanizomenon flos-aquae and Chlorella vulgaris are carried out in an air-conditioned room where the temperature is regulated at 25±1°C.

[0067] The reactor used is of the "raceway" type with a liquid height of up to 1.5 m. It is equipped with a paddle agitator (marine propeller type agitator, diameter of the agitator = 200 mm, agitation speed = 20-200 rpm).

[0068] The light supply is provided by 2 light-emitting diode panels (Supplier: Grocruiser, reference: GL-CR600 65W, irradiance of the synthetically active photo radiation measured at the liquid surface = 100 pmol.s *.m2).

[0069] The inoculation of the cultures was carried out so as to obtain an initial concentration in AFA close to 150 mg DM.L. The inoculation with chlorella was carried out so as to achieve an initial mass ratio chlorella / AFA+chlorella of between 27% and 55%.

[0070] Unit operations and operating conditions for the separation of AFA / chlorella operated on as part of the cyclotrophy treatment

[0071] The separation between AFA, chlorella and other microorganisms present is carried out by sieving using a vibrating sieve (Supplier: Russell Finex, reference: Eco-separator 40').

[0072] The system comprises different separation stages allowing for the selective recovery of an AFA-enriched phase, a chlorella-enriched phase, and a pass-through containing the majority of the other microorganisms. Each separation stage is characterized by the use of a sieving support with a chosen porosity.

[0073] For the first stage (refuse = phase enriched in AFA), the sieve porosity is between 30 µm and 300 µm. For the second stage (refuse = phase enriched in chlorella, passing = culture must containing the majority of the other microorganisms), the sieve porosity is between 1 µm and 30 µm.

[0074] The operating conditions applied make it possible to obtain a volumetric concentration factor (ratio between the feed flow rate of the stage and the flow rate of the retained phase) greater than or equal to 2, and preferably greater than or equal to 10.

[0075] Since the mass concentrations of microorganisms in each retained phase (rejection from the first stage and rejection from the second stage) are known, it is possible to re-inoculate a culture with a defined AFA / chlorella ratio from determined volumes of the 2 rejection phases.

[0076] Cultivation of AFA alone or in the presence of chlorella and in autotrophy

[0077] High-scale AFA culture (> m3) requires obtaining a sufficient culture volume of inoculum beforehand (> 100 L). The increase in volume is gradual: starting with a stock strain (approximately 20 mL), which is progressively transferred into increasingly larger volumes (200 mL; 1 L; 3 L; 5 L; 25 L; 100 L).

[0078] For practical reasons, small volume cultures (20 mL - 3 L) are carried out under sterile conditions, while larger volumes (5 L - 100 L) are carried out under open conditions (i.e. without special attention to sterility).

[0079] This increase in volume occurs under autotrophic conditions, that is, in the absence of organic carbon and solely under the condition of illumination. This condition allows a priori to limit the development of bacteria, whose growth is dependent on the presence of organic substrate, mineral nutrients and predominantly mesophilic physico-chemical conditions (between 20°C and 40°C) and pH between 6 and 10 pH units (preferably alkaline between 8.5 and 9.5 pH units).

[0080] Figure 1 shows the evolution of the reconstituted biomass (X) (in g) over time for five different AFA cultures grown in volume. This reconstituted biomass is calculated from the estimated concentration multiplied by the volume. Since the cell concentration is kept relatively constant (around an optical density equivalent of around 0.3), the lowest reconstituted biomass values ​​are representative of the small culture volumes (between 20 mL and 1 L).

[0081] Cultures A and B were grown in a maximum volume of 2 L, while cultures C and D were progressively increased in volume up to 25 L. Culture E, on the other hand, was grown up to a maximum volume of 3 L.

[0082] During this routine volume increase procedure, it is observed that cultivating AFA at low volumes, i.e. less than 2 L, under autotrophic conditions is possible over extended periods of culture, up to nearly 140 days for culture B without contamination or collapse of biomass.

[0083] In the case of cultures C and D, an AFA culture in autotrophy could be maintained for nearly 45 days in volumes between 3 L and 10 L. On the other hand, a collapse of biomass was observed a few days after the increase in volume to 10 L (culture C) or 25 L (culture D), materialized by the black arrows on the graphs.

[0084] Finally, while the increase in volume between 3 L and 10 L was possible for these two cultures, they constitute an exception. Indeed, in most cases, we observed a collapse in biomass as soon as the volume increased above 3 L. This is illustrated by culture E, which, after increasing its volume beyond 3 L, saw its biomass gradually decline at approximately 120 days. In most of the other cultures that increased in volume (data not shown), we observed this same, more or less rapid, decline in biomass. Microscopic observations generally indicate the absence of AFA and significant bacterial contamination (leading to cell lysis) or other microalgal species (mainly Nanochloropsis sp.).

[0085] In the two cultures that could be maintained in volumes greater than 3 L (cultures C and D), the presence of Chlorella was detected throughout the culture. Chlorella therefore appears to have a stabilizing effect on the growth of AFA.

[0086] Two other AFA cultures were therefore initiated, with a controlled addition of chlorella ([Fig. 2]). This addition of chlorella is carried out by adding a specific volume of axenic chlorella culture broth to the AFA culture. The volume added is determined to obtain an initial AFA / chlorella ratio of approximately 2:1 (mass concentration ratio). Culture A was successfully increased in volume to 18 L in less than 30 days. The culture is still ongoing. Culture B, on the other hand, was successfully increased in volume to more than 30 L in about 20 days.

[0087] In conclusion, while autotrophic AFA culture is feasible when sterile conditions are maintained, switching to open culture leads to biomass collapse, primarily due to contamination by exogenous microorganisms. When Chlorella is present, a stable consortium is established, allowing for prolonged AFA culture without significant microbial contamination. Thus, the presence of Chlorella helps stabilize the culture.

[0088] Stabilization of AFA cultures during the cyclotrophy process in the presence of chlorella

[0089] The result presented in the preceding paragraph, valid in autotrophy, was also observed during a cyclotrophy process.

[0090] Thus, AFA production by cyclotrophy with an organic substrate X was implemented. To do this, three successive culture / separation cycles were carried out in an open 1000 L tank.

[0091] The first cycle (cycle 1) was carried out with a nearly pure culture of AFA. This culture could not be maintained, and a collapse in the AFA mass was observed (explaining the observed negative productivity). This collapse was accompanied by a drop in pH throughout the culture, with a decrease of approximately 0.91 pH units / h. Despite regular adjustment to 8.3 pH units, a large portion of the culture settled to the bottom of the tank. The supernatant was yellowish, indicative of high bacterial activity.

[0092] Considering these results and the results obtained in autotrophy, it was decided to cultivate AFA in consortium with chlorella (cycle 2 and cycle 3). The ratio was arbitrarily set at approximately 70% AFA to 30% chlorella.

[0093] In these cases, the AFA culture was successfully maintained, with high productivity during cycle 2 of nearly 8.9 g / h, yielding 247 g of AFA. For comparison, the autotrophic productivity obtained under similar conditions is 0.16 g / h.

[0094] The productivity obtained in cycle 3 is indeed lower, but remains nearly 20 times higher than the autotrophic productivity. It is likely that this lower productivity is due to a very high initial concentration of AFA.

[0095] We observed that, in cycles 2 and 3, the rate of pH change was nearly 2.3 times lower (approximately -0.4 pH units / h). The presence of chlorella therefore had a stabilizing effect on the pH. This stabilization certainly had a positive effect on the growth of AFA, resulting in improved productivity.

[0096] [Tables5] Cycle 1 Cycle 2 Cycle 3 AFA Productivity (g / h) -470 8.9 2.5 Total AFA Mass (g) 62.8 247 289.7 %AFA 99.5 72.3 66.5 Delta pH (upH / h) -0.91 ±0.18 -0.39 +0.18 -0.37 +0.13

[0097] Table 5: AFA productivity data over several cyclotrophic cycles. References

[0098] [1] Debella, 2005, “Mass culture of Aphanizomenon flos -aquae Ralfs ex Bom. And Flah. Var. flos -aquae (cyanobacteria) from Klamath falls, Oregon, USA, in closed chamber bioreactors”, Ethiop. J. Biol. Sci., vol. 4(2), pages 135-145.

[0099] [2] French patent application FR 3 085 960 Al in the name of Kyanos Biotechnologies published March 20, 2020.

Claims

Demands

1. A process for producing cyanobacteria of the genus Aphanizomenon comprising the following steps: a) inoculating, in an aqueous culture medium, cyanobacteria of the genus Aphanizomenon and microalgae of the genus Chlorella in quantities such that the initial dry mass ratio (microalgae of the genus Chlorella) / (microalgae of the genus Chlorella + cyanobacteria of the genus Aphanizomenon) is between 5% and 70%; b) maintaining the co-culture under illumination whereby the cyanobacteria of the genus Aphanizomenon and the microalgae of the genus Chlorella grow in the aqueous culture medium; then c) physically separating the cyanobacteria of the genus Aphanizomenon from the microalgae of the genus Chlorella and recovering the cyanobacteria of the genus Aphanizomenon.

2. A method according to claim 1, characterized in that said cyanobacteria of the genus Aphanizomenon are cyanobacteria of the species Aphanizomenon flos-aquae (AFA).

3. A method according to claim 1 or 2, characterized in that said microalgae of the genus Chlorella are microalgae of the species Chlorella vulgaris.

4. A method according to any one of claims 1 to 3, characterized in that the mass concentration of cyanobacteria of the genus Aphanizomenon inoculated during step a) is greater than or equal to 100 mg dry matter / L of culture medium, in particular greater than or equal to 125 mg dry matter / L of culture medium and, in particular, in the order of 150 mg dry matter / L of culture medium (i.e. 150 mg dry matter / L ± 10 mg dry matter / L of culture medium).

5. A method according to any one of claims 1 to 4, characterized in that said aqueous culture medium is devoid of organic carbon.

6. A method according to any one of claims 1 to 4, characterized in that said aqueous culture medium comprises organic carbon.

7. A method according to any one of claims 1 to 4, characterized in that said aqueous culture medium is initially devoid of organic carbon and the cyanobacteria of the genus Aphanizomenon and the microalgae of the species Chlorella are left in autotrophic culture, during step b), before an input of organic carbon.

8. A method according to any one of claims 1 to 7, characterized in that said step c) comprises a sieving step.

9. A process according to any one of claims 1 to 8, characterized in that said process has an additional step, following said step c), consisting of recovering microalgae of the genus Chlorella.

10. A process according to claim 9, characterized in that said process comprises an additional step of recovering contaminating microorganisms following the recovery of microalgae of the genus Chlorella.

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