Method and system for selecting aquatic photosynthetic organisms.

Abstract

Method and system for selecting aquatic photosynthetic organisms. The invention relates to a method for selecting aquatic photosynthetic organisms, comprising the following steps: i / inoculation of photosynthetic organisms into an aquatic medium of a predetermined initial volume; ii / lowering the initial volume level at least once so as to progressively increase the thermal and light stress of the photosynthetic organisms; iii / application of a chemostat or turbidostat regulation method to the aquatic medium, so as to further select a population of organisms exhibiting the highest level of thermal and light stress; iv / harvesting the population of organisms exhibiting the highest level of thermal and light stress for use and / or conservation. Figure for the abstract: Fig. 1

Description

Title of the invention: Method and system for selecting aquatic photosynthetic organisms. technical field

[0001] The present invention relates to the field of aquatic photosynthetic organisms, in particular photosynthetic microorganisms.

[0002] By "photosynthetic microorganisms", we mean the usual meaning, as given in publication [1], namely algae and cyanobacteria, i.e. eukaryotic or prokaryotic organisms whose growth is mainly by photosynthesis, and of microscopic size, typically between 1 and 100 sqm.

[0003] The present invention aims primarily to provide a solution for selecting a population, preferably a consortium, of photosynthetic organisms based on criteria of resilience to culture conditions, such as temperature and light intensity, which can be extreme and which often lead to the destruction of the culture.

[0004] Although described with reference to a selection of photosynthetic microorganisms, the invention can also be applied to any type of macroalgae. Prior art

[0005] Microalgae and cyanobacteria represent an exceptional diversity: [2].

[0006] In their natural state, microalgae (photosynthetic eukaryotic or prokaryotic microorganisms of one of the following families: Chlorophyceae, Diatoms, Coccolithophyceae, Chrysophyceae, Rhodophyceae, Trebouxiophyceae, Euglenophyceae or cyanobacteria) are unicellular photosynthetic organisms, the main components of phytoplankton, which have populated the oceans and waterways for more than three and a half billion years.

[0007] The cultivation of microalgae on an industrial scale has recently experienced a strong surge in popularity due to the numerous fields of application among which we can cite, in a decreasing scale of valorization, pharmaceuticals and cosmetics, human food, animal feed, fertilizers, bio-based materials, bioremediation and biofuels (liquid or gaseous).

[0008] For the cultivation of microalgae, two main families of culture systems are distinguished [1]:

[0009] - open-type architecture systems, also known as "Raceways," when optimized (agitation by paddle wheels and the typical oval shape of the basin), consist of open-air basins or lagoons. Their main advantage is generating production costs. relatively low. Their major drawbacks lie in low volumetric productivity, i.e., a lower concentration of microalgae at the end of this culture stage, and poor control of culture conditions,

[0010] - closed-type architecture systems, known as "photo" "Bioreactors" consist of one or more chambers, meaning their interior is not in contact with the ambient air. Although requiring a significant initial investment, photobioreactors, compared to open bioreactors, allow for higher volumetric productivity and cultivation under more controlled conditions, notably because contamination and pollution are avoided, and because CO2 can be injected while limiting CO2 losses to the atmosphere.

[0011] As the production costs of microalgal biomass are currently high, current industrial applications of microalgae are primarily focused on the production of high-value molecules by very specific strains with particular metabolic characteristics. These high costs are mainly due to the need to maintain a population of a single species, selected for its productivity in target molecule(s), using closed-architecture culture systems.

[0012] To date, there is a lack of equipment enabling the selection of microalgae strains that can be domesticated, i.e. with robustness and high growth kinetics, on a large scale, in order to produce biomass that can serve as a commodity, i.e. a basic product with a relatively low commercial value.

[0013] Numerous laboratory techniques have been developed to improve the productivity of particular compounds.

[0014] Publication [3], for example, offers a recent review of the various techniques used to evolve a single-strain population in conventional laboratory systems. The stresses usually applied are thus of the thermal, pH, salinity, or oxidation type.

[0015] In order to accelerate the emergence of individuals resistant to these stresses, a mutation-promoting agent, for example UV radiation [4], [5], or a methanesulfonate-type chemical agent (EMS [6],) is applied to cause a target population to diverge more rapidly into different subpopulations capable of resisting the applied stresses. To ensure the emergence within these new (sub)populations of the individuals dividing most rapidly under the selected stress conditions, regular dilution is applied, for example with continuous chemostat or turbidostat-type controls. It should be noted here that in a chemostat, the bioreactor is continuously fed with a complete nutrient medium containing all the nutrients. necessary for the needs of microorganisms and by applying a constant dilution rate; the equivalent of the fresh medium added continuously is withdrawn. In a turbidostat, the feeding is controlled by the measurement of biomass concentration by turbidimetry, that is to say by optical measurements of turbidity or optical density.

[0016] The fully automated device proposed in patent application WO2018 / 055282A1 effectively synthesizes the systems classically developed in the prior art for microalgae selection. In this application, the bioreactor that houses the population to be developed is equipped with thermal regulation that allows for the control of thermal cycles by defining high and low extrema on the one hand and the application of artificial light on the other. Population selection is carried out by regular dilution while maintaining a targeted cell density in the bioreactor using turbidostat control.

[0017] This principle of applying a particular stress to select a population trained to resist that stress has been applied to many species and different selection factors, as illustrated in the table in [3].

[0018] There is a need to improve the methods and devices for selecting a population, enabling it to undergo directed evolution, preferably while controlling costs.

[0019] The object of the invention is to meet at least partially this need. Description of the invention

[0020] To this end, the invention relates, in one of its aspects, to a method for selecting aquatic photosynthetic organisms, comprising the following steps:

[0021] i / inoculation of photosynthetic organisms in an aquatic medium of a predetermined initial volume;

[0022] ii / lowering at least once the initial volume level so as to progressively increase the thermal and light stress of the photosynthetic organisms;

[0023] iii / application of a chemostat or turbidostat mode of regulation to the aquatic environment, so as to continue the selection of a population of organisms exhibiting the highest level of thermal and light stress;

[0024] iv / sampling of the population of organisms exhibiting the highest level of thermal and light stress for use and / or conservation purposes.

[0025] Preferably step ii / is carried out once a week.

[0026] According to an advantageous embodiment, the process includes, during at least one step ii / , a step iil / of limiting the entry of light and thermal amplitudes so as to adapt the population more gradually to stresses.

[0027] According to an advantageous embodiment, the process includes, during at least one step of step ii / , a step ii2 / an automatic leveling of the lowered water level so as to compensate for thermal evaporation.

[0028] Advantageously, step ii2 / is carried out once a day.

[0029] According to an advantageous embodiment, the process includes, later in step iv / , a step v / of preserving the population on agar gel or at cryogenic temperatures.

[0030] Preferably, the population is a consortium or a single species of photosynthetic organisms.

[0031] The invention also relates to a system for selecting aquatic photosynthetic organisms, comprising: - a photobioreactor, intended to be filled with a predetermined initial volume of an aquatic medium in which a culture of photosynthetic organisms is inoculated or to be inoculated; - means to lower the level of the predetermined initial volume within the photobioreactor; - means of measuring cell density within the photobioreactor; - control mechanisms to apply a chemostat or turbidostat mode to culture.

[0032] According to an advantageous embodiment, the system includes at least one sensor for measuring the level of the volume of an aquatic medium within the photobioreactor.

[0033] Preferably, the wall(s) of the photobioreactor is / are made of borosilicate glass. For cost reasons, the wall(s) of the photobioreactor may be made of PMMA (Plexiglas®).

[0034] According to an advantageous embodiment, the means for lowering the level of the predetermined initial volume within the photobioreactor include: - a withdrawal duct preferably opening into the bottom of the photobioreactor; - at least one withdrawal pump, preferably arranged below the photobioreactor, and connected to the withdrawal line.

[0035] Advantageously, the system includes at least one solenoid valve arranged between the extraction line and the extraction pump, the solenoid valve being controllable to be opened when the extraction pump is actuation.

[0036] The dispensing pump can be a dosing pump.

[0037] According to another advantageous embodiment, the means for measuring cell density within the photobioreactor include at least one turbidity and / or optical density probe.

[0038] According to an advantageous embodiment, the control means include a controller and a human-machine interface (HMI) connected to the controller to control the latter.

[0039] The system may advantageously include further one or more of the following elements:

[0040] - at least one opaque and / or outwardly reflective wall of fixed height corresponding to a volume level of the aquatic environment or deployable according to at least a height corresponding to a volume level of the aquatic environment, to be arranged around the wall(s) of the photobioreactor;

[0041] - at least one pH regulation sensor for the aquatic environment within the photobioreactor;

[0042] - at least one sensor for measuring the temperature of the aquatic environment within the photobioreactor; - at least one sensor for measuring sunlight / brightness of the aquatic environment within the photobioreactor. - a reservoir and means for injecting water, and / or an acid or a base, and / or aquatic medium containing photosynthetic organisms.

[0043] According to an advantageous embodiment, the injection means comprise:

[0044] - an injection conduit preferably opening into the bottom of the photobioreactor;

[0045] - at least one injection pump, preferably arranged below the photobioreactor, and connected to the injection conduit.

[0046] According to another advantageous embodiment, the system includes at least one solenoid valve arranged between the injection line and the injection pump, the solenoid valve being controllable to be opened when the injection pump is actuation.

[0047] The injection conduit can advantageously be the withdrawal conduit.

[0048] According to another advantageous embodiment, the system comprises means of injecting gas, containing air and CO2 into the photobioreactor.

[0049] Advantageously, the gas injection means include at least a rotameter and / or a mass flow meter.

[0050] The system may finally include at least one battery suitable for electrically powering the active means of the system, in particular the controller, and at least one photovoltaic solar panel connected to a battery to recharge it and make the system electrically autonomous.

[0051] The invention essentially consists of a method and a semi-automatic implementation system, according to which the inoculation of a first population is initially carried out, preferably a consortium, but possibly also a single species, in a culture reactor, usually called a photobioreactor (PBR, filled with a nutrient solution adapted to the growth of the inoculum).

[0052] In parallel with the increase in cell density, the amount of light and the thermal variations applied are progressively increased by decreasing in step(s) the height of the water column (level of the aquatic volume from its predetermined initial volume, in the culture reactor.

[0053] This reduction in the height of the water column in the reactor makes it possible to obtain both a lower thermal inertia and an increase in the ratio of illuminated surface area to culture volume.

[0054] This reduction in water column height is preferably accompanied by the addition or modification of the height of an opaque or reflective wall, which can be done manually. In an advantageous embodiment, preparation is made beforehand for launching the selection of opaque walls for the different target water column heights.

[0055] Thus, in combination with the preceding steps, a chemostat or turbidostat type reactor control mode is applied, allowing the thermal and light stresses applied to aquatic photosynthetic organisms to be increased to values ​​representative of those that can be applied in the case of cultures grown in open architecture systems of the "raceway" type. These systems can be installed in locations highly exposed to solar radiation, particularly coastal deserts.

[0056] Compared to state-of-the-art methods and systems, the semi-automatic method and system according to the invention allow:

[0057] - the performance of a selection of a population from among organisms synthetic photos based on their growth kinetics, by controlling the height of the water column in a photobioreactor which therefore produces a leaching of the slowest-growing organisms;

[0058] - the simultaneous application of thermal and light stress, using solar energy alone, these stressors being representative of a real environment, allows for a population more robust to that environment;

[0059] - the possibility of automatic compensation for water evaporation from the medium aquatic culture, evaporation which can be significant due to agitation of the culture by bubbling where appropriate and by the application of relatively high temperatures for stress);

[0060] - agitation to homogenize the exposure of microorganisms contents in the photobioreactor.

[0061] In addition, the invention allows:

[0062] - obtaining ratios between illuminated surface area and water volume comparable to those encountered in open architecture systems, and ranging from the most intensified "thin layer" type system to the raceway type system, which allows for the development of consortia well adapted to open cultivation systems, currently used for large-scale production;

[0063] - the possibility of developing different consortia depending on the seasons in reproducing conditions representative of extremes in different seasons;

[0064] - with a photobioreactor made of glass or untreated plastic to absorb UV, We increase the probability of obtaining new genetic profiles (generation of mutants by the application of UV rays naturally present in sunlight).

[0065] The method and system according to the invention are perfectly suited for selecting a population of unicellular organisms such as microalgae or cyanobacteria. The invention can also be implemented to optimize a population of macroalgae, by adapting the culture tank (photobioreactor).

[0066] In addition, it is possible to add pH regulation by a strong acid or base, which allows an additional factor to be applied in the selection pressure or to better support the growth of the culture.

[0067] The semi-automatic system according to the invention can be made electrically autonomous by coupling a rechargeable battery system with photovoltaic panels. Such autonomy would allow the deployment of this type of system in areas without a stable electrical grid and / or in isolated environments.

[0068] In conclusion, the semi-automatic implementation method and system according to the invention offers a number of advantages, among which we can mention: - the possibility of selecting a consortium which can be cultivated into a system of diverse architectures; - the selection of consortia of robust photosynthetic organisms for the production of commodity biomass, including biofuels or bioplastics, or more generally, the selection of a population aimed at the production of high value-added molecules at more competitive prices.

[0069] Other advantages and features of the invention will become clearer from the detailed description of examples of implementation of the invention given by way of illustration and not limitation with reference to the following figures. Brief description of the drawings

[0070] [Fig-1] [Fig. 1] is a schematic view of a photobioreactor equipped with various sensors and conduits for a semi-automatic system for selecting aquatic photosynthetic organisms, according to the invention.

[0071] [Fig.2] [Fig.2] is a schematic view illustrating a semi-automatic system for selecting aquatic photosynthetic organisms, according to the invention.

[0072] [Fig.3] [Fig.3] is a synoptic diagram of the essential steps of the process for selecting aquatic photosynthetic organisms, according to the invention.

[0073] [Fig.4] [Fig.4] shows in screen form an example of a human-machine interface of a semi-automatic system according to the invention.

[0074] [Fig. 5A], [Fig. 5B] Figures 5A and 5B illustrate in the form of graphs respectively the thermal amplitudes measured during the sequence of variation of water column height carried out according to the process of the invention during a test period and the measurements of the corresponding solar resource.

[0075] [Fig. 6A], [Fig. 6B] Figures 6A and 6B are observations made under a microscope optics of samples taken on two separate dates during the testing period. Detailed description

[0076] Throughout this application, the terms "inlet", "outlet", "upstream", "downstream" are to be understood by reference to the direction of flow of a fluid within a semi-automatic system according to the invention.

[0077] The aquatic photosynthetic organism selection system 100 according to the invention is shown with reference to Figures 1 and 2.

[0078] It comprises, firstly, a photobioreactor 1, the tank 10 of which is intended to be filled with a predetermined initial volume of an aquatic medium in which photosynthetic organisms are inoculated or to be inoculated. In other words, the tank 10 is intended to hold a culture, and the photobioreactor 1 includes all the controls necessary to maintain the culture under good growth conditions.

[0079] The tank 10 can have different shapes. In a preferred embodiment, the tank will be made of borosilicate glass. However, in order to reduce the initial investment, it can also be made of plastic, for example PMMA, which is the most common plastic. A plastic tank will, however, have a shorter lifespan, especially if it is not UV-treated.

[0080] The tank to which an opaque wall 13 is added, having the same height as the level of the water column during a part of the test sequence, arranged outside the tank.

[0081] The tank 10 is first equipped with a sensor 11 to measure the level of the volume of an aquatic environment within the photobioreactor 1.

[0082] A draining conduit 12 opens into the bottom of the tank 10.

[0083] An opaque or reflective wall 13 can be arranged around the tank 10 with a fixed height corresponding to a predetermined water column level.

[0084] A turbidity probe 14 as a means of measuring cell density within the photobioreactor 1 is also fitted to the tank 10.

[0085] The tank 10 is also equipped with a pH measurement sensor 15 for the aquatic environment and a temperature measurement sensor within the photobioreactor.

[0086] A gas injection conduit 16, containing air, possibly enriched with CO2, in the photobioreactor 1 may be provided.

[0087] A sunlight / brightness measurement sensor, not shown, also allows the solar resource of the aquatic environment to be measured within the photobioreactor.

[0088] The withdrawal line 12 is connected to a withdrawal pump 17, preferably arranged below the photobioreactor. The withdrawal pump 17 is connected to a drain tank 19, which can also be connected to the top of the tank 10 to discharge any overflow and form a vent.

[0089] A solenoid valve 18 is arranged between the extraction line and the extraction pump, the solenoid valve being controllable to be opened when the extraction pump is actuation.

[0090] A water reservoir 20 and an injection pump 21 which are connected can bring water through the bottom of the photobioreactor.

[0091] An acid or base reservoir 30 and an injection pump 31 which are connected can bring an acid or a base, through the bottom of the photobioreactor.

[0092] A reservoir of the complete nutrient medium 40 and an injection pump 41 which are connected can bring the complete nutrient medium containing the nutrients enabling the growth of photosynthetic organisms.

[0093] A non-return valve 22, 32, 42 can be provided for each of the injection lines.

[0094] Each of the injection pumps 21, 31, 41, and preferably 17, is preferably arranged below the photobioreactor. Thus, with an arrangement below the photobioreactor, the withdrawal pump 17 is constantly "under pressure" due to gravity. The pumps 21, 31, and 41 will also preferably be placed below their respective reservoirs (20, 30, and 40) to avoid priming problems due to gravity.

[0095] A solenoid valve 50 is arranged between the injection line and the delivery line, the solenoid valve being controllable to be opened when the injection pump is actuation.

[0096] In a preferred embodiment, a single port 12 is used on the culture tank, serving both for the injection of the different solutions and for the withdrawal of a culture volume. This allows for easy rinsing of the different channels and limits the risk of leaks.

[0097] In order to obtain the selection of a population based on criteria of resistance to thermal and light extremes, the system according to the invention implements a process comprising the following essential steps:

[0098] Step i / j. Inoculation of the culture, which is placed in a suitable nutrient medium; reactor 10 is filled to obtain a significant culture volume. Typically, the tank can be filled with approximately 20 L of culture, corresponding to a water column height of about 40 cm. This volume allows for a large water mass and therefore small temperature variations and less light available per cell.

[0099] Step ii / _: The culture is grown by regularly lowering, preferably once a week, the water column level in the reactor so as to progressively increase the culture's exposure to higher light intensities and temperatures. Preferably, the side walls of the tank are protected by the opaque wall 13 so as to limit light entry and temperature fluctuations, and thus adapt the population even more gradually to the targeted stresses. During this phase, evaporation is preferably compensated for by regular automatic top-ups, for example, once a day.

[0100] Step iii / : a phase of maintaining the population at a constant density is carried out by applying a chemostat type regulation (constant daily dilution) or a turbidostat type regulation (constant maintenance of cell density), with the objective of progressively leaching out the slowest-growing individuals in the population,

[0101] Step iv / j. The selected population is finally recovered for later use in cultivation systems or to continue other selection steps.

[0102] Preferably, the resulting population is preserved at cryogenic temperatures so that it can be used to start again from the selected population after a significant period of time. To maintain the specific characteristics of the selected population, it is also possible to preserve it on agar gel, by performing monthly subculturing.

[0103] As previously stated, the entire process control is based on measuring the water column level. The process flow diagram is illustrated in [Fig. 3].

[0104] Temperature and solar resource measurements are not used for regulation. They are used to precisely determine the conditions for obtaining the population, which gives an idea of ​​the intended use context for the ultimately obtained population.

[0105] The injection of gas (air enriched with CO2) into the tank 10 is carried out in a controlled manner, preferably with flow regulators (electronic or mechanical mass flow meter). (For example, a rotameter) allows for the adjustment of the CO2 concentration in the incoming air. CO2 regulation can be achieved using a mass flow meter to control, to some extent, the pH increase by injecting a higher concentration of CO2. A peristaltic pump 31 can also be advantageously used to regulate the pH with a dedicated solution. For example, a basic solution would be preferred if nitrogen is supplied via ammonium, or an acidic solution if nitrogen is supplied via nitrates.

[0106] In a preferred embodiment, the process will be controlled by a water level probe, which allows:

[0107] - automatic compensation for evaporation, which is particularly important when using thermal stresses involving high temperatures, typically >30°C,

[0108] - easy control of the phases of crop volume reduction, in order to reduce its thermal inertia and therefore increase the amplitudes of thermal stresses,

[0109] - easy automation of the culture dilution phase during the activation of the chemostat mode (for cylindrical, square or rectangular tanks which are the most common, the dilution factor is proportional to the variation in height of the water column, which greatly simplifies regulation).

[0110] The automation of system 100 (photobioreactor) is achieved by all the pumps and solenoid valves as well as by a controller, ensuring efficient and safe operation.

[0111] Since the system is intended to be easily deployed outdoors, the different entities will be integrated into a compact design that is easily movable.

[0112] For a system installed outdoors, a human-machine interface protected from the elements is preferred, such as a laptop PC, which is removed between maintenance operations. Assembly must be carried out according to best practices to ensure that all connections are watertight, typically with a protection rating higher than IP64.

[0113] The automation is preferably carried out using a Human Machine Interface (HMI) allowing the automaton to be controlled in an intuitive manner.

[0114] An example of an advantageous HMI interface is shown in [Fig.4]. A main window provides access to all the parameters of the process in progress: sensor measurements, automation setpoints, such as chemostat control mode or turbidostat mode, and whether automation is in operation.

[0115] Then tabs (or sub-windows) allow access to the details of the settings, as follows: - The "pH-CO2" tab allows you to adjust the CO2 injection and pH control parameters; the Water Level tab allows you to control evaporation compensation and the injection or withdrawal of medium. - The "Chemo / Turbidostat" tab allows activation of the dilution mode and entry of parameters (dilution rate or optical density target for Chemo / Turbidostat modes respectively), - The "Auxiliaries" tab allows manual control of all actuators (pumps and solenoid valves) and adjustment of the fine parameters of the automated process (pumping speed, ramps to the target speed for peristaltic pumps, calibration of the various sensors...), - The "History" tab allows visualization of acquired data, - The "System" tab allows fine adjustments of the acquisition system (selection of channels to record, acquisition parameters - such as recording delays between measurements, remaining storage space before saturation).

[0116] This human-machine interface will be developed according to the chosen automation system.

[0117] In order to validate the proof of concept, the inventors carried out a test sequence of four consecutive water column heights in the photobioreactor 1, followed by the removal of the opaque wall 13 at the end of the sequence. The variation in water column height can be from 42 to 5 cm.

[0118] Table 1 below summarizes this test sequence, with the dates indicated corresponding to the actual dates.

[0119] [Tables 1] No. Period Wall Level (cm) Diameter (cm) Illuminated Area (cm²) Volume (L) Volume / Area Ratio (dm³) AT (°C) 1 From 3 to 11 / 09 / 2023 Yes 44 23.5 434 19 4 7 2 From 11 to 19 / 09 / 2023 Yes 20 434 9 2 11 3 From 19 to 28 / 09 / 2023 Yes 10 434 4 1 14 4 From 28 / 09 to 03 / 10 / 2023 Yes 5 434 2 0.5 17 5 From 03 / 10 to 16 / 10 / 2023 No 5 618 2 0.35 19

[0120] It is clear from this table that the process according to the invention makes it possible to gradually decrease the volume-to-surface ratio (second-to-last column of the table) and thus increase the amplitude of the temperatures measured during the different periods.

[0121] The graph in [Fig.5A] shows the measurements used to calculate the thermal amplitudes during the five stages of the sequence, as well as the water level; the graph in [Fig.5B] shows the corresponding solar resource.

[0122] These graphs show that, despite the decrease in day length between early September and mid-October, temperature ranges increased in parallel with the decrease in water column height. The removal of the opaque wall 13 from 03 / 10 / 23 further accentuates this range, in addition to significantly increasing the crop's access to light with an increase of slightly less than 50% in the illuminated surface area.

[0123] During the test, the inventors were able to observe an evolution of the population with a disappearance of the filamentous cells initially present in suspension in the reactor: following the strong thermal amplitudes, these preferentially grouped together in the form of biofilms adhering to the walls of the photobioreactor tank.

[0124] This is reproduced in Figures 6A and 6B, which are optical microscope observations using a x20 objective with an interference contrast filter, of samples taken on 19 / 09 / 2023 and 16 / 10 / 2023 respectively.

[0125] The invention is not limited to the examples just described; in particular, features of the illustrated examples can be combined in unillustrated variants.

[0126] Other variants and embodiments may be envisaged without departing from the scope of the invention.

[0127] In order to be easily integrated into various locations, preference will be given to using an automaton that can be powered directly by an autonomous system consisting of a battery recharged by photovoltaic solar panels, as illustrated below.

[0128] A peak power of 1200 Wp supplied by four photovoltaic panels is thus sufficient to recharge a 200 Ah battery under 24V allowing to power a conventional automation system in sunny regions (typically in geographical situations similar to or better exposed than the south of France).

[0129] In order to be self-sufficient in air, it is proposed to use an air blower for agitation; a 10-20 W blower has thus proven sufficient to achieve agitation in a 25 cm diameter tank, using diffusers made with a perforated polymer membrane.

[0130] CO2, generally used as a carbon source with low mass flow rates (0.1 L / min), can be supplied by pressurized gas cylinders. List of references cited

[0131] [1]: J. Pruvost et al. “Industrial production of microalgae and cyanobacteria”, Techniques de l’ingénieur, IN 200, 11 / 2011 https: / / doi.org / 10.51257 / a-vl-chv4030.

[0132] [2]: Andersen, R.A. (1992) «Diversity of eukaryotic algae», Biodiversity & Conservation, 1(4), pp. 267-292. https: / / doi.org / 10.1007 / BF00693765.

[0133] [3]: LaPanse, A. J.; Krishnan, A.; Posewitz, M. C. Adaptive Laboratory Evolution for Algal Strain Improvement: Méthodologies and Applications. Algal Research 2021, 53, 102122. https: / / doi.Org / 10.1016 / j.algal.2020.102122.

[0134] [4]: Cazzaniga, S. et al. (2014) «Domestication ofthe green alga Chlorella sorokiniana : réduction ofantenna size improves light-use efficiency in a photobioreactor», Biotechnology for Biofuels, 7(1), p. 157.: https: / / doi.org / 10.1186 / sl3068-014-0157-z.

[0135] [5]: Takouridis, S.J. et al. (2015) «The sélective breeding of the freshwater microalga Chlamydomonas reinhardtii for growth in salinity», Bioresource Technology, 184, pp. 18-22. https: / / doi.Org / 10.1016 / j.biortech.2014.10.120.

[0136] Kassen, R. (2002) «The experimental évolution of specialists, generalists, and the maintenance of diversity», Journal of Evolutionary Biology, 15(2), pp. 173-190. https: / / doi.Org / 10.1046 / j.1420-9101.2002.00377.x.

[0137] [6]: Sandesh Kamath, B. et al. (2008) «Enhancement of carotenoids by mutation and stress induced carotenogenic genes in Elaematococcus pluvialis mutants», Bioresource Technology, 99(18), pp. 8667-8673. https: / / doi.Org / 10.1016 / j.biortech. 2008.04.013.

Claims

Demands

1. A method for selecting aquatic photosynthetic organisms, comprising the following steps: i / inoculating photosynthetic organisms into an aquatic medium of a predetermined initial volume; ii / lowering the level of the initial volume at least once so as to progressively increase the thermal and light stress of the photosynthetic organisms; iü / applying a chemostat or turbidostat mode of regulation to the aquatic medium, so as to continue the selection of a population of organisms exhibiting the highest level of thermal and light stress; iv / collecting the population of organisms exhibiting the highest level of thermal and light stress for use and / or conservation.

2. Method according to claim 1, steps i / to iv / being carried out within a photobioreactor.

3. Method according to claim 1 or 2, step ii / being carried out once a week.

4. A method according to any one of the preceding claims, comprising during at least one step of step ii / , a step iil / of limiting light input and thermal amplitudes so as to adapt the population more gradually to stresses.

5. A method according to any one of the preceding claims, comprising during at least one step of step ii / , a step ii2 / an automatic leveling of the lowered water level so as to compensate for thermal evaporation.

6. Method according to claim 5, step ii2 / being carried out once a day.

7. A method according to any one of the preceding claims, comprising, subsequent to step iv / , a step v / of preserving the population on agar gel or at cryogenic temperatures.

8. A method according to any one of the preceding claims, the population being a consortium or a single species of photosynthetic organisms.

9. A system for selecting aquatic photosynthetic organisms, comprising:

10.

11.

12.

13.

14.

15.

16.

17. - a photobioreactor, intended to be filled with a predetermined initial volume of an aquatic medium in which a culture of photosynthetic organisms is inoculated or to be inoculated; - means to lower the level of the predetermined initial volume within the photobioreactor; - means of measuring cell density within the photobioreactor; - means of regulation to apply a chemostat or turbidostat mode to the culture. System according to claim 9, comprising at least one sensor for measuring the level of the volume of an aquatic medium within the photobioreactor. System according to claim 9 or 10, the wall(s) of the photobioreactor being made of borosilicate glass. A system according to any one of claims 9 to 11, the means for lowering the level of the predetermined initial volume within the photobioreactor comprising: - a withdrawal duct preferably opening into the bottom of the photobioreactor; - at least one withdrawal pump, preferably arranged below the photobioreactor, and connected to the withdrawal line. System according to claim 12, comprising at least one solenoid valve arranged between the extraction line and the extraction pump, the solenoid valve being controllable to be opened when the extraction pump is actuation. System according to claim 12 or 13, the dispensing pump being a dosing pump. System according to any one of claims 9 to 14, the means for measuring cell density within the photobioreactor comprising at least one turbidity and / or optical density probe. System according to any one of claims 9 to 15, the control means comprising a controller and a human-machine interface (HMI) connected to the controller to control the latter. A system according to any one of claims 9 to 16, further comprising at least one opaque and / or outwardly reflective wall, of fixed height corresponding to a volume level of the aquatic environment or deployable according to at least a height corresponding to a volume level of the aquatic environment, to be arranged around the wall(s) of the photobioreactor.

18. System according to any one of claims 9 to 17, further comprising at least one pH regulation sensor for the aquatic environment within the photobioreactor.

19. System according to any one of claims 9 to 18, further comprising at least one sensor for measuring the temperature of the aquatic environment within the photobioreactor.

20. System according to any one of claims 9 to 19, further comprising at least one sensor for measuring sunlight / brightness of the aquatic environment within the photobioreactor.

21. System according to any one of claims 9 to 20, further comprising a reservoir and means for injecting water, and / or an acid or a base, and / or aquatic medium containing photosynthetic organisms.

22. System according to claim 21, the injection means comprising: - an injection conduit opening preferably into the bottom of the photobioreactor; - at least one injection pump, preferably arranged below the photobioreactor, and connected to the injection conduit.

23. System according to claim 22, comprising at least one solenoid valve arranged between the injection duct and the injection pump, the solenoid valve being controllable to be opened when the injection pump is actuation.

24. System according to claim 22 or 23, the injection conduit being the withdrawal conduit.

25. System according to any one of claims 9 to 24, comprising means for injecting gas, containing air and CO2 into the photobioreactor.

26. System according to claim 25, the means comprising at least one rotameter and / or one mass flow meter.

27. ​​A system according to any one of claims 9 to 26, comprising at least one battery suitable for electrically powering the active means of the system, in particular the controller, and at least one solar panel photovoltaic system connected to a battery to recharge it and make the system electrically self-sufficient.

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