METHOD FOR CULTIVATING PHOTOSYNTHETIC MICROORGANISMS AND ASSOCIATED DEVICE

Abstract

The present invention relates to a method for culturing photosynthetic microorganisms in a biological reactor comprising a culture medium in which the carbon source comprises at least one inorganic carbon, said inorganic carbon being entirely in the form of bicarbonate ions (HCO3-) or in the form of HCO3- and carbon dioxide ions. In particular, the method according to the invention comprises a step of enzymatic conversion of CO2 to HCO3- by means of enzymes immobilized on a support.

Description

Title of the invention: METHOD FOR CULTIVATING PHOTOSYNTHETIC MICROORGANISMS AND ASSOCIATED DEVICE. FIELD OF THE INVENTION

[0001] The present invention relates to a method for culturing photosynthetic microorganisms in a biological reactor comprising a culture medium in which the carbon source comprises at least one inorganic carbon, said inorganic carbon being entirely in the form of bicarbonate ions (HCO3) or in the form of HCO3 ions and carbon dioxide. In particular, the method according to the invention comprises a step of enzymatic conversion of CO2 to HCO3 by means of enzymes immobilized on a support.

[0002] BACKGROUND OF THE INVENTION

[0003] The methods for culturing microorganisms are well known to those skilled in the art. Culture can be carried out on a laboratory scale in Petri dishes or Erlenmeyer flasks, but also on a larger industrial scale in biological reactors of 50,000 liters and more.

[0004] Many photosynthetic microorganisms, particularly microalgae, are cultivated for the intrinsic properties of the biomass obtained. For example, the cultivation of algae or certain microalgae makes it possible to produce biomass rich in lipids and / or proteins. This biomass is then used for the preparation of nutritional, cosmetic, and pharmaceutical compositions, or for the preparation of biofuel or biofertilizer.

[0005] These photosynthetic microorganisms use inorganic carbon, particularly in the form of carbon dioxide (CO2) and bicarbonate ions (HCO3) to grow and multiply.

[0006] However, the ability to develop reactors enabling industrial culture of photosynthetic microorganisms with efficient absorption of inorganic carbon supplied in the culture medium, in particular the ability to supply a sufficient quantity of inorganic carbon assimilable by photosynthetic microorganisms without affecting culture conditions (in particular pH variations), remains to be improved.

[0007] The present invention aims to meet this need.

[0008] BRIEF DESCRIPTION OF THE INVENTION

[0009] The present invention relates to a method for culturing photosynthetic microorganisms with bicarbonate ions (HCO3) as the main carbon source in a culture system comprising a biological reactor, a feeding device carbon dioxide (CO2) and an enzymatic CO2-to-HCO3 ion conversion module coupled to the reactor, said process comprising the steps of: - Enrichment of the culture medium with CO2 via the CO2 supply device, to obtain a CO2-enriched culture medium, and - contacting the CO2-enriched culture medium obtained with CO2-to-HCO3 conversion enzymes in the conversion module to obtain a culture medium enriched in HCO3 ions, wherein the CO2-to-HCO3 conversion enzymes are immobilized on a support.

[0010] The invention also relates to a photosynthetic microorganism culture system comprising a biological reactor, a carbon dioxide (CO2) supply device, and an enzymatic CO2-to-bicarbonate (HCO3) conversion module coupled to the reactor, said conversion module comprising CO2-to-HCO3 conversion enzymes immobilized on a support. Brief description of the drawings

[0011] [Fig-1]: Method of culturing microorganisms with a reactor (1), a device CO2 supply (2) located between the reactor and the enzymatic conversion module (3).

[0012] [Fig.2]: Membrane contactor (figure 2B) and cross-sectional view of a membrane of the membrane contactor (figure 2A).

[0013] [Fig. 3A]: Enzyme conversion module according to an embodiment of the invention in which the enzymes are immobilized on a support in the form of enzyme-coated plates.

[0014] [Fig. 3B]: A more specific embodiment of the invention, in which the enzyme-coated plates are integrated into a tube.

[0015] [Fig. 4]: An embodiment of the invention in which the enzymes are immobilized on a support placed in an independent enclosure (53), the enclosure being disposed between the CO2 supply device (52) and the biological reactor (51). DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention relates to a method for culturing photosynthetic microorganisms with HCO3 ions as the main carbon source in a culture system comprising a biological reactor, a carbon dioxide (CO2) supply device, and an enzymatic CO2-to-HCO3 ion conversion module coupled to the reactor, said method comprising the steps of: - Enrichment of the culture medium with CO2 via the CO2 supply device, to obtain a CO2-enriched culture medium, and - contacting the CO2-enriched culture medium obtained with CO2-to-HCO3 conversion enzymes in the conversion module to obtain a culture medium enriched in HCO3 ions, in which the conversion modules for the enzymes converting CO2 to HCO3 are immobilized on a support.

[0017] Definitions

[0018] The term "carbon source" refers to a nutrient added to the culture medium that provides the carbon necessary for the formation of new organic molecules and for the chemical reactions of molecular synthesis in the cultured microorganism. This carbon source can be inorganic (CO2, HCO3-) or organic (sugars, fatty acids).

[0019] The term "main carbon source" means a carbon source representing more than 50% of the carbon source in the culture medium, in particular, more than 60%, more than 70%, more than 80%, more than 90%, or even about 100%.

[0020] The term "HCO3 ions as the main source of carbon" means that in the culture medium, more than 50% of the carbon source is in the form of HCO3 ions, in particular, more than 60%, more than 70%, more than 80%, more than 90%, or even about 100%.

[0021] The term “biological reactor” means a reactor in which biological phenomena develop, such as the growth of a single type of microorganism or a consortium of microorganisms (in particular microalgae), in very diverse fields such as the treatment of effluents, the production of biomass containing biomolecules of interest (i.e. biomolecules that can be used) and / or metabolite products.

[0022] For the purposes of the present invention, a device is said to be "coupled" to the biological reactor when it constitutes a separate element of the reactor but is connected to this reactor by means of a fluidic network in which the culture medium included in the reactor can circulate.

[0023] The term "culture medium" refers to a liquid medium containing the essential nutrients for the development, growth, and / or multiplication of the cultured microorganisms. The essential nutrients for the development of photosynthetic microorganisms are well known to those skilled in the art, who will be able to determine and adapt their quantity and nature according to, in particular, the microorganism(s) being cultured, the size of the reactor, and the desired amount of biomass. These nutrients include, for example, sources of carbon, nitrogen, oxygen, phosphorus, etc.; minerals such as calcium, iron, magnesium, potassium, sodium, sulfur, etc.; and trace elements (boron, zinc, copper, cobalt, etc.).

[0024] By "CO2-enriched culture medium" is meant a culture medium whose dissolved CO2 content is between 200 and 600 mg / L.

[0025] By "HCO3-enriched culture medium" is meant a culture medium in which the The dissolved HCO3 content is between 200 and 600 mg / L.

[0026] The term "photosynthetic microorganisms" refers to eukaryotic or prokaryotic microorganisms that use light as an energy source and inorganic carbon, in the form of CO2 or HCO3, as their primary carbon source. These include bacteria, protists such as yeasts, and microalgae.

[0027] The term "protist" means all unicellular eukaryotic microorganisms, including microalgae, yeasts, and protozoa. "Autotrophic culture conditions" means a culture medium comprising water, an inorganic carbon source, and light. "Inorganic carbon" means carbon species including carbon dioxide, carbonic acid, and the anions bicarbonate and carbonate.

[0028] The term “hydrogel” means a matrix comprising a network of polymers which swells on contact with water and which comprises between 75 and 90% water by weight.

[0029] Microorganisms

[0030] The microorganisms cultivated according to the invention are photosynthetic microorganisms, eukaryotic such as protists or prokaryotic such as bacteria.

[0031] In one embodiment, the cultured microorganisms are bacteria, in particular cyanobacteria and especially cyanobacteria of the genera Anabaena, Nostoc, Microcistis, Arthrospira and Spirulina.

[0032] According to a preferred embodiment, the cultured microorganisms are protists, even more preferably microalgae.

[0033] In one embodiment, the microorganisms according to the invention are microalgae belonging preferentially to the divisions of Chlorophytes, Rodophytes, Bigyra and Haptophytes.

[0034] When microorganisms belong to the division of Chlorophytes, these are chosen from microorganisms belonging to the class of Chlorophyceae, Ul-vophyceae, Trebouxiophyceae, Prasinophyceae, Pedinophyceae and Charophyceae.

[0035] In one embodiment, the microorganisms belong to the class Chlorophyceae, and more particularly to the order Sphaeropleales. The microorganisms are then preferentially of the genus Scenedesmus.

[0036] When the microalgae are of the Scenedesmus genus, they can be chosen from the species S. abandons, S. aciculatus, S. aculeolatus, S. aculeotatus, S. acuminatus, S. acutiformis, S. acutus, S. aldavei, S. alternons, S. ambuehlii, S. an-huiensis, S. anomalus, S. antennatus, S. antillarum, S. apicaudatus, S. apiculatus, S. arcuatus, S. aristatus, S. armatus, S. arthrodesmiformis, S. arvemensis, S. asym-metricus, S. bacillaris, S. baculiformis, S. bajacalifomicus, S. balatonicus, S. ba-siliensis, S. bernardii, S. bicaudatus, S. bicellularis, S. bidentatus, S. bijuga, S. bijugatus, S. bijugus, S. brasiliensis, S. breviaculeatus, S. brevispina, S. caribeanus, S. carinatus, S. caudato-aculeolatus, S. caudatus, S. chlorelloides, S. circumfusus, S. coalitus, S. costatogranulatus, S. crassidentatus, S. curvatus, S. decorus, S. den-ticulatus, S. deserticola, S. diagonalis, S. dileticus, S. dimorphus, S. disciformis, S. dispar, S. distentus, S. ecomis, S. ellipsoideus, S. ellipticus, S.falcatus, S. fenestratus, S. flavescens, S. flexuosus, S. furcosus, S. fuscus, S. fusiformis, S. gracilis, S. grae-venitzii, S. grahneisii, S. granulatus, S. gujaratensis, S. gutwinskii, S. hanleyi, S. helveticus, S. heteracanthus, S. hindakii, S. hirsutus, S. hortobagyi, S. houlensis, S. huangshanensis, S. hystrix, S. incrassatulus, S. indianensis, S. indicus, S. inermis, S. insignis, S. intermedius, S. javanensis, S. jovais, S. jugalis, S. kerguelensis, S. kissii, S. komarekii, S. lefevrei, S. linearis, S. littoralis, S. longispina, S. longus, S. luna, S. lunatus, S. magnus, S. maximus, S. microspina, S. minutus, S. mirus, S. morzinensis, S. multicauda, ​​S.multiformis, S. multispina, S. multistriatus, S. naegelii, S. nanus, S. notatus, S. nygaardii, S. oahuensis, S. obliquus, S. obtusiusculus, S. obtusus, S. ol-valtemus, S. oocystiformis, S. opoliensis, S. omatus, S. ovalternus, S. pannonicus, S. papillosum, S. parisiensis, S. parvus, S. pecsensis, S. pectinatus, S. perforatus, S. planctonicus, S. plarydiscus, S. platydiscus, S. pleiomorphus, S. polessicus, S. polyden-ticulatus, S. polyglobulus, S. polyspinosus, S. praetervisus, S. prismaticus, S. producto-capitatus, S. protuberans, S. pseudoarmatus, S. pseudobemardii, S. pseudoden-ticulatus, S. pseudogranulatus, S. pseudohystrix, S. pyrus, S. quadrialatus, S. quadricauda, ​​S. quadricaudata, S. quadricaudus, S. quadrispina, S. raciborskii, S. ralfsii, S. reginae, S. regularis, S. reniformis, S. rostrato-spinosus, S. rotundus, S. rubescens, S. scenedesmoides, S. schnepfii, S. schroeteri, S. securiformis, S. semicristatus, S. semipulcher, S. sempervirens, S. senilis, S.serrato-perforatus, S. serratus, S. serrulatus, S. setiferus, S. sihensis, S. smithii, S. soli, S. sooi, S. spicatus, S. spinoso-aculeolatus, S. spinosus, S. spinulatus, S. striatus., S. subspicatus, S. tenuispina, S. terrestris, S. tetra-desmiformis, S. transilvanicus, S. tricostatus, S. tropicus, S. tschudyi, S. vacuolatus, S. variabilis, S. velitaris, S. verrucosus, S. vesiculosus, S. westii, S. weberi, S. wiscon-sinensis, S. wuhanensis, S. wuhuensis. Advantageously according to the invention the algae of the genus Scenedesmus may be algae chosen from among the species S. obliquus or S. abundans. .

[0037] In another embodiment, the microorganisms belong to the class of Chlorophyceae, and more particularly to the order of Chlamydomonadales. The microorganisms are then preferentially of the genus Haematococcus. When the microalgae are of the Haematococcus genus, they may be chosen from the species: H. buetschlii, H. capensis, H. carocellus, H. droebakensis, H. grevilei, H. insignis, H. lacustris, H. murorum, H. pluvialis, H. salinus, H. sanguineis, H. thermalis, H. zimbabwiensis.

[0039] In another embodiment, microorganisms belong to the class of Trebouxiophyceae, and more specifically the order Chlorellales. The microorganisms are preferentially of the genus Chlorella.

[0040] Lorsque les microalgues sont du genre Chlorella, elles poignant être choisies parmi les species C. acuminata, C. angustoellipsoidea, C. anitrata, C. antarctica, C. au-reoviridis, C. autotrophica, C. botryoides, C. caldaria, C. candida, C. capsulata, C. chlorelloides, C. cladoniae, C. coelastroides, C. colonialis, C. commuais, C. conductor, C. conglomerata, C. desiccata, C. ellipsoidea, C. elongata, C. emersonii, C. faginea, C. fusca, C. glucotropha, C. homosphaera, C. infusionum, C. kessleri, C. koettlitzii, C. lacustris, C. lewinii, C. lichina, C. lobophora, C. luteo-viridis, C. marina, C. miniata, C. minor, C. minute, C. mirabilis, C. mucosa, C. mutabilis, C. noctuma, C. nordstedtii, C. oblonga, C. oocystoides, C. ovalis, C. paramecii, C. pa-rasitica, C. parva, C. peruviana, C. photophila, C. pituita, C. pringsheimii, C. proto-thecoides, C. pulchelloides, C. pyrenoidosa, C. regularis, C. reisiglii, C. reniformis, C. rotunda, C. rubescens, C. rugosa, C. saccharophila, C.salina, C. simplex, C. singularis, C. sorokiniana, C. spaerckii, C. sphaerica, C. stigmatophora, C. sub-sphaerica, C. terricola, C. trebouxioides, C. vannielii, C. variabilis, C. viscosa, C. volutis, C. vulgaris, C. zopfingiensis. Advantageously according to the invention, the Chlorella genus may be chosen from the species C. sorokiniana or C. vulgaris. .

[0041] In another embodiment, the microorganisms belong to the division of Chlorophytes, but to the class of Chlorodendrophyceae. In this embodiment, the microorganisms are of the genus Tetraselmis, and are chosen from the species: T. alacris, T. apiculata, T. arnoldii, T. ascus, T. astigmatica, T. bichlora, T. bilobata, T. bolosiana, T. chui, T. contracta, T. convolutae, T. cordiformis, T. desi-kacharyi, T. elliptica, T. fontiana, T. gracilis, T. hazenii, T. helgolandica, T. im-pellucida, T. incisa, T. inconspicua, T. indica, T. levis, T. maculata, T. marina, T. me-diterranea, T. micropapillata, T. rubens, T. striata, T. subcordiformis, T. suecica, T. te-trabrachia, T. tetrathele, T. verrucosa, T. viridis, T. wettsteinii.

[0042] When microorganisms belong to the division Rhodophyta, they are preferentially chosen from among microorganisms belonging to the class Cyaniophyceae. Examples include microalgae belonging to the order Cyanidiales, and more specifically to the families Cyanidiaceae and Galdieriaceae. Examples of microorganisms are those of the genus Cyanidioschyzon, such as Cyandidioschyzon merolae; Cyanidium, such as Cyanidium caldarium, Cyanidium daedalum, Cyanidium maximum, Cyanidium partitum, and Cyanidium rumpens; Galdieria, such as Galdieria daedala, Galdieria maxima, Galdieria partita, and Galdieria sulphuraria; and Pluto, including Pluto calderium.

[0043] Microorganisms can also be selected by microorganisms belonging to the superclass Dinoflagellates. Among these microorganisms, one can in particular, mention microorganisms of the genus Amphidinium, such as the species Amphidinium acutum, Amphidinium aloxalocium, Amphidinium asymmetricum, Amphidinium bipes, Amphidinium carbunculus, Amphidinium carterae, Amphidinium carteri, Amphidinium celestinum, Amphidinium coeruleum, Amphidinium conradi, Amphidinium corallinum, Amphidinium crassum, Amphidinium cyaneoturba, Amphidinium dubium, Amphidinium elegans, Amphidinium extensum, Amphidinium flagellans, Amphidinium flexum, Amphidinium fusiforme, Amphidinium glaucum, Amphidinium globosum, Amphidinium hoefleri, Amphidinium hyalinum, Amphidinium in-coloratum, Amphidinium inflatum Amphidinium kesslitzi, Amphidinium klebsii, Amphidinium lacustre, Amphidinium lanceolatum, Amphidinium latum ,Amphidinium lilloense Amphidinium longum Amphidinium luteum Amphidinium machapungarum ,Amphidinium macrocephalum ,Amphidinium mammillatum,Amphidinium massartii ,Amphidinium microcephalum ,Amphidinium operculatum ,Amphidinium omitho-cephalum, Amphidinium ovoideum,Amphidinium ovum, Amphidinium pellucidum, Amphidinium phthartum Skuja, , Amphidinium psammophilum, Amphidinium pseudo-galbanum, Amphidinium purpureum, Amphidinium salinum Ruinen, Amphidinium schroederi, Amphidinium scissoides, Amphidinium sphenoides, Amphidinium steini, Amphidinium stellatum, Amphidinium subsalsum, Amphidinium tortum, Amphidinium trochodinoides, Amphidinium turbo, Amphidinium vasculum, Amphidinium vittatum, and Amphidinium wislouchii. ,

[0044] Dans un autre mode de réalisement, les microorganismes sont choisis parmi la classe des Euglenoidea, et plus particulier parmi les microalgues du genre Euglena. Elles poignant être choisies entre autres parmi les species E. viridis, E. gracilis, E. limosa, E. globosa, E. prowsei, E. polomorpha.

[0045] Lorsque les microalgues sont des diatomées, elles poignant être choisies notably parmi les genres Nitzschia, Navicula, Gyrosigma, Phaeodactylum, Thalassiosira.

[0046] These microalgues are of the Nitzschia genre, which are specifically chosen for the specific species N. abbreviata, N. abonuensis, N. abridia, N. accessens, N. accommodata, N. aciculariformis, N. acicularioides, N. acicularis (including all varieties), N. acidoclinata, N. actinastroides, N. actydrophila, N. acula, N. acuminata (comprenant all ces variétés), N. acuta, N. adamata, N. adamatoides, N. adapta, N. adducta, N. adductoides, N. admissa, N. admissoides, N. aequalis, N. ae-quatorialis, N. aequora, N. aequorea, N. aerophila, N. aerophiloides, N. aestuari, N. affinis, N. africana, N. agnewii, N. agnita, N. alba, N. albicostalis, N. alexandrina, N. alicae, N. allanssonii, N. alpina, N. alpinobacillum, N. amabilis, N. ambigua, N. americana, N. amisaensis, N. amphibia, N. amphibia (including all varieties), N. amphibioides, N. amphicephala, N. amphilepta, N. amphioxoides, N. amphioxys (including all varieties), N. amphiplectans, N. amphiprora, N.embracing, N. . amuadoaii, N. anassae, N. andicola, N. aagularis (comprenant all ces variétés), N. aagulata, N. aagustata (comprenant all ces variétés), N. aagustatula, N. angustifo-raminata, N. aniae, N. antarctica, N. aatillarum, N. apiceconica, N. apiculata, N. ar-chibaldii, N. arcuata, N. arcula, N. arcus, N. ardua, N. aremoaica, N. areaosa, N. areolata, N. armoricana, N. asperula, N. astridiae, N. atomus, N. atteauata, N. au-rantiaca, N. aurariae, N. aurica, N. auricula, N. australis, N. austriaca, N. bacata (including all varieties), N. bacillariaeformis, N. bacilliformis, N. bacillum, N. balatoais, N. balcanica, N. baltica, N. barbieri (including all varieties), N. barkleyi, N. barroaii, N. barrowiana, N. bartholomei, N. bathursteasis, N. bavarica, N. behrei, N. bergii, N. beyeri, N. biacrula, N. bicapitata (comprenant all ces variétés), . bicuaeata, N. bifurcata, N. bilobata (comprenant all ces variétés), N. birostrata, N. bisculpta, N. bita, N. bizerteasis, N.blaakaarteasis, N. bombiformis, N. borealis, N. bo-sumtwieasis, N. braarudii, N. brebissoaii (including all these varieties), N. bremeasis (including all these varieties), N. brevior, N. brevirostris, N. brevissima (including all these varieties), N. brevistriata, N. brightwellii, N. brittoaii, N. . bruaoi, N. bryophila, N. buceros, N. bukeasis, N. bulaheimiaaa, N. buschbeckii, N. calcicola, N. caledoaeasis, N. calida (including all varieties), N. californica, N. campechiaaa, N. capeasis, N. capitata, N. capitellata (including all varieties), N. capuluspalae, N. carnicobarica, N. camico-barica, N. challeageri, N. chalonii, N. chaadoleasis, N. chardezii, N. chasei, N. chauhaaii, N. chuagara, N. chutteri, N. circumsuta, N. clarissima, N. clausii, N. clemeatei, N. clemeatia, N. clevei, N. closterium (including all varieties), N. coarctata, N. coccoaeiformis, N. commuais (including all varieties), N. commutata, N. commutatoides, N. compacta, N. compressa (including all varieties), N. concordia, N. confiais, N. coaformata, N. coafusa, N. coagoleasis, N. coastricta (including all varieties), N. coasummata, N. corpuleata, N. costei, N. coutei, N. creticola, N. cucumis, N. cursoria, N. curta, N. curvata, N. curviliaeata, N. curvipuactata, N.curvirostris (including all these varieties), N. curvula (including all these varieties), N. cuspidata, N. cyliadriformis, N. cyliadrus, N. dakariasis, N. davidsoaii, N. dealpiaa, N. debilis, N. decipieas, N. delauaeyi, N. delicatissima, N. delicatula, N. delogaei, N. deaticula (including all these varieties), N. deaticuloides, N. desertorum, N. diaaae, N. diaphaaa, N. diducta, N. didyma, N. dietrichii, N. dilatata, N. diluviaaa, N. dippelii, N. directa, N. deserta, N. disputata, N. dissipata (comprising all these varieties), N. dissipatoides, N. distaas (comprising all these varieties), N. distaatoides, N. di-varicata, N. divergeas, N. diversa, N. diversostata, N. doljeiasis, N. draveilleasis, N. droebakeasis, N. dubia (including all these varieties), N. dubiformis, N. dubioides, N. ebroiceasis, N. eglei, N. elegaas, N. elegaatula, N. elegeas, N. elliptica, N. eloagata, N. eatomoa, N. epiphytica, N. epiphyticoides, N. epithemiformis, N. epithemioides, N. . epithemoides (including all these varieties), N. epsilon, N. erlandssonii, N. erosa, N. etoshensis, N. examinanda, N. eximia, N. famelica, N. fasciculata, N. febigeri, N. ferox, N. ferrazae, N. fibula-fissa, N. filinanformis (including all . varieties), N. flexa, N. flexoides, N. fluminensis, N. fluorescens, N. fluvialis, N. fogedii, N. fonticola (including all these varieties), N. fonticoloides, N. fonticula, N. fontifuga, N. forfica, N. formosa, N. fossalari, N. fragilis, N. fragilis, N. fossilis. franconica, N. fraudulenta, N. frauenfeldii, N. frequens, N. frickei, N. frigida (including all these varieties), N. frustuloides, N. frustulum (including all these varieties), N. fruticosa, N. fundi, N. fusiformis, N. gaardnera, N. gaerneraeimi, N. gandereimi, N. N. garrensis, N. gazellae, N. geitleri, N. geitlerii, N. gelida (including all these varieties), N. geniculata, N. gessneri, N. gieskesii, N. gigantea, N. gisela, N. glabra, N. glacialis (including all these varieties), N.glandiformis, N. goetzeana (including all these varieties), N. gotlandica, N. graciliformis, N. gracilis (including all these varieties), N. . gracillima, N. graciloides, N. gradifera, N. graeffii, N. grana, N. grandis, N. granii (including all these varieties), N. granulata (including all these varieties), N. granulosa, N. groenlandica, N. grossestriata, N. grovei, N. gruendleri, N. grunowii, N. guadalupensis, N. guineensis, N. guttula, N. gyrosigma, N. habirshawii, N. habishawii, N. hadriatica, N. halteriformis, N. hamburgiensis, N. hantzschiana (including all these varieties), N. harderi, N. harrissonii, N. hassiaca, N. heidenii, N. heimii, N. he-mistriata, N. heteropolica, N. heuflerania, N. heufleriana (including all these varieties), N. hiemalis, N. hiengheneana, N. hierosolymitana, N. hoehnkii, N. holastica, N. hollerupensis, N. holsatica, N. hamburgiensis, N. hudsonii, N. hummii, N. hungarica (including all these varieties), N. hustedti, N. hustedtiana, N. hyalina, N. hybrida (including all these varieties), N. hybridaeformis, N. ignorata (including all these varieties), N. iltisii, N. impressa, N. improvisa, N.incerta, N. incognito, N. in-conspicua, N. incrustans, N. incurva (including all these varieties), N. indica, N. in-distincta, N. inducta, N. inflatula, N. ingenua, N. inimasta, N. innominata, N. insecta, N. insignis (including all these varieties), N. intermedia (including all these varieties), N. intermissa, N. interrupta, N. interruptestriata, N. invicta (including all these varieties), N. invisa, N. invisitata, N. iranica, N. irregularis, N. irremissa, N. irrepta, N. irresoluta, N. irritons, N. italica, N. janischii, N. jelineckii, N. johnmartinii, N. juba, N. jucunda, N. jugata (including all these varieties), N. jugiformis, N. . kahlii, N. kanakarum, N. kanayae, N. kavirondoensis, N. kerguelensis, N. kim-berliensis, N. kittlii, N. kittonii, N. knysnensis, N. kolaczeckii, N. kotschyi, N. kowiensis, N. krachiensis, N. krenicola, N. kuetzingiana (including all varieties), N. kuetzingii, N. kuetzingioides, N. kurzeana, N. kurzii, N. kützingiana (including all these varieties), N. labella, N. labuensis, N. lacrima, N. lacunarum, N. lacunicola, N. locus-karluki, N. lacustris, N. lacuum, N. laevis, N. laevissima, N. lagunae, N. la- gunensis, N. lamprocampa (including all these varieties), N. lanceola (including all these varieties), N. lanceolata (including all these varieties), N. lancettula, N. lancettuloid.es, N. lange-bertalotii, N. latens, N. latestriata, N. latestriata, N. latiuscula. lauen-bergiana, N. lauenburgiana, N. lecointei, N. leehyi, N. legleri, N. lehyi, N. leistikowii, N. lesbia, N. lesinensis, N. lesothensis, N. leucosigma, N. levidensis (including all these varieties), N. lice truth (including all varieties), N. lice N. ligowskii, N. limicola, N. limulus, N. linearis (including all these varieties), N. lineata, N. lineola, N. linkei, N. lionella, N. littoralis (including all these varieties), N. littorea, N. longa, N. lon-gicollum, N. longino, N. longstrit (including all these varieties). these varieties), N. lorenziana (including all these varieties), N. lucisensibilis, N. lunaris, N. lunata, N. lurida, N. luzonensis, N. macaronesica, N. macedonica, N. macéra, N. machardyae, N.macilenta (including all these varieties), N. magnacarina, N. mahihaensis, N. mahoodii, N. maillardii, N. major, N. majuscula (including all these varieties), N. makarovae, N. manca, N. mancoides, N. manguini, N. marginata, N. marginulata (including all these varieties), N. marina, N. martiana, N. maxima, N. media, N. medioconstricta, N. mediocris, N. mediterranea, N. metzeltinii, N. microcephala (including all these varieties), N. migrons, N. minuta, N. minutissima, N. minutula, N. miramarensis, N. mi-serabilis, N. mitchelliana, N. modesta, N. moissacensis (including all these varieties), N. mollis, N. monachorum, N. monoensis, N. montanestris, N. morosa, N. multistriata, N. nana, N. natalensis, N. natans, N. nathorsti, N. navicularis, N. navis-varingica, N. navrongensis, N. neglecta, N. nelsonii, N. neocaledonica, N. neo-constricta, N. neofrigida, N. neogena, N. neotropica, N. nereidis, N. nicobarica, N. nienhuisii, N. normannii, N. notabilis, N. nova, N. novae-guineaensis, N.novae-guineensis, N. novaehollandiae, N. nova-zealandia, N. nyassensis, N. oberheimiana, N. obesa, N. obliquecostata, N. obscura, N. obscurepunctata, N. obsidialis, N. obsoleta, N. obsoletiformis, N. obtusa (including all varieties), N. obtusangula, N. oceanica, N. ocellata, N. oliffi, N. oméga, N. osmophila, N. ossiformis, N. ostenfeldii, N. ovalis, N. paaschei, N. pacifica, N. palacea, N. palea (including all varieties), N. paleacea, N. paleaeformis, N. paleoides, N. palustris, N. pamirensis, N. panduriformis (including all varieties), N. paleacea, N. paleaeformis, N. paleoides, N. palustris, N. pamirensis, N. panduriformis (including all varieties), N. paleaeformis, N. paleoides, N. palustris, N. pamirensis, N. panduriformis (including all varieties), N. paleaea, N. paleaeformis, N. paleoides, N. palustris, N. pamirensis, N. panduriformis (including all varieties), N. paleaea, N. paleaeformis, N. paleoides, N. palustris, N. pamirensis, N. panduriformis (including all varieties), N. paleaea, N. obsidialis ... all varieties), N. pantocsekii, N. paradoxa (including all varieties), N. parallela, N. pararostrata, N.partita, N. parvula (including all varieties), N. parvuloides, N. paxillifer, N. peisonis, N. pelagica, N. pellucida, N. pennata, N. peragallii, N. perindistincta, N. perminuta, N. perpusilla (comprenant all ces variétés), N. perspicua, N. persuadens, N. pertica, N. perversa, N. petitiana, N. philippinarum, N. pilum, N.pinguescens, N. piscinarum, N. plana (including all these varieties), N. planktonica, N. plicatula, N. plioveterana, N. polaris, N. polymorpha, N. ponciensis, N. praecurta, N. praefossilis, N. praerinholdii, N. princeps, N. procera, N. prolongata (including all these varieties), N. proion- . gatoides, N. promare, N. propinqua, N. pseudepiphytica, N. pseudoamphioxoides, N. pseudoamphioxys, N. pseudoamphyoxys, N. pseudoatomus, N. pseudobacata, N. pseudocapitata, N. pseudocarinata, N. pseudocommunis, N. pseudocylindrica, N. pseudo-delicatissima, N. pseudofonticola, N. pseudohungarica, N. pseudohybrida, N. pseudonana, N. pseudoseriata, N. pseudosigma, N. pseudosinuata, N. pseudos-tagnorum, N. pubens, N. pulcherrima, N. pumila, N. punctata (including all varieties), N. pungens (including all varieties), N. pungiformis, N. pura, N. pu-riformis, N. pusilla (including all varieties), N. putrida, N. quadrangula, N. quickiana, N. rabenhorstii, N. radicula (comprehent toutes ces varieties), N. rau-tenbachiae, N. recta (including all these varieties), N. rectiformis, N. rectilonga, N. rectirobusta, N. rectissima, N. régula, N. reimeri, N. reimerii, N. reimersenii, N. retusa, N. reversa, N. rhombica, N. rhombiformis, N. rhopalodioides, N. richterae, N.difficile (including all varieties), N. ritscheri, N. robusta, N. rochensis, N. rolandii, N. Romana, N. romanoides, N. romanowiana, N. rorida, N. rosenstockii, N. rostellata, N. rostrata, N. ruda, N. rugosa, N. rupestris, N. rusingae, N. ruttneri, N. salinarum, N. salinicola, N. salpaespinosae, N. salvadoriana, N. sansimoni, N. sar-cophagum, N. scabra, N. scalaris, N. scaligera, N. scalpelliformis, N. schoenfeldii, N. schwabei, N. schweikertii, N. scutellum, N. sellingii, N. semicostata, N. semirobusta, N. separanda, N. serieta (comprenant all varieties), N. serpenticola, N. ser-pentiraphe, N. serrata, N. sibula (including all varieties), N. sigma (including all varieties), N. sigmaformis, N. sigmatella, N. sigmoidea (including all varieties), N. silica, N. silicula (including all varieties), N. silica, N. silicula (including all varieties), variétés), N. siliqua, N. similis, N. simplex, N. simpliciformis, N. sinensis, N. sinuata (comprenant all ces variétés), N. smithii, N. sociabilis, N.socialis (including all varieties), N. solgensis, N. solida, N. solda, N. soratensis, N. sp., N. spathulata (including all varieties), N. speciosa, N. spectabilis (including all varieties), N. sphaerophora, N. spiculoides, N. spiculum, N. spinarum, N. spinifera, N. giganterum, N. steenbergensis, N. stellata, N. steynii, N. stimulus, N. stoliczkiana, N. stompsii (comprenant all ces variétés), N. strelnikovae, N. stricto, N. strigillata, N. striolata, N. subaccommodata, N. subacicularis, N. subacuta, N. subamphioxioides, N. subapiculata, N. subbacata, N. subcapitata, N. subcapitellata, N. subcohaerens (comprenant all ces variétés), N. subcommunis, N. subconstricta, N. subcurvata, N. subdenticula, N. subfalcata, N. subfraudulenta, N. subfrequens, N. subfrustulum, N. subgraciloides, N. subinflata, N. subinvicta, N. sublaevis, N. sublanceolata, N. sublica, N. sublinearis, N. sublongirostris, N. submarina, N. submediocris, N. subodiosa, N. subpacifica, N.subpunctata, N. subromana, N. subrostrata, N. subrostratoides, N. su-brostroides, N. subsalsa, N. subtilioides, N. subtilis (including all varieties), N. subtubicola, N. subvitrea, N. suchlandtii, N. sulcata, N. sundaensis, N. supralitorea, . N. tabellaria, N. taenia, N. taeniiformis, N. tantata, N. tarda, N. taylorii, N. temperei, N. tenella, N. tenerifa, N. tenuiarcuata, N. tenuirostris, N. tenais (including all these varieties), N. tenuissima, N. tergestina, N. terrestris, N. terricola, N. thermalis (including all these varieties), N. thermaloides, N. tibetana, N. tirstrupensis, N. tonoensis, N. towutensis, N. translucida, N. tropica, N. tryblionella (including all these varieties), N. tsarenkoi, N. tubicola, N. tumida, N. turgidula, N. turgiduloides, N. umaoiensis, N. umbilicata, N. umbonata, N. vacillata, N. vacua, N. valdecostata, N. valdestriata, N. valens, N. valga, N. valida (including all these varieties), N. van-heurckii, N. vanoyei, N. vasta, N. ventricosa, N. vermicularioides, N. vermicularis (including all these varieties), N. vermicularoides, N. vexans, N. victoriae, N. vi-dovichii, N. vildaryana, N. villarealii, N. virgata, N. visurgis, N. vitrea (including all these varieties), N.vivax (including all these varieties), N. vixnegligenda, N. vonhauseniae, N. vulga, N. weaveri, N. weissflogii, N. westii, N. williamsiii, N. wip-plingeri, N. witkowskii, N. wodensis, N. woltereckii, N. woltereckoides, N. wuel-lerstorfii, N. wunsamiae, N. yunchengensis, N. zebuana, N. zululandica. .

[0047] According to a particular embodiment of the invention, the protists are chosen from the genera Chlorella, Galdieria and Euglena or cyanobacteria and diatoms.

[0048] Biological reactor

[0049] Biological reactors are well known to those skilled in the art and generally consist of a vessel in which a stirring or agitation element (for example, one or more paddles) may be mounted to promote the homogenization of the vessel's contents. Homogenization of the vessel's contents can also be achieved via a liquid pump that induces a circulation flow of the culture medium within the vessel.

[0050] The biological reactor according to the invention is suitable for the culture of photosynthetic microorganisms, in particular for a culture comprising at least one culture stage under autotrophic culture conditions comprising both water, an inorganic carbon source and a light source.

[0051] The invention is particularly suited for a culture comprising at least one stage under autotrophic culture conditions including water, an inorganic carbon source and a light source.

[0052] In the context of the invention, the biological reactor includes means for illumination. The supply of light can be continuous or cyclical (WO2019 / 034792, WO2021 / 160776).

[0053] It is understood within the framework of the present invention that a person skilled in the art will be able to adapt the speed of agitation and the quantity of light as well as the mode of lighting to obtain optimal culture conditions according to the geometry of the reactor and the microorganism(s) being cultured.

[0054] It should be noted that the reactor according to the invention can be equipped with other devices or apparatus used for the control and regulation of the culture conditions of microorganisms. These include, in particular, the use of probes or sensors for measuring various culture parameters such as temperature, pH, and pressure at the inlet and outlet of the reactor.

[0055] Enzyme conversion module

[0056] Enzymatic conversion means the transformation of carbon dioxide (CO2) into bicarbonate ions (HCO3) by an enzyme having the capacity to carry out such a transformation, called converting enzymes.

[0057] The conversion module is a device coupled to the biological reactor and comprising enzymes for converting CO2 into HCO3 immobilized on a support.

[0058] Advantageously, controlling the proportion of CO2 dissolved in the form of HCO3 ions allows control of the pH of the reaction medium.

[0059] The conversion module is coupled to the biological reactor by any suitable means allowing the circulation of the CO2-enriched culture medium in the conversion module and then to the reactor, once the culture medium has been enriched in HCO3.

[0060] The reactor and the conversion module form a circuit in which the CO2-enriched culture medium circulates in the conversion module where the CO2 is converted into HCO3 and then from the conversion module to the reactor.

[0061] According to a preferred embodiment, the reactor and the conversion module form a circuit in which the CO2-enriched culture medium flows from the reactor to the conversion module where the CO2 is converted into HCO3 and then from the conversion module back to the reactor once the CO2 has been converted into HCO3.

[0062] According to a further preferred embodiment, the reactor, the conversion module and the CO2 supply device form a circuit in which the culture medium flows from the reactor to the CO2 supply device, then once enriched in CO2 flows from the reactor to the conversion module where the CO2 is converted into HCO3 and finally once enriched in HCO3 flows from the conversion module back to the reactor once the CO2 has been converted into HCO3.

[0063] The enzyme conversion module (53) corresponds to a closed enclosure comprising at least one inlet and one outlet for the circulation of the CO2 receiving fluid, or, in one embodiment, of the culture medium, in which the conversion enzymes are immobilized on a support. It is understood that the enclosure may comprise one or more supports containing said immobilized enzymes, said supports being arranged randomly or according to a particular arrangement within the enclosure.

[0064] A person skilled in the art will be able to determine the dimensions of the enzyme conversion module, and in particular the quantity of enzyme required to obtain a medium of culture enriched in HCO3 ions, depending on the process parameters including the enzymatic activity of the enzymes used, the CO2 content of the CO2-enriched culture medium and the circulation speed of the culture medium.

[0065] Enzyme-converting enzymes

[0066] Conversion enzymes are known, including carbonic anhydrases (CA), and more particularly cadmium carbonic anhydrases (cadmium-carbonic anhydrase or CDCA) and receptor-type tyrosine phosphatases (TPTR) [3 and y.

[0067] Carbonic anhydrases are enzymes known for their ability to catalyze the reversible hydration reaction of carbon dioxide (CO2) to bicarbonate ions (HCO3). There are 7 classes: α-AC, γ-AC, γ-AC, β-AC, α-AC, β-AC, θ-AC and β-AC.

[0068] The carbonic anhydrases used in the invention are preferably carbonic anhydrases from the family of α-carbonic anhydrases.

[0069] Such converting enzymes are described in particular in application WO 2013 / 022348.

[0070] In the context of this application, the enzymes used according to the invention are selected from cytoplasmic and membrane carbonic anhydrases. Some carbonic anhydrases are commercially available, such as bovine erythrocyte carbonic anhydrases marketed by Sigma-Aldrich® (CAS 9001-03-0) or mouse carbonic anhydrases marketed by Acro-biosystems® (mouse carbonic anhydrase IX).

[0071] Like most enzymes, carbonic anhydrases have a limited lifespan. To increase their lifespan, these enzymes can, according to a preferred embodiment of the invention, be used in association with a proton acceptor. A proton acceptor allows the regeneration of a carbonic anhydrase via the formation of a complex; thus, the carbonic anhydrase can release a proton from its active site and carry out a new cycle of CO2 to HCO3 conversion.

[0072] Said proton acceptor is preferably chosen from asparagine, glutamine, histidine, serine, threonine, tyrosine, aspartic acid, and glutamic acid; more preferably, said proton acceptor is aspartic acid or glutamic acid. Those skilled in the art will know how to adjust the quantity of proton acceptor to be used, in particular to ensure that the proportions of proton acceptors and enzymes are stoichiometric.

[0073] Immobilization of conversion enzymes

[0074] The techniques for immobilizing enzymes are well known to those skilled in the art and include adsorption, covalent bonding, imprisonment or membrane confinement.

[0075] Several types of supports are suitable for immobilizing enzymes. These include, in particular, polymers, especially in the form of a hydrogel or fibers, such as carbon fiber reinforced polymer (CFRP), polyvinylidene fluoride (PVDF), polypropylene (PP), polyacrylonitrile (PAN), silicone and its derivatives, cellulose and its derivatives, such as cellulose acetate, as well as inorganic compounds such as aluminum oxide (Al2O3) or glass or even carbon nanotubes.

[0076] In a particular embodiment, the enzymes are immobilized in the form of an enzyme precipitate on a fiber-type support, and more specifically on nanofibers.

[0077] Nanofibers can be of natural origin such as nanofibers based on collagen, cellulose, keratin or gelatin, or of artificial origin such as nanofibers based on poly(lactic-co-glycolic acid) (PLGA), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) or poly(ethylene co-vinyl acetate).

[0078] In another embodiment, the support on which the enzymes are immobilized is an inorganic support, preferably glass. The support can be in various geometric shapes: square, rectangle, circle, triangle, polygon, or helical. The support is possibly a rectangular glass plate.

[0079] In the case of a glass support, such as a glass plate, enzymes can be immobilized by disulfide bonds, notably via cysteine ​​molecules using a silanization process. Trimethoxysilane is then deposited on the glass surface, and silver nanoparticles capped with cysteine ​​are added to form a cysteine ​​layer capable of binding to the enzymes. The enzyme conversion module can then comprise one or more plates, particularly glass plates, on which the enzymes are immobilized. The plates are preferably arranged parallel to each other and placed inside the enclosure.

[0080] The enclosure may include one or more supports containing said immobilized enzymes. With reference to [Fig. 3B], the glass plates may in particular be arranged parallel to each other within the enclosure of the conversion module.

[0081] CO2 supply device

[0082] The CO2 supply device allows CO2 to be supplied to the culture medium.

[0083] The CO2 supply device can be disposed in the reactor, or outside the reactor on the circulation circuit of the culture medium, upstream or downstream of the conversion module in the direction of circulation.

[0084] According to a preferred mode, the CO2 supply device is arranged between the reactor and the enzymatic conversion module, upstream of the conversion module in the direction of circulation of the culture medium ([Fig.1]).

[0085] Many CO2 supply devices exist, including membranes, membrane bubblers or bubble columns, ceramic bubblers, or even bubble diffuser stones.

[0086] According to a particular embodiment, the CO2 supply device is a membrane contactor.

[0087] According to a preferred embodiment of the invention, when the CO2 supply device is a membrane contactor, it is arranged, in the direction of circulation of the culture medium, after the reactor and before the conversion module.

[0088] A person skilled in the art will be able to determine the implementation parameters of the CO2 supply device necessary to obtain a culture medium enriched in CO2 as a function of the process parameters including the CO2 content of the culture medium before enrichment, the kinetics of conversion of CO2 to HCO3 and the circulation speed of the culture medium.

[0089] Membrane contactor

[0090] A membrane contactor is a device comprising at least two fluid circulation circuits separated by a transfer membrane. The fluids circulating in said membrane contactor are also referred to as the circulating fluid(s). The membrane contactor thus comprises a first circulation circuit of a first circulating fluid for supplying carbon dioxide (CO2), called the CO2 supply fluid, in contact with a first face of the transfer membrane, and a second circulation circuit of a second circulating fluid for receiving the CO2, called the CO2 receiving fluid, in contact with the second face of the transfer membrane. Each fluid circulation circuit is independent of the other and can be closed or open.

[0091] In one embodiment, the CO2 receiving fluid circulation circuit is a closed circuit. In another embodiment, the CO2 receiving fluid circulation circuit and the CO2 supply fluid circulation circuit are closed circuits to ensure optimal growth conditions for microorganisms.

[0092] In one embodiment, the CO2 feed fluid is in gaseous form, preferably an air / CO2 mixture comprising at least 5% CO2, or at least 25% CO2, more preferably at least 50% CO2, and even more preferably at least 75% CO2. Advantageously, the feed fluid is gaseous CO2 with a content of 100% CO2.

[0093] In another embodiment, the CO2 supply fluid is in the form of a liquid comprising a CO2-absorbing solvent in which CO2 is dissolved.

[0094] A CO2-absorbing solvent is defined as a solvent whose chemical composition allows the absorption of CO2 molecules. These solvents are generally solutions comprising carbonate ions (CO32), bicarbonate ions (HCO3), primary, secondary, or tertiary amines, or amino acids. Such solvents are, for example, solutions comprising 3-alanine, sarcosine, taurine, 6-aminohexanoic acid, monoethanolamine (MEA), diethanolamine (DEA), methyl diethanolamine (MDEA), or carbonate compounds such as potassium carbonate (K2CO3), sodium carbonate (Na2CO3), or ammonium bicarbonate (NH4HCO3). These compounds are preferentially present in CO2-absorbing solutions at concentrations between 0.1 and 1.0 M.

[0095] The CO2 receiving fluid is the culture medium. Said CO2 receiving fluid may also include the cultured photosynthetic microorganisms and the nutrients necessary for their growth.

[0096] Thus, according to the invention, the membrane contactor makes it possible to ensure the exchange of CO2 molecules between a CO2 supply fluid, preferably in gaseous form, and the CO2 receiving fluid, which is the culture medium, through a membrane, also called a transfer membrane.

[0097] Within the membrane contactor, the two fluids, the CO2 supply fluid and the CO2 receiving fluid, are in circulation.

[0098] The circulation of the CO2 supply fluid can be ensured by any means known to those skilled in the art, in particular by using a CO2 source already under pressure or by means of a gas compressor which ensures a regular flow in the membrane contactor. In parallel, the circulation of the CO2 receiving fluid, i.e., the culture medium, is achieved by means of a mixing or agitation element in the biological reactor (for example, by the rotation of blades in the biological reactor) or by pumping the culture medium, which is then reinjected into the biological reactor by means of a pump. A vacuum pump is preferably used in this case.

[0099] Advantageously, the use of a membrane contactor according to the invention allows a CO2 injection process that does not generate deleterious shear stresses in the reactor, thus avoiding the formation of foam in the culture.

[0100] In one embodiment, the flows of the CO2 supply fluid and the CO2 receiving fluid are parallel. In another embodiment, the flows are perpendicular. Yet, in yet another embodiment, the flows are crossed. The flows of the CO2 supply fluid and the CO2 receiving fluid can be crossed, that is to say, the CO2 supply fluid and the CO2 receiving fluid circulate in opposite directions.

[0101] With reference to [Fig. 2], the membrane contactor thus comprises an inlet (210) or (211) and an outlet (210) or (211) for a first circulating fluid, as well as an inlet (220) or (221) and an outlet (220) or (221) for a second circulating fluid. These two fluids are separated by a transfer membrane (20). The membrane contactor therefore also comprises two circulation circuits: a first circulation circuit for a first circulating fluid in contact with a first face of the membrane and a second circulation circuit for the second circulating fluid in contact with a second face of the membrane.

[0102] The membrane thus promotes contact between the two circulating fluids without direct physical contact. The properties of the membrane determine the nature and characteristics of the exchanges between the two circulating fluids.

[0103] Membrane contactors are thus defined as systems comprising one or more transfer membranes and whose objective is to promote contact between two circulating fluids, without having direct contact between the two circulating fluids.

[0104] The membrane can be porous or non-porous, also called a dense or pervaporation membrane. Dense membranes are membranes without created porosity. They are divided into two categories: hydrophilic membranes and organophilic membranes.

[0105] Hydrophilic membranes allow the passage of water and thus, for example, the dehydration of a solvent. Typically, these membranes are made of a polymer material (polyvinyl alcohol) deposited on a porous support. Their geometric characteristics are identical to those of ultrafiltration membranes, for example. These membranes are implemented primarily in flat modules, with the "permeate" compartment connected to a partially vacuum chamber to maintain the acting force and remove the separation products.

[0106] Organophilic membranes are essentially silicone-based (PDMS polydimethylseloxane), and in this case, it is the affinity of the silicone material for one or more of the components of the mixture that allows for selective transfer across the membrane. This results in very high selectivities, while the transfer fluxes generally remain low.

[0107] Membrane contactors are characterized by the transfer membranes which compose them, more particularly by the geometric configuration and structure of these membranes.

[0108] Membranes can be of planar, spiral, tubular or hollow fiber geometric configuration (i.e. pipes or tubes).

[0109] According to a preferred embodiment, the membrane(s) have a hollow fiber geometric configuration. Advantageously, the membrane contactor comprises several fibers dispersed in bundles of tubes. The external diameter of the membranes is greater than 50 pm, preferably greater than 60 pm, and even more preferably greater than 70 pm. In another embodiment, the membranes with a hollow fiber configuration have an external diameter of at least 100 pm. In yet another embodiment, the external diameter of the membranes is less than 2 mm, preferably less than 1.5 mm, and more preferably less than 1 mm. Advantageously, the external diameter of the membranes with a hollow fiber configuration is between 100 pm and 1 mm. The internal diameter of the membranes according to the invention is preferably less than 900 pm, or even less than 850 pm.In a preferred embodiment, the external diameter of the membranes with a hollow fiber configuration is between 100 and 800 pm.

[0110] The internal diameter of membranes with a hollow fiber configuration is generally less than 1 mm, preferably less than 900 pm, less than 800 pm, less than 700 pm, less than 600 pm, less than 500 pm, less than 400 pm, less than 300 pm, less than 200 pm, or even less than 100 pm. In one embodiment, the internal diameter of membranes with a hollow fiber configuration according to the invention is between 1 and 100 pm. In another embodiment, the internal diameter is between 5 and 100 pm, preferably between 7 and 100 pm, and even more preferably between 10 and 100 pm. Advantageously, the internal diameter of membranes with a hollow fiber configuration according to the invention is between 20 and 90 pm.

[0111] With reference to Figure 2B and according to one embodiment, the membrane contactor comprises a transfer membrane in a hollow fiber configuration, the fibers being dispersed in the form of a bundle of tubes. With reference to Figure 2A, the membrane (20) is in a hollow fiber (or tube) configuration with an internal diameter and an external diameter. An internal face and an external face of the membrane can thus be defined, corresponding respectively to the inner face of the fiber (or tube) (21) and the outer face of the fiber (or tube) (22).

[0112] In one embodiment, the CO2 supply fluid circulates inside the fibers constituting the membrane and is therefore in contact with the inner face of the membrane, while the CO2 receiving circulation fluid is in contact with the outer face of the membrane. In a second preferred embodiment, the CO2 supply circulation fluid is in contact with the outer face of the membrane and the CO2 receiving circulation fluid is inside the fibers and is in contact with the inner face of the membrane.

[0113] Within the framework of this application, transfer membranes with a confi Hollow fiber structures are microporous membranes or non-porous membranes.

[0114] In one embodiment, the transfer membrane of the membrane contactor is a porous membrane comprising pores with a diameter of less than 10 pm. Advantageously, the porous membrane is microporous and has pores with a diameter of less than 8 pm, preferably less than 5 pm, or even less than 3 pm, and more preferably less than 2 pm. In another embodiment, the microporous membrane comprises pores with a diameter greater than 0.001 pm, 0.002 pm, 0.003 pm, 0.004 pm, or even greater than 0.005 pm. Advantageously, the pore diameter of the membrane is thus between 0.005 and 2 pm. In one embodiment, the pore diameter is less than 1 pm, or even less than 0.5 pm, and preferably less than 0.2 pm or even less than 0.1 pm. In another embodiment, the diameter of the membrane pores is between 0.005 and 0.1 pm.In another embodiment, the pore diameter is between 0.008 and 0.09 pm, preferably between 0.009 and 0.07 pm, or even preferably between 0.01 and 0.06 pm, and more preferably between 0.01 and 0.05 pm. Advantageously, the membrane pore diameter is approximately 0.01 pm, 0.02 pm, 0.03 pm, 0.04 pm, or 0.05 pm.

[0115] In a preferred embodiment, the transfer membrane of the membrane contactor is a dense membrane. The diffusion of molecules is then characterized by a diffusion phenomenon across the membrane via the adsorption of molecules with a high affinity for the membrane. This embodiment has the advantage of reducing or even preventing the clogging of the membrane contactor membrane by the various elements transported in the liquid medium (such as cell debris) compared to a porous membrane which has potential adhesion zones on its structure, and thus allows the same gas exchange performance across the membrane to be maintained throughout the entire culture process.

[0116] According to one embodiment, the transfer membrane is a non-porous membrane with an internal diameter of 150 to 450 pm and an external diameter of 250 to 550 pm.

[0117] According to one embodiment, the transfer membrane is a porous membrane with an internal diameter of 150 to 250 pm and an external diameter of 250 to 350 pm and comprising pores with a diameter of 20 to 40 pm.

[0118] According to another embodiment, the transfer membrane is a dense membrane with an internal diameter of 350 to 450 pm and an external diameter of 5 to 100 mm.

[0119] Advantageously, the transfer membrane, porous or dense, particularly dense, has a thickness of less than 1000 pm, preferably from 100 pm to 1000 pm.

[0120] The use of a membrane contactor according to the invention allows, in particular to ensure all or part of the exchange between two circulating fluids via the presence of a transfer membrane as defined above. The membrane contactor can therefore also be characterized by the exchange surface area between the two circulating fluids.

[0121] According to one embodiment of the invention, the membrane contactor has an exchange surface area between the two circulating fluids of at least 0.003 m2 / L of CO2 receiving circulating fluid, preferably of at least 0.006 m2 / L. In parallel, the exchange surface area between the two circulating fluids is less than or equal to 0.020 m2 / L per liter of CO2 receiving circulating fluid, preferably less than or equal to 0.017, or even less than or equal to 0.015 and preferably less than or equal to 0.012 m2 / L of CO2 receiving circulating fluid. In another embodiment, the membrane contactor has an exchange surface of between 0.003 and 0.012 m2 / L of CO2 receiving circulation fluid, preferably between 0.006 and 0.012 m2 / L of CO2 receiving circulation fluid.

[0122] The materials used for the production of transfer membranes are generally polymers, such as polyethylene (PE), polypropylene (PP), polysulfone (PSu), polyethersulfone (PES), polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE), polyimide (PI), polyamide (PA), polycarbonate (PC), polyacrylonitrile (PAN), and cellulose acetate (CA). Transfer membranes may also be made from silicone or silicone derivatives. For the purposes of this application, the term silicone includes inorganic compounds formed from a chain of silicon and oxygen atoms, and in which the silicon atoms may bear subunits.Examples include polymers belonging to the siloxane family, and more specifically polydimethylsiloxane (PDMS) or compositions comprising polydimethylsiloxane (PDMS), particularly a ternary system composed of PDMS, water, and tetrahydrofuran (THF). Advantageously, the transfer membranes of this application are made from silicone, preferably PDMS; in particular, these membranes are thin PDMS membranes with a thickness between 0.01 and 1.00 cm in sheets or rolls. Examples of commercial transfer membranes are the thin silicone membranes of type SSP-M823 from SSP® or PDMS from Saint-Gobain. Such membrane contactors and their operating principles are known to those skilled in the art for other applications. They are commercially available, notably under the brand names Pervelys and 3M™ Liqui-Cel.

[0123] The combination of the use of a converting enzyme and a membrane contactor makes it possible to reduce the CO2 concentration, or even to lead to the absence of CO2 molecules, in the culture medium, thus accentuating the differences in CO2 concentration and pressure between the culture medium constituting the fluid of CO2 reception and CO2 feed fluid. In this way, the diffusion of CO2 molecules from the CO2 feed fluid to the culture medium is promoted.

[0124] Oxygen evacuation device (OJ

[0125] In a preferred embodiment of the invention, the reactor is included in a closed circuit so as to isolate the culture from external environmental hazards to the reaction, such as temperature variations or variations in the gas composition of the ambient atmosphere.

[0126] In this case, it is necessary to remove the oxygen (O2) produced during photosynthesis by means of an oxygen removal device to promote the growth of photosynthetic microorganisms and prevent potential overpressures within the reactor enclosure. This may consist of one or more valves associated with the reactor to allow the release of excess gases.

[0127] In the case where the CO2 supply means is a membrane contactor, and in particular when the CO2 supply fluid is a gas, the oxygen is evacuated at the level of the membrane contactor membrane, by a path opposite to that of the CO2.

[0128] Where the CO2 supply means is a membrane contactor, and in particular where the CO2 supply fluid is a liquid, the oxygen removal shall be carried out by a suitable means, independent of said CO2 supply membrane contactor, for example by means of one or more valve(s) or with another membrane contactor adapted for removing oxygen from the culture medium. A membrane contactor adapted for removing O2 comprises the same structure as a CO2 supply membrane contactor as described above but with an O2 removal circulating fluid, i.e. a fluid in which the O2 concentration at the inlet of the contactor is lower than that in the culture medium, instead of the CO2 supply fluid.

[0129] Conversion enzyme regeneration device

[0130] In a particular embodiment, the culture system further comprises an enzyme regeneration device (531). This regeneration device, placed in parallel with the main circuit of the culture medium, consists of a proton acceptor supply module, said proton acceptor being preferably selected from asparagine, glutamine, histidine, serine, threonine, tyrosine, aspartic acid, and glutamic acid; even more preferably, said proton acceptor is aspartic acid or glutamic acid. By means of valves (55), the circulation of the culture medium is interrupted while the circulation of the proton acceptor solution (531) is activated. The solution then passes through the enzyme conversion module (53) in order to regenerate the converting enzymes. The cycle of The circulation of the proton acceptor solution can be repeated several times in the enzyme conversion module instead of the culture medium. Those skilled in the art will be able to determine the number of regeneration cycles to apply based on the various culture parameters and the characteristics of the enzymes used. The circulation of the proton acceptor solution is achieved by means of a pump (54) positioned, in the direction of solution flow, between the enzyme conversion module (53) and the proton acceptor solution supply module (531), or between the proton acceptor solution supply module (531) and the enzyme conversion module (53).

[0131] Cleaning device

[0132] In another particular embodiment, the culture system further comprises a cleaning device (56) for the culture medium circulation circuit. This device is placed in parallel with the main culture medium circuit and consists of a series of cleaning operations comprising one or more washes with water, a wash with an alkaline solution, and a wash with an acidic solution. The alkaline solution is preferably a sodium hydroxide-based solution, containing from 0.5% to 5% sodium hydroxide. The acidic solution is an aqueous nitric acid-based solution used to remove limescale and sodium hydroxide deposits from the circuit. In one embodiment, cleaning the device consists of first injecting water, then an alkaline solution and rinsing the device with water, before injecting an acidic solution and rinsing the device again with water.It is understood that the operations can be repeated several times as needed, individually or sequentially. The cleaning solutions circulate through the various components of the culture system, such as the biological reactor (51), the CO2 supply unit (52), or the enzyme conversion module (53), notably by means of pumps (54) and valves (55). An appropriate arrangement of valves and pumps within the culture system can thus allow the cleaning of one or more components independently or in combination. Those skilled in the art will know how to arrange the system components optimally according to the objectives pursued.

[0133] Cropping system

[0134] The invention also relates to a photosynthetic microorganism culture system comprising a culture system including a biological reactor, a carbon dioxide (CO2) supply device and an enzymatic CO2 to HCO3 ion conversion module coupled to the reactor, said conversion module comprising CO2 to HCO3 conversion enzymes immobilized on a support.

[0135] In one embodiment, the culture medium is first supplied with CO2 at the CO2 supply device before passing through the module of enzymatic conversion where the catalysis of CO2 into HCO3 ions occurs.

[0136] In particular, with reference to [Fig.1], the culture system according to the invention comprises a biological reactor (1), a CO2 supply device (2), said CO2 supply device being preferably a membrane contactor, and an enzyme conversion module (3) in which the enzymes are immobilized on a support.

[0137] The CO2 supply device is advantageously a membrane contactor. With reference to [Fig. 2] and according to one embodiment, the membrane contactor comprises a transfer membrane (20) in a hollow fiber configuration, the fibers being dispersed in the form of a tube bundle within the membrane contactor. According to one embodiment, the CO2 supply fluid flows in a first space (21) corresponding to the interior of the hollow fibers, the fluid flowing between the inlet (210) or (211) and the outlet (210) or (211) of the hollow fiber bundle. According to this embodiment, the CO2 receiving fluid circulates in a second space (22) delimited by the watertight walls of the membrane contactor, the fluid circulating in a flow between the inlet (220) or (221) and the outlet (220) or (221) of the membrane contactor.

[0138] In another embodiment, the CO2 supply fluid flows within the membrane contactor, i.e., within a space (22) delimited by the watertight walls of the membrane contactor, the CO2 supply fluid then flowing between the inlet (220) or (221) and the outlet (220) or (221) of the membrane contactor. In parallel, according to this embodiment, the CO2 receiving fluid flows inside the hollow fibers (21) from the inlet (210) or (211) to the outlet (210) or (211).

[0139] With reference to the embodiment of the invention illustrated in [Fig. 4], the microorganism culture system according to the invention comprises a biological reactor (51), a membrane contactor as a CO2 supply device (52), and a module for the enzymatic conversion of CO2 into HCO3 ions (53). The system presented is preferably a closed circuit.

[0140] Preferably, the various elements of the device are arranged as shown in [Fig. 4], so that the culture medium first passes through the CO2 supply device (52) and then the enzymatic conversion module (53). According to one embodiment, the culture medium thus flows from the biological reactor to the membrane contactor (52) where CO2 is supplied, then the CO2-enriched culture medium passes through the enzymatic conversion module (53) where the catalysis of CO2 molecules into HCO3 ions takes place, before the culture medium returns to the biological reactor (51).

[0141] Preferably, the system may further comprise one or more pumps (54) to ensure the continuous circulation of fluids and valves (55). It is understood that probes, sensors and other elements for measuring cultivation parameters or safety systems may be added to the system.

[0142] It should be noted that the above embodiments characterized by the presence of a cleaning device and an enzyme regeneration device can be combined with each other and associated with different CO2 supply devices, including but not limited to a membrane contactor.

[0143] In particular, [Fig.4] represents a specific embodiment comprising as a CO2 supply device a membrane contactor, a cleaning device and an enzyme regeneration device.

[0144] It is understood that the culture system according to the invention, comprising a biological reactor (1), a CO2 supply device (2), and an enzyme conversion module (3), is used for the culture of photosynthetic microorganisms, comprising at least one stage under autotrophic culture conditions, preferably only stages under autotrophic culture conditions. EXAMPLES

[0145] 5 separate cultures of Chlorella sorokiniana are carried out in a photobioreactor of 2.5 L, under artificial lighting of wavelength between 400 and 460 nm and between 620 and 700 nm and intensity of 350 pmol / m2 / s.

[0146] These cultures differ from one another by the use of different CO2 supply devices (bubbler or membrane contactor coupled to the reactor) and by the use or not of an alpha-type carbonic anhydrase converting enzyme from Sigma, CAS: 9001-03-0 at a dosage of 0.1 g / L. The carbonic anhydrase is either free in the culture medium or immobilized on a support in an enzyme conversion module coupled to the biological reactor. The different conditions are summarized in Table 1 below.

[0147] [Tables] Culture CO2 Supply Device Carbonic Anhydrase A Bubbler No B Membrane Contactor No C Bubbler Yes, free in the culture medium D Bubbler Yes, supplied via conversion module E Membrane Contactor Yes, supplied via conversion module

[0148] When a device coupled to the reactor is used (conversion module or membrane contactor (i.e., experiments B, D, and E) and that there is therefore a circuit in which the culture medium circulates; the flow of the culture medium is induced by a paddle shaker with a speed of 500 revolutions per minute (rpm). Note that the homogenization of the medium is ensured using the same stirring paddle at 500 rpm.

[0149] Optical density (OD) measurements are made in duplicate at 750 nm with a spectrophotometer (7315, JENWAY).

[0150] In addition, the daily productivity is calculated from the OD and a correlation coefficient (CC) specific to the cultured microorganism according to the following formula in particular when the biomass concentration is too low to have an accurate value of the dry mass.

[0151] Productivity (DO) (g.Lhday1) = ((DO(day J)-DO (day J1))x 0.6) / (time (day J)-time (day J1))x24

[0152] Or for the present culture of Chlorella sorokinina:

[0153] Productivity (DO) (g.LAday1) = ((DO(day J)-DO (day Jl))x 0.6) / (time (day J)-time (day Jl))x24.

Claims

Demands

1. A method for culturing photosynthetic microorganisms with HCO3 ions as the main carbon source in a culture system comprising a biological reactor, a carbon dioxide (CO2) supply device and an enzymatic CO2-to-HCO3 ion conversion module coupled to the reactor, said method comprising the steps of - enriching the culture medium with CO2 via the CO2 supply device, to obtain a CO2-enriched culture medium, and - contacting the CO2-enriched culture medium obtained with CO2-to-HCO3 conversion enzymes in the conversion module to obtain an HCO3-enriched culture medium, wherein the CO2-to-HCO3 conversion enzymes are immobilized on a support, the method being characterized in that the support is a glass plate.

2. A process according to claim 1, characterized in that the converting enzymes are carbonic anhydrases.

3. A method according to any one of claims 1 or 2, characterized in that the CO2 supply device is a membrane contactor.

4. A method according to any one of claims 1 to 3, characterized in that the culture medium meets the conditions for autotrophic culture.

5. A photosynthetic microorganism culture system comprising a biological reactor, a carbon dioxide (CO2) supply device and an enzymatic CO2 to HCO3 ion conversion module coupled to the reactor, said conversion module comprising CO2 to HCO3- conversion enzymes immobilized on a support, characterized in that the support is a glass plate.

6. System according to claim 5, characterized in that the enzymes are carbonic anhydrases.

7. System according to any one of claims 5 or 6, characterized in that the flow of the culture medium is directed from the biological reactor to the CO2 supply device, and then from the CO2 supply device to the enzymatic conversion module.

8. System according to any one of claims 5 to 7, characterized in that the carbon dioxide (CO2) supply device is a membrane contactor.

9. System according to any one of claims 5 to 8, characterized in that the device further comprises an enzyme regeneration device and / or a cleaning device.

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