Method for producing 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 cyanobacteria of the genus Aphanizomenon and microalgae of the genus Chlorella into an aqueous culture medium in amounts such that the initial weight ratio of dry matter (microalgae of the genus Chlorella) / (microalgae of the genus Chlorella + cyanobacteria of the genus Aphanizomenon) is between 5% and 70%; (b) keeping the co-culture under illumination, whereby the cyanobacteria of the genus Aphanizomenon and the microalgae of the genus Chlorella develop 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

[0001] PROCESS FOR THE PRODUCTION OF CYANOBACTERIA OF THE GENUS APHANIZOMENON

[0002] TECHNICAL FIELD

[0003] The present invention applies to the general field of microorganism production and, more particularly, to the production of cyanobacteria of the genus Aphanizomenon and especially of cyanobacteria of the species Aphanizomenon flos-aquae.

[0004] Indeed, the present invention proposes a process for the mass production of cyanobacteria of the genus Aphanizomenon and in particular of cyanobacteria of the species Aphanizomenon flos-aquae by implementing a co-culture of the latter with microalgae of the genus Chlorella and this, in autotrophy or in cyclotrophy.

[0005] PREVIOUS STATE OF THE ART

[0006] Among the known cyanobacteria, the species Aphanizomenon flos-aquae (AFA) is the subject of increasing interest.

[0007] Indeed, this algae has a composition rich in vitamins, particularly vitamin B12 and vitamin C, minerals, essential fatty acids, proteins containing all the essential amino acids, pigments such as chlorophylls and phycocyanins, potential antioxidants.

[0008] This composition offers significant industrial potential in the food, pharmaceutical, and cosmetic sectors. For this reason, AFA is often sold as a dietary supplement. AFA is also being studied for its potential bioactive properties, with preliminary studies suggesting immunomodulatory, antioxidant, and anti-inflammatory effects.

[0009] 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.

[0010] However, the current state of research reports few attempts at controlled culture of this cyanobacterium outside its natural environment. Furthermore, the available data in the literature only describe culture in small volumes and with poor growth performance (maximum concentration = 170 rng). ma dry heat / L) [1].

[0011] Furthermore, as illustrated in the experimental section below, the inventors' work has shown that, while the culture of AFA in autotrophy 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.

[0012] It therefore seems that the culture, particularly the non-axenic culture of AFA, is not easy and that achieving large volumes of implementation represents a real challenge.

[0013] Therefore, the inventors set themselves the goal of proposing a process that would allow the mass production of AFAs without the drawbacks of the processes currently used.

[0014] DESCRIPTION OF THE INVENTION

[0015] 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.

[0016] 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 provide strict containment from the external environment (so-called "open culture systems"), without significant microbial contamination. Thus, the presence of chlorella stabilizes the culture. Furthermore, the presence of chlorella also stabilizes AFA cultures during the cyclotrophy process.

[0017] 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) to acquire energy and grow, the two types of phototrophic microorganisms being cyanobacteria of the genus Aphanizomenon and microalgae of the genus Chlorella.

[0018] More particularly, the present invention relates to 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 Ch / ore / / a) / (microalgae of the genus Chlorella + cyanobacteria of the genus Aphanizomenon) is between 5% and 70% (inclusive); 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.

[0019] By "process for producing cyanobacteria of the genus Aphanizomenon", we mean a process enabling the production in large quantities of cyanobacteria of the genus Aphanizomenon i.e. volumes used greater than 100 litres of culture medium and concentrations of cyanobacteria of the genus Aphanizomenon greater than 170 mg dry matter / L of culture medium.

[0020] 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).

[0021] In the process according to the present invention, any species of microalgae of the genus Chlorella may be used. In one particular embodiment, the Chlorella microalgae used in the process according to the invention are selected from the group consisting of Chlorella luteoviridis, Chlorella pyrenoidosa, Chlorella vulgaris, and mixtures thereof. In a more specific embodiment, the Chlorella microalgae used in the process according to the invention are Chlorella vulgaris.

[0022] To take advantage of the presence of Chlorella microalgae in the aqueous culture medium containing cyanobacteria of the genus Aphanizomenon, it is necessary to have neither too little nor too much Chlorella microalgae.

[0023] Indeed, using an initial dry matter mass ratio (microalgae of the genus Ch / ore / / a) / (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.

[0024] Similarly, using an initial mass ratio of dry matter (microalgae of the genus Ch / ore / / a) / (microalgae of the genus Chlorella + cyanobacteria of the genus Aphanizomenon) greater than 70%, we observe a prevalence of the microalga which will quickly dominate the co-culture.

[0025] 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 Ch / ore / / a) / (microalgae of the genus Chlorella + cyanobacteria of the genus Aphanizomenon) is between 5% and 65%, more preferably between 10% and 65%, in particular between 20% and 60%, and, in particular, between 27% and 55%.

[0026] Furthermore, the mass concentration of cyanobacteria of the genus Aphanizomenon inoculated during step a) is typically greater than or equal to 100 rng dry matter / L of culture medium, in particular greater than or equal to 125 rng dry matter / L of culture medium and, in particular, in the order of 150 rng dry matter / L of culture medium (i.e. 150 rng dry matter / L ± 10 rng dry matter / L of culture medium).

[0027] 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 sequentially or simultaneously. Advantageously, the cyanobacteria of the genus Aphanizomenon and the microalgae of the genus Chlorella are introduced into the culture medium simultaneously. "Aqueous culture medium" means an aqueous medium containing the nutrients necessary for 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 (MgSC₂), calcium chloride (CaCb), citric acid, EDTA, sodium carbonate (Na₂CO₃), iron ammonium citrate, boric acid (H₃BO₃), manganese chloride (MnC₂), zinc sulfate (ZnSC₂), sodium molybdate (Na₂MoU₄), copper sulfate (CuSC₂), and cobalt nitrate (CojNC₂H). As a specific example of an aqueous culture medium usable within the scope of the invention, BG-11 culture medium, also known as "modified ATCC medium 616," may be cited.

[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 suitable for the aqueous culture medium used in the invention. Illustrative and non-limiting examples of usable organic carbon sources in the invention include 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, particularly powdered whey, or peptones such as soy peptone. In this second embodiment, step b) of the process according to the invention is carried out under mixotrophy.This mode of nutrition for living organisms is characterized by their ability to feed 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. 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) for a time t before the addition of organic carbon as previously defined.

[0030] 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.

[0031] 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.

[0032] 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).

[0033] 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, is usable 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 specific example, light-emitting diodes, and in particular light-emitting diodes exhibiting an irradiance, in the photosynthetically active radiation spectrum, measured at the liquid surface, equal to 100 pmol.s Tnr. 2 can be used during step b) of the process according to the invention.

[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 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.By "moderate speed", we mean, within the framework of the present invention, 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.

[0035] Typically, step b) of the process according to the invention lasts for a sufficient time to achieve 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.

[0036] 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.

[0037] 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 involve a difference in size, shape, surface properties, density, or propensity for aggregation or flocculation.

[0038] According to a particular embodiment, step c) of the 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. Filtration can be carried out using a membrane. Advantageously, step c) of the physical separation of the process according to the invention comprises a sieving step. In particular, step c) of the physical separation of the process according to the invention comprises 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.

[0039] Once the Aphanizomenon cyanobacteria have been recovered, they can be used for any of the current applications in the food, pharmaceutical, and cosmetic industries. 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.

[0040] Furthermore, the process according to the invention may include an additional step following step c) 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).

[0041] 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, specifically between 1 µm and 30 µm. At the end of this additional step, a phase rich in Chlorella microalgae is obtained, along with a second culture wash free of Aphanizomenon cyanobacteria and Chlorella microalgae, and potentially containing contaminating microorganisms.

[0042] 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 or reintroduce an aqueous culture medium, this inoculation or reintroduction occurs simultaneously with the inoculation or reintroduction of Aphanizomenon cyanobacteria, whether recovered or not.

[0043] 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 the contaminating microorganisms following the recovery of the Chlorella microalgae.

[0044] The term "contaminating microorganisms" refers to 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 Nanochloropsis.

[0045] 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 must free of microorganisms are obtained.

[0046] The contaminating microorganisms recovered after this additional step can be lysed, thus providing 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.

[0047] 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 this 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 offers no containment from the surrounding environment and is mixotrophic (i.e., in the presence of both organic substrate and light), while the separation steps allow for (i) harvesting and concentrating the AFA and (ii) specifically separating the other microorganisms in order to lyse them before the resulting nutrient stream is returned to the culture basin.

[0048] 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.

[0049] BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 shows the evolution of 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 failing. The black arrows indicate culture termination due to observed lysis or the complete absence of AFA in the culture. Figure 2 shows the evolution of 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.

[0051] Figure 3 shows the evolution of microorganism concentrations in two cultures grown under mixotrophy in the presence of a carbohydrate substrate. Triangles represent absorbance values ​​for the AFA / chlorella co-culture, and squares represent absorbance values ​​for the AFA culture alone.

[0052] Figure 4 shows the evolution of the growth rate of AFA for different cultures carried out in cyclotrophy, with initial AFA ratios ranging from 62% to 100% (100% corresponding to the culture of AFA without the addition of chlorella).

[0053] DETAILED DESCRIPTION OF SPECIFIC METHODS OF IMPLEMENTATION

[0054] Materials and Methods

[0055] Microorganisms used

[0056] The Aphanizomenon flos-aquae strain used in this study was 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 pre-culture inoculum when culture trials are performed.

[0057] 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 pre-culture inoculum in cases where culture trials are performed.

[0058] Growing environments

[0059] The culture medium used is BG11 medium (detailed composition provided in the appendix, Tables 1 to 4 below), native for autotrophic culture. [Table 1]

[0060] Table 1: Composition of stock solution 1

[0061] [Table 2]

[0062] Table 2: Composition of stock solution 2

[0063] [Table 3]

[0064] Table 3: Composition of stock solution 3

[0065] [Table 4]

[0066] Table 4: Composition of the BG11 nutrient medium solution. This medium is used with the addition of soy peptone (supplier: Cari Roth, reference 2365.4) to a final concentration of 1.0 gL -1 , for cyclotrophic culture™.

[0067] For the sake of experimental simplification, the organic substrate consisting of the soybean peptone solution is added by scoops into the culture medium.

[0068] Operating conditions of the cultures

[0069] The co-cultures of Aphanizomenonflos-aquae and Chlorella vulgaris are carried out in an air-conditioned room where the temperature is regulated at 25±1°C.

[0070] 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 impeller = 200 mm, agitation speed = 20-200 rpm).

[0071] Light is provided by two LED panels (Supplier: Grocruiser, reference: GL-CR600 65W, irradiance of photosynthetically active radiation measured at the liquid surface = 100 pmoI.s Lnr) 2 ).

[0072] The cultures were inoculated in such a way as to obtain an initial AFA concentration of approximately 150 mg DM.L -1 . Chlorella inoculation was carried out in such a way as to achieve an initial mass ratio of chlorella / AFA+chlorella between 27% and 55%.

[0073] Unit operations and operating conditions for AFA / chlorella separation carried out within the framework of cyclotrophy

[0074] 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').

[0075] The system comprises several separation stages, allowing for the selective recovery of an AFA-enriched phase, a chlorella-enriched phase, and a pass-through containing the majority of other microorganisms. Each separation stage is characterized by the use of a sieving medium with a chosen porosity.

[0076] For the first stage (refuse = AFA-enriched phase), the sieve porosity is between 30 µm and 300 µm. For the second stage (refuse = Chlorella-enriched phase, pass-through = culture must containing the majority of the other microorganisms), the sieve porosity is between 1 µm and 30 µm. The operating conditions applied allow for a volumetric concentration factor (ratio between the stage feed rate and the retained phase flow rate) greater than or equal to 2, and preferably greater than or equal to 10.

[0077] Since the mass concentrations of microorganisms in each phase retained (rejection from the first stage and rejection from the second stage) are known, it is possible to reinoculate a culture with a defined AFA / chlorella ratio from determined volumes of the 2 rejection phases.

[0078] Culture of AFA alone or in the presence of chlorella and in autotrophy

[0079] High-scale AFA culture (> m3 ) requires obtaining a sufficient culture volume of inoculum beforehand (> 100 L). The increase in volume is progressive: we start 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).

[0080] 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).

[0081] 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, the limitation of bacterial development, whose growth is dependent on the presence of organic substrate, mineral nutrients, and predominantly mesophilic physicochemical conditions (between 20°C and 40°C) and a pH between 6 and 10 pH units (preferably alkaline between 8.5 and 9.5 pH units).

[0082] Figure 1 shows the evolution of reconstituted biomass (X) (in g) over time for five different AFA cultures grown to 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 0.3), the lowest reconstituted biomass values ​​are representative of the small culture volumes (between 20 mL and 1 L). Cultures A and B were grown to a maximum volume of 2 L, while cultures C and D were progressively grown to 25 L. Culture E was grown to a maximum volume of 3 L.

[0083] 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.

[0084] In the case of cultures C and D, an AFA culture in autotrophy was able to 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.

[0085] Finally, while the volume increase between 3 L and 10 L was possible for these two cultures, they represent 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, experienced a gradual decline in biomass at approximately 120 days. In most of the other cultures that underwent volume increase (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 the presence of other microalgal species (mainly Nanochloropsis sp.).

[0086] In both cultures that could be maintained in volumes greater than 3 L (cultures C and D), the presence of Chlorella was detected throughout the entire growth cycle. Chlorella therefore appears to have a stabilizing effect on the growth of AFA.

[0087] Two additional AFA cultures were therefore initiated, with a controlled addition of chlorella (Figure 2). This chlorella addition is achieved by introducing a specific volume of axenic chlorella culture broth into 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 to 18 L in less than 30 days and is still ongoing. Culture B, on the other hand, was successfully increased to over 30 L in approximately 20 days.

[0088] 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.

[0089] Stabilization of AFA cultures during the cyclotrophy process in the presence of chlorella

[0090] The result presented in the previous paragraph, valid in autotrophy, was also observed during a cyclotrophy process.

[0091] Thus, the production of AFA using cyclotrophy with an organic substrate X was implemented. To this end, three successive culture / separation cycles were carried out in a 1000 L open tank. The organic substrate X, a liquid industrial by-product containing a mixture of glucose and fructose, was added to the culture medium to achieve a glucose equivalent concentration of 39 g / L. The duration of each of cycles 1, 2, and 3 was set at 24 hours. The culture was carried out in a climate-controlled room with a temperature regulated at 25 ± 1°C, in a raceway-type reactor agitated by a paddle. Light was provided during the culture, and the irradiance of the photosynthetically active radiation measured at the liquid surface during the culture was approximately 100 pmol·s⁻². -2 .

[0092] The first cycle (cycle 1) was performed 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.

[0093] Considering these results and the results obtained in autotrophy, it was decided to cultivate AFA in a consortium with Chlorella (cycles 2 and 3). The initial ratio at the beginning of cycles 2 and 3 was arbitrarily set at approximately 70% AFA to 30% Chlorella. Between cycles 1 and 2, and between cycles 2 and 3, the entire medium was treated by filtration, and the fraction required for inoculation of the next trial, based on AFA mass concentration criteria, was reintroduced into the culture system.

[0094] For cycles 2 and 3, 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.

[0095] While the productivity achieved in cycle 3 is lower, it remains nearly 20 times greater than autotrophic productivity. This lower productivity is likely due to a very high initial concentration of AFA.

[0096] We observed that, in cycles 2 and 3, the rate of pH change was nearly 2.3 times slower (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.

[0097] [Table 5]

[0098] Cycle 1 Cycle 2 Cycle 3

[0099] Productivity AFA (g / h) -470 8.9 2.5

[0100] Total mass AFA (g) 62.8 247 289.7

[0101] %AFA 99.5 72.3 66.5

[0102] Delta pH (upH / h) -0.91 ± 0.18 -0.39 ± 0.18 -0.37 ± 0.13

[0103] Table 5: AFA productivity data over several cyclotrophic cycles.

[0104] Culture of AFA alone and in the presence of chlorella, under mixotrophy (supply of a glycosidic substrate)

[0105] The inventors demonstrated improved robustness and productivity in a culture containing AFA and chlorella during mixotrophic cultures after the introduction of a carbohydrate substrate. In this case, the carbohydrate substrate used is an industrial by-product consisting of a mixture of glucose and fructose.

[0106] The inventors carried out two mixotrophic cultures in Erlenmeyer flasks in the presence of a carbohydrate substrate: a first culture of AFA in the presence of chlorine and a second of AFA alone. The first culture was inoculated with AFA at a concentration of 0.060 g / L and inoculated with Chlorella at a concentration of 0.060 g / L, resulting in an AFA / Chlorella mass ratio of 50% / 50%. The second culture was inoculated with AFA at a concentration of 0.124 g / L. The other culture parameters used were identical for the first and second cultures, including the addition of a carbohydrate substrate to the medium to achieve an equivalent glucose concentration of 39 g / L and cultivation under illumination with photosynthetically active radiation of 100 pmol / s _1 .nT 2 , at a temperature maintained between 20°C and 30°C, preferably around 25°C and under continuous stirring at 80 rpm. The stirring of the medium is ensured by an orbital shaker.

[0107] The evolution of microorganism concentrations in these two cultures was assessed by measuring the absorbance of the culture medium at 620 nanometers. The results of these absorbance measurements are shown in Figure 3. In Figure 3, the triangles represent the absorbance values ​​for the AFA / chlorella co-culture, and the squares represent the absorbance values ​​for the AFA culture alone. From these absorbance measurements, the growth rate of both cultures was calculated.

[0108] During these cultures, the inventors observed that, despite the presence of a carbohydrate substrate, the AFA culture alone did not appear to exhibit a growth phase. Microscopic observation of the cells revealed short cells and a significant presence of material resulting from cell lysis. The final AFA concentration was 0.126 g / L, compared to an initial concentration of 0.124 g / L.

[0109] The AFA / chlorella co-culture, on the other hand, showed marked microbial growth. The final AFA concentration was 0.489 g / L, and the chlorella concentration was 0.094 g / L, resulting in an AFA / chlorella mass ratio of 84% / 16%. In terms of the specific growth rate of AFA, this represents a value of 7.2 x 10⁻³ -4 h 1 for the cultivation of AFA alone and 9.5.10 -2 h 1 for AFA / chlorella co-culture. The growth rate observed for AFA alone is very low compared to those classically obtained for AFA alone autotrophic cultures and is due to the rapid appearance of microbial contamination which will lower the pH and slow down, or even inhibit, the growth of AFA.

[0110] These results show the positive influence of an initial fraction of chlorella on the growth of AFA during a mixotrophic culture.

[0111] Culture of AFA alone and in the presence of chlorella, under cyclotrophy with the addition of plant peptone

[0112] Nineteen AFA culture trials, both alone and in the presence of Chlorella, were also conducted by the inventors under cyclotrophic conditions, with the addition of plant peptone at a mass concentration of 0.2 g / L. The reactor used for these tests was a raceway-type reactor with a liquid head of up to 1.5 m. The initial mass ratio of AFA to AFA+chlorella inoculation varied from 62% to 100%, with 100% corresponding to an AFA culture alone, without Chlorella inoculation. The other culture parameters used in the 19 trials were identical, including culture under illumination with 100 pmol·s⁻¹·nRH photosynthetically active radiation. 2 , at a temperature of around 25°C and under stirring at 20-200 rpm. The stirring of the medium is ensured by a paddle shaker.

[0113] Each trial lasted 24 hours. The growth rate (average growth rate) was calculated over the entire 24-hour culture period. The growth performance of AFA was estimated by calculating the specific growth rate of AFA. Data from the 19 trials are shown in Figure 4, which presents the AFA growth rate observed in each trial. As illustrated in the graph in Figure 4, the initial AFA / AFA+chlorella inoculation ratios tested were 62%, 76%, 79%, 90%, 95%, and 100% (AFA alone, 14 trials).

[0114] It is therefore possible to see that for cultures of AFA alone, the growth rate varies from a value of zero (no growth) to a value of 0.01h -1 It is also possible to deduce that the initial addition of chlorella promotes AFA growth, since for ratios below 100%, AFA growth rate values ​​increase up to a value of 0.031h~ 1, which is 3 times the maximum value observed for the culture of AFA alone.

[0115] These trials highlight the positive influence of an initial fraction of chlorella on the growth of AFA during cyclotrophic culture. In particular, a beneficial effect is observed for initial mass ratios of AFA / AFA+chlorella inoculation of 95% or less, i.e., for initial mass ratios of chlorella / AFA+chlorella inoculation greater than or equal to 5%. References

[0116] [1] Debella, 2005, "Mass culture of Aphanizomenon flos-aquae Ralfs ex Born. And Flah. Var. flos-aquae (cyanobacteria) from Klamath falls, Oregon, USA, in closed chamber bioreactors", Ethiop. J. Biol. Sci., vol. 4(2), pp. 135-145.

[0117] [2] French patent application FR 3 085 960 Al in the name of Kyanos Biotechnologies published on 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 Ch / ore / / a) / (microalgae of the genus Chlorella + cyanobacteria of the genus Aphanizomenon) is between 5% and 70%; b) maintaining the co-culture under illumination by means of which 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, wherein the microalgae of the genus Chlorella used 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.

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

5. A method according to any one of claims 1 to 4, characterized in that the mass concentration of cyanobacteria of the genus Aphanizomenon inoculated in step a) is greater than or equal to 100 ng mat dry matter / L of culture medium, in particular greater than or equal to 125 ng matdry 1 / L of culture medium and, in particular, of 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).

6. A process according to any one of claims 1 to 5, wherein, at step a), the initial mass ratio of dry matter (microalgae of the genus Ch / ore / / a) / (microalgae of the genus Chlorella + cyanobacteria of the genus Aphanizomenon) is between 5% and 65%, in particular between 20% and 60%, and, in particular, between 27% and 55%.

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

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

9. A method according to any one of claims 1 to 8, characterized in that said aqueous culture medium is initially devoid of organic carbon and cyanobacteria of the genus Aphanizomenon and microalgae of the species Chlorella are left in autotrophic culture, during step b), before an input of organic carbon.

10. A method according to any one of claims 1 to 9, characterized in that said step c) comprises a sieving step.

11. A process according to any one of claims 1 to 10, characterized in that said process has an additional step, following said step c), consisting of recovering microalgae of the genus Chlorella.

12. A process according to claim 11, characterized in that said process comprises an additional step consisting of recovering contaminating microorganisms following the recovery of microalgae of the genus Chlorella.

13. A method according to any one of claims 1 to 12, wherein in step b), the culture medium is subjected to agitation, preferably by pumping, by blades, by propellers or by magnetic stirrer, most preferably by blade.

14. A method according to any one of claims 1 to 13, wherein in step a), cyanobacteria of the genus Aphanizomenon and microalgae of the genus Chlorella are introduced simultaneously into the culture medium.

15. A method according to any one of claims 1 to 14, wherein step b) lasts for a sufficient time to obtain at least one doubling of the cyanobacteria of the genus Aphanizomenon, preferably a time longer than the doubling time of the cyanobacteria of the genus Aphanizomenon, in particular, 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.

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