Method for culturing 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 source of carbon comprises at least one inorganic carbon, the inorganic carbon being entirely in the form of bicarbonate (HCO3-) ions or in the form of HCO3- ions and carbon dioxide.<sb / > In particular, the method according to the invention comprises a step of enzymatic conversion of CO2 into HCO3- by means of enzymes immobilised on a support.

Description

METHOD FOR CULTIVATING PHOTOSYNTHETIC MICROORGANISMS AND ASSOCIATED DEVICE

[0001] FIELD OF THE INVENTION

[0002] 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 into HCO3' by means of enzymes immobilized on a support.

[0003] BACKGROUND OF THE INVENTION

[0004] The cultivation of microorganisms 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 or more.

[0005] Many photosynthetic microorganisms, including microalgae, are cultivated for the intrinsic properties of the resulting biomass. For example, the cultivation of algae or certain microalgae makes it possible to produce biomass rich in lipids and / or proteins. These biomasses are then used for the preparation of nutritional, cosmetic and pharmaceutical compositions, or for the preparation of biofuel or biofertilizer.

[0006] These photosynthetic microorganisms use inorganic carbon, particularly in the form of carbon dioxide (CO2) and bicarbonate ions (HCO3) to grow and multiply.

[0007] However, the ability to develop reactors allowing industrial cultivation 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 the culture conditions (in particular pH variations), remains to be improved.

[0008] The present invention aims to meet this need.

[0009] BRIEF DESCRIPTION OF THE INVENTION

[0010] 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 carbon dioxide (CO2) supply device and a module for enzymatic conversion of CO2 into HCO3' ions coupled to the reactor, said method comprising the steps of: enriching the culture medium with CO2 via the CO2 supply device, to obtain a culture medium enriched in CO2, and contacting the obtained CO2-enriched culture medium with CO2-to-HCO3' conversion enzymes in the conversion module to obtain a culture medium enriched in HCO3' ions, in which conversion module the CO2-to-HCO3' conversion enzymes are immobilized on a support.

[0011] The invention also relates to a system for cultivating photosynthetic microorganisms comprising a biological reactor, a carbon dioxide (CO2) supply device and a module for enzymatic conversion of CO2 into bicarbonate ions (HCO3) coupled to the reactor, said conversion module comprising enzymes for converting CO2 into HCO3' ions immobilized on a support.

[0012] BRIEF DESCRIPTION OF THE DRAWINGS

[0013] [Fig. 1]: Method of cultivating microorganisms with a reactor (1), a CO2 supply device (2) placed between the reactor and the enzymatic conversion module (3).

[0014] [Fig. 2]: Membrane contactor (Figure 2B) and cross-sectional view of a membrane of the membrane contactor (Figure 2A).

[0015] [Fig. 3 A]: Enzymatic 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.

[0016] [Fig. 3B]: More specific embodiment of the invention, in which the enzyme-coated plates are integrated into a tube.

[0017] [Fig. 4]: Embodiment of the invention in which the enzymes are immobilized on a support placed in an independent enclosure (53), the enclosure being arranged between the CO2 supply device (52) and the biological reactor (51).

[0018] DETAILED DESCRIPTION OF THE INVENTION

[0019] 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 a module for enzymatic conversion of CO2 into HCO3' ions coupled to the reactor, said method comprising the steps of: enriching the culture medium with CO2 via the CO2 supply device, to obtain a culture medium enriched in CO2, and contacting the obtained CO2-enriched culture medium with CO2-to-HCO3' conversion enzymes in the conversion module to obtain a culture medium enriched in HCO3' ions, in which conversion module the CO2-to-HCO3' conversion enzymes are immobilized on a support.

[0020] Definitions

[0021] A "carbon source" is a nutrient introduced into the culture medium and providing the carbon necessary for the formation of new organic molecules and for the chemical reactions of molecular synthesis of the cultured microorganism. This carbon source can be inorganic (CO2, HCO3-) or organic (sugars, fatty acids).

[0022] “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 approximately 100%.

[0023] "HCO3 ions as the main carbon source" 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%.

[0024] 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 (notably microalgae), in very varied fields such as effluent treatment, biomass production containing biomolecules of interest (i.e. biomolecules that we know can be used) and / or metabolite products.

[0025] For the purposes of the present invention, a device is said to be “coupled” to the biological reactor when it constitutes a separate element from the reactor but is connected to this reactor by means of a fluid network in which the culture medium included in the reactor can circulate.

[0026] By "culture medium" is meant a liquid medium comprising the nutrients essential for the development, growth and / or multiplication of the cultivated microorganisms. The nutrients essential for the development of photosynthetic microorganisms are known per se, and can be adapted in quantity and nature depending in particular on the microorganism(s) cultivated, the size of the reactor, the quantity of biomass desired. Among these nutrients, we find for example sources of carbon, nitrogen, oxygen, phosphorus, etc.; minerals, such as calcium, iron, magnesium, potassium, sodium, sulfur, etc.; trace elements (boron, zinc, copper, cobalt, etc.).

[0027] “CO2-enriched culture medium” means a culture medium with a dissolved CO2 content of between 200 and 600 mg / L.

[0028] By "culture medium enriched with HCO.v" we mean a culture medium whose dissolved HCO3' content is between 200 and 600 mg / L.

[0029] Photosynthetic microorganisms are 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, or microalgae.

[0030] The term "protist" refers to all unicellular eukaryotic microorganisms, including microalgae, yeasts, and protozoa. The term "autotrophic culture conditions" refers to a culture medium that includes water, an inorganic carbon source, and light. The term "inorganic carbon" refers to carbon species including carbon dioxide, carbonic acid, and bicarbonate and carbonate anions.

[0031] 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.

[0032] Microorganisms

[0033] The microorganisms cultivated according to the invention are photosynthetic microorganisms, eukaryotic such as protists or prokaryotes such as bacteria.

[0034] In one embodiment, the cultured microorganisms are bacteria, in particular cyanobacteria and in particular cyanobacteria of the genera Anabaena, Nostoc, Microcistis, Arthrospira and Spirulina.

[0035] According to a preferred embodiment, the cultivated microorganisms are protists, even more preferably microalgae.

[0036] In one embodiment, the microorganisms according to the invention are microalgae preferably belonging to the divisions of Chlorophytes, Rodophytes, Bigyra and Haptophytes.

[0037] When the microorganisms belong to the division of Chlorophytes, they are chosen from among the microorganisms belonging to the class of Chlorophyceae, Ulvophyceae, Trebouxiophyceae, Prasinophyceae, Pedinophyceae and Charophyceae.

[0038] 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.

[0039] When 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. anhuiensis, S. anomalus, S. antennatus, S. antillarum, S. apicaudatus, S. apiculatus, S. arcuatus, S. aristatus, S. armatus, S. arthrodesmiformis, S. arvernensis, S. asymmetricus, S. bacillaris, S. baculiformis, S. bajacalifomicus, S. balatonicus, S. basiliensis, 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. circumfiisus, S. coalitus, S. costatogranulatus, S. crassidentatus, S. curvatus, S. decorus, S. denticulatus, S. deserticola, S. diagonalis, S. dileticus, S. dimorphus, S. disciformis, S. dispar, S. distentus, S. ecornis, S. ellipsoideus, S. ellipticus, S.falcatus, S. fenestratus, S. flavescens, S. flexuosus, S. furcosus, S. fuscus, S. fusiformis, S. gracilis, S. graevenitzii, 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. indiens, S. inermis, S. insignis, S. intermedis, S. javanensis, S. jovais, S. jngalis, S. kerguelensis, S. kissii, S. komarekii, S. lefevrei, S. linearis, S. littoralis, S. longispina, S. longus, S. inna, S. lunatus, S. magnns, S. maximns, S. microspina, S. minntns, S. mirus, S. morzinensis, S. mnlticanda, S. multiformis, S. multispina, S. multistriatus, S. naegelii, S. nanus, S. notatus, S. nygaardii, S. oahuensis, S. obliquus, S. obtusiusculus, S. obtusus, S. olvalternus, S. oocystiformis, S. opoliensis, S. ornatus, S. ovaltemus, S. pannonicus, S. papillosum, S. parisiensis, S. parvus, S. peesensis, S. pectinatus, S. perforatus, S. planctonicus, S. plarydiscus, S. platydiscus, S. pleiomorphus, S. polessicus, S. polydenticulatus, S. polyglobulus, S. polyspinosus, S. praetervisus, S. prismaticus, S. producto-capitatus, S.protuberans, S. pseudoarmatus, S. pseudobernardii, S. pseudodenticulatus, S. pseudogranulatus, S. pseudohystrix, S. pyrus, S. quadrialatus, S. quadricauda, ​​S. quadric audata, 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. s errato -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. tetradesmiformis, S. transilvanicus, S. tricostatus, S. tropicus, S. tschudyi, S. vacuolatus, S. variabilis, S. velitaris, S. verrucosus, S. vesiculosus, S. westii, S. weberi, S. wisconsinensis, S. wuhanensis, S. wuhuensis.Advantageously according to the invention, the algae of the genus Scenedesmus may be algae chosen from the species S. obliquus or S. abundans.

[0040] In another embodiment, the microorganisms belong to the class Chlorophyceae, and more particularly to the order Chlamydomonadales. The microorganisms are then preferentially of the genus Haematococcus.

[0041] When the microalgae are of the genus Haematococcus, they can 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.

[0042] In another embodiment, the microorganisms belong to the class Trebouxiophyceae, and more particularly to the order Chlorellales. The microorganisms are preferably of the genus Chlorella.

[0043] When microalgae are of the genus Chlorella, they can be chosen from the species C. acuminata, C. angustoellipsoidea, C. anitrata, C. antarctica, C. aureoviridis, C. autotrophica, C. botryoides, C. caldaria, C. Candida, C. capsulata, C. chlorelloides, C. cladoniae, C. coelastroides, C. colonialis, C. communis, C. conducttrix, 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. minutissima, C. mirabilis, C. mucosa, C. mutabilis, C. nocturna, C. nordstedtii, C. oblonga, C. oocystoides, C. ovalis, C. paramecii, C. parasitica, C. parva, C. peruviana, C. photophila, C. pituita, C. pringsheimii, C. protothecoides, 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. subsphaerica, 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.

[0044] In another embodiment, the microorganisms belong to the division Chlorophyta, but to the class 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. desikacharyi, T. elliptica, T. fontiana, T. gracilis, T. hazenii, T. helgolandica, T. impellucida, T. incisa, T. inconspicua, T. indica, T. levis, T. maculata, T. marina, T. mediterranea, T. micropapillata, T. rubens, T. striata, T. subcordiformis, T. suecica, T. tetrabrachia, T. tetrathele, T. verrucosa, T. viridis, T. wettsteinii.

[0045] When the microorganisms belong to the division of Rhodophytes, they are preferentially chosen from among the microorganisms belonging to the class of Cyaniophyceae. We can notably cite the microalgae belonging to the order of Cyanidiales and more particularly to the family of Cyanidiaceae and to the family Galdieriaceae. Examples of microorganisms are microorganisms 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, Pluto including Pluto calderium.

[0046] Les microorganismes peuvent par ailleurs être choisis par les microorganismes appartenant à la super-classe des Dinoflagellés. Among these microorganisms, we can mention in particular the microorganisms of the genus Amphidinium, such as the species Amphidinium acutum, Amphidinium aloxalocium, Amphidinium asymmetricum, Amphidinium bipes, Amphidinium carbunculus, Amphidinium carterae, Amphidinium carte ri, Amphidinium ce le stinum, Amphidinium coeruleum, Amphidinium conradi, Amphidinium corallinum, Amphidinium crassum, Amphidinium cyaneoturba, Amphidinium dubium, Amphidinium elegans, Amphidinium extensum, Amphidinium flagellons, Amphidinium flexum, Amphidinium fusiforme, Amphidinium glaucum, Amphidinium globosum, Amphidinium hoefleri, Amphidinium hyalinum, Amphidinium incoloratum, 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 ornithocephalum , 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.,

[0047] In another embodiment, the microorganisms are chosen from the class Euglenoidea, and more particularly from the microalgae of the genus Euglena. They may be chosen, among others, from the species E. viridis, E. gracilis, E. limosa, E. globosa, E. prowsei, E. polomorpha.

[0048] When the microalgae are diatoms, they can be chosen in particular from the genera Nitzschia, Navicula, Gyrosigma, Phaeodactylum, Thalassiosira.

[0049] 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. aequatorialis, 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.amplectens, N. amundonii, N. anassae, N. andicola, N. angularis (comprenant all ces variétés), N. angulata, N. angustata (comprenant all ces variétés), N. angustatula, N. angustiforaminata, N. aniae, N. antarctica, N. antillarum, N. apiceconica, N. apiculata, N. archibaldii, N. arcuata, N. arcula, N. arcus, N. ardua, N. aremonica, N. arenosa, N. areolata, N. armoricana, N. asperula, N. astridiae, N. atomus, N. attenuata, N. aurantiaca, N. aurariae, N. aurica, N. auricula, N. australis, N. austriaca, N. bacata (comprenant toutes ces variétés), N. bacillariaeformis, N. bacilliformis, N. bacillum, N. balatonis, N. balcanica, N. baltica, N. barbieri (including all ces variétés), N. barkleyi, N. barronii, N. barrowiana, N. bartholomei, N. bathurstensis, N. bavarica, N. behrei, N. bergii, N. beyeri, N. biacrula, N. bicapitata (including all varieties), . bicuneata, N. bifurcata, N. bilobata (comprenant all ces variétés), N. biros trata, N. bisculpta, N. bita, N.bizertensis, N. blankaartensis, N. bombiformis, N. borealis, N. bosumtwiensis, N. braarudii, N. brebissonii (including all these varieties), N. bremensis (including all these varieties), N. brevior, N. brevirostris, N. brevissima (including all these varieties), N. brevistriata, N. brightwellii, N. brittonii, N. brunoi, N. bryophila, N. buceros, N. bukensis, N. bulnheimiana, N. buschbeckii, N. calcicola, N. caledonensis, N. calida. (including all these varieties), N. californica, N. campechiana, N. capensis, N. capitata, N. capitellata (including all these varieties), N. capuluspalae, N. carnicobarica, N. camico-barica, N. challenged, N. chalonii, N. chandolensis, N. chardezii, N. chasei, N. chauhanii, N. chungara, N. chutteri, N. circumsuta, N. clarissima, N. clausii, N. clementei, N. dementia, N. clevei, N. closterium (including all these varieties), N. coarctata, N. cocconeiformis, N. communis (including all these varieties), N. commutata, N. commutatoides, N. compacta, N. compressa (including all these varieties), N. concordia, N. confinis, N. conformata, N. confusa, N. congolensis, N. constricta (including all these varieties), N. consummata, N. corpulenta, N. costei, N. coutei, N. creticola, N. cucumis, N. cursoria, N. curta, N. curvata, N. curvilineata, N. curvipunctata, N. curvirostris (including all these varieties), N. curvula (including all these varieties), N. cuspidata, N. cylindriformis, N.cylindrus, N. dakariensis, N. davidsonii, N. dealpina, N. debilis, N. decipiens, N. delauneyi, N. delicatissima, N. delicatula, N. delognei, N. denticula (including all varieties), N. denticuloides, N. desertorum, N. dianae, N. diaphana, N. diducta, N. didyma, N. dietrichii, N. dilatata, N. diluviana, N. dippelii, N. directa, N. disperta, N. disputata, N. dissipata (comprising all varieties), N. dissipatoides, N. distans (comprising all varieties), N. distantoides, N. divaricata, N. divergens, N. diversa, N. diversostata, N. doljensis, N. draveillensis, N. droebakensis, N. dubia (comprising all varieties), N. dubiformis, N. dubioides, N. ebroicensis, N. eglei, N. elegans, N. elegantula, N. elegens, N. elliptica, N. elongata, N. entomon, 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. filiformis (including all these varieties), N.flexa, N.flexoides, N.fluminensis, N.fluorescens, N.fluvialis, N.fogedii, N.fonticola (including all varieties), N.fonticola (including all varieties). N.fonticoloides, N. fonticula, N. fontifuga, N. forfica, N. formosa, N. fossalis, N. fossilis, N. fragilariiformis, N. franconica, N. fraudulenta, N. frauenfeldii, N. frequens, N. frickei, N. frigida (including all varices), N. varices, N. fruits frustulum (including all these varieties), N. fruticosa, N. fundi, N. fusiformis, N. gaarderi, N. gaertnerae, N. gandersheimiensis, N. garrensis, N. gazellae, N. goats, N. goats, 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. hemistriata, 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. incognita, N. inconspicua, N. incrustons, N. incurva (including all these varieties), N. indica, N. indistincta, 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. kimberliensis, 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 (comprenant all ces variétés), N. labella, N. labuensis, N. lac rima, N. lacunarum, N. lacunicola, N. lacus-karluki, N. lacustris, N. lacuum, N. laevis, N. laevissima, N. lagunae, N. lagune nsis, N. lamprocampa (comprenant all varieties), N. lanceola (including all varieties), N. lanceolata (including all varieties), N. lancettula, N. lancettuloides, N. lange-bertalotii, N. latens, N. latestriata,. N. latiuscula, N. lauenbergiana, N. lauenburgiana, N. lecointei, N. leehyi, N. legleri, N. lehyi, N. leistikowii, N. lesbia, N. lesinensis, N. lesothensis, N. leucosigma, N. levidensis (including all variéces), N. liebetruth all these varieties), 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. longicollum, N. longistris. longissima (including all 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. macedonica (including all 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. miserabilis, N. mitchelliana, N. modesta, N. moissacensis (including all these varieties), 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. neoconstricta, 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 these varieties), N. obtusangula, N. oceanica, N. ocellata, N. oliffi, N. omega, N. osmophila, N. ossiformis, N. ostenfeldii, N. ovalis, N. paaschei, N. pacifica, N. palacea, N. pale (including all varieties), N. pale. paleacea, N. paleaeformis, N. paleoides, N. palustris, N. pamirensis, N. panduriformis (including all these varieties), N. pantocsekii, N. paradoxa (including all these varieties), N. parallela, N. pararostrata, N. partita, N. partitévula (including all these varieties), N. parvuloides, N. paxillifer, N. peisonis, N. pelagica, N. pellucida, N. pennata, N. peragallii, N. perindistincta, N. perminuta, N. perpusilla (including all these varieties), 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. praereinholdii, N. princeps, N. procera, N. prolongata (including all these varieties), N. prolongatoides, 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. pseudodelicatissima, N. pseudofonticola, N. pseudohungarica, N. pseudohybrida, N. pseudonana, N. pseudoseriata, N. pseudosigma, N. pseudosinuata, N. pseudo stagnorum, N. pubens, N. pulcherrima, N. pumila, N. punctata (include all varieties), N. pungens (include all varieties), N. pungiformis, N. pura, N. puriformis, N. pusilla (include all varieties), N. putrida, N. quadrangula, N.quickiana, N. rabenhorstii, N. radicula (including all these varieties), N. rautenbachiae, 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. rigida (including all these varieties), N. ritscheri, N. robusta, N. roche nsis, 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. sarcophagum, 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. seriata (including all varieties), N. serpenticola, N. serpentiraphe, N. serrata, N.sibula (including all these varieties), N. sigma (including all these varieties), N. sigmaformis, N. sigmatella, N. sigmoidea (including all these varieties), N. silica, N. silicula (including all these varieties), N. siliqua, N. similis, N. simplex, N. simpliciformis, N. sinensis, N. sinuata (including all these varieties), N. smithii, N. sociabilis, N. socialis (including all these varieties), N. solgensis, N. solida, N. solita, N. soratensis, N. sp., N. spathulata (including all these varieties), N. speciosa, N. spectabilis (including all these varieties), N. sphaerophora, N. spiculoides, N. spiculum, N. spinarum, N. spinifera, N. stagnorum, N. steenbergensis,. N. stellata, N. steynii, N. stimulus, N. stoliczkiana, N. stompsii (including all these varieties), N. strelnikovae, N. stricta, N. strigillata, N. striolata, N. subaccommodata, N. subacicularis, N. subacutaxio, N. subacutaxio, N. subapiculata, N. subapiculata. subbacata, N. subcapitata, N. subcapitellata, N. subcohaerens (including all these varieties), N. subcommunis, N. subconstricta, N. subcurvata, N. subdenticula, N. subfalcata, N. subfraudulenta, N. subfrequens, N. subfrustulum, N. subgracida, N. subgracida, N. fl. subinvicta, N. sublaevis, N. sublance olata, N. sublica, N. sublinearis, N. sublongirostris, N. submarina, N. submediocris, N. subodiosa, N. subpacifica, N. subpunctata, N. subromana, N. subrostrata, N. subro stratoides, N. subrosalides, N. subrosaliides, N. subrosaliides, N. subrosaliides, N. submarina. N. subtilis (including all these 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. tenuis (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. vanheurckii, N. vanoyei, N. vasta, N. ventricosa, N. vermicularioides, N. vermicularis (including all these varieties), N. vermicularoides, N. vexans, N. victoriae, N. vidovichii, 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. wipplingeri, N. witkowskii, N. wodensis, N. woltereckii, N. woltereckoides, N. wuellerstorfii, N. wunsamiae, N. yunchengensis, N. zebuana, N. zululandica.

[0050] According to a particular embodiment of the invention, the protists are chosen from the genera Chlorella, Galdieria and Euglena or cyanobacteria and diatoms.

[0051] Biological reactor

[0052] Biological reactors generally consist of a tank in which a stirring or agitation element (for example one or more blades) is possibly mounted to promote homogenization of the contents of the tank. Homogenization of the contents of the tank can also be done via a liquid pump inducing a circulation flow of the culture medium inside the tank.

[0053] The biological reactor according to the invention is suitable for the cultivation of photosynthetic microorganisms, in particular for a culture comprising at least one culture step under autotrophic culture conditions comprising both water, a source of inorganic carbon and a source of light.

[0054] The invention is particularly suitable for cultivation comprising at least one step under autotrophic cultivation conditions comprising water, an inorganic carbon source and a light source.

[0055] Within the scope of the invention, the biological reactor comprises illumination means. The supply of light can be continuous or cyclical (WO20 19 / 034792, WO2021 / 160776).

[0056] The stirring speed and the quantity of light as well as the lighting mode can be adapted by the operator in order to obtain optimal culture conditions depending on the geometry of the reactor and the microorganism(s) cultivated.

[0057] It should be noted that the reactor according to the invention may be equipped with other devices or apparatus used for controlling and regulating the conditions for culturing microorganisms. Examples include the use of probes or sensors for measuring various culture parameters such as temperature, pH, pressure at the inlet and outlet of the reactor.

[0058] Enzymatic conversion module

[0059] Enzymatic conversion means the transformation of carbon dioxide (CO2) into bicarbonate ions (HCO3) by an enzyme capable of carrying out such a transformation, called conversion enzymes.

[0060] The conversion module is a device coupled to the biological reactor and comprising enzymes for converting CO2 into HCO3' immobilized on a support.

[0061] Advantageously, controlling the proportion of CO2 dissolved in the form of HCO3' ions makes it possible to control the pH of the reaction medium.

[0062] The conversion module is coupled to the biological reactor by any suitable means allowing the circulation of the culture medium enriched with CO2 in the conversion module and then towards the reactor, once the culture medium is enriched with HCO3'.

[0063] 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.

[0064] According to a preferred embodiment, the reactor and the conversion module form a circuit in which the CO2-enriched culture medium circulates from the reactor to the conversion module where the CO2 is converted into HCO3' and then from the conversion module to the reactor once the CO2 has been converted into HCO3'. This embodiment makes it possible to control more precisely all the reactions, namely the dissolution of the CO2 in the reactor and the conversion of the CO2 into HCO3' in the conversion module, so as to be able to optimize each parameter such as the temperature or the flow rate. In addition, in the case where the dissolution of the CO2 is separated from the culture medium, it is necessary to provide an additional tank for dissolving the CO2. The present embodiment, in which the CO2 is directly dissolved in the culture medium, makes it possible to overcome this drawback.

[0065] According to a further preferred embodiment, the reactor, the conversion module and the CO2 supply device form a circuit in which the culture medium circulates from the reactor to the CO2 supply device, then once enriched in CO2 circulates from the reactor to the conversion module where the CO2 is converted into HCO3' and finally once enriched in HCO3' circulates from the conversion module to the reactor once the CO2 has been converted into HCO3'.

[0066] The enzymatic 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 comprising said immobilized enzymes, said supports then being arranged randomly or according to a particular organization in the enclosure.

[0067] The dimensions of the enzymatic conversion module, and in particular the quantity of enzyme sufficient to obtain a culture medium enriched in HCO3' ions, in 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 can be adjusted by the operator.

[0068] Enzyme conversion enzymes

[0069] Conversion enzymes are known, including carbonic anhydrases (CAs), and more specifically cadmium carbonic anhydrases (CDCAs) and P and γ receptor-type tyrosine phosphatases (TPTRs).

[0070] Carbonic anhydrases are enzymes known for their ability to catalyze the hydration reaction of carbon dioxide (CO2) to bicarbonate ions (HCO3) in a reversible manner. There are 7 classes: a-AC, P-AC, y-AC, ô-AC, Ç-AC, Î]-AC, 0-AC and i-AC.

[0071] The carbonic anhydrases used in the invention are preferably carbonic anhydrases from the family of α-carbonic anhydrases.

[0072] Such conversion enzymes are described in particular in application WO 2013 / 022348.

[0073] In the context of the present application, the enzymes used according to the invention are chosen from cytoplasmic and membrane carbonic anhydrases. Certain 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 Acrobiosystems® (mouse carbonic anhydrase IX).

[0074] Like most enzymes, carbonic anhydrases have a limited lifespan. In order to increase their lifespan, these enzymes can, according to a preferred embodiment of the invention, be used in combination 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 conversion of CO2 into HCO3'.

[0075] Said proton acceptor is 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. The amount of proton acceptor to be used may be adapted by the operator, in particular so that the proportions of proton acceptors and enzymes are stoichiometric. Under such circumstances, all enzymes are regenerated without further interactions between proton acceptors and third elements being able to take place.

[0076] Immobilization of conversion enzymes

[0077] Enzyme immobilization techniques can be based on adsorption, covalent bonds, entrapment or even membrane confinement.

[0078] Several types of supports are suitable for immobilizing enzymes. These include 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 (AI2O3) or glass or carbon nanotubes.

[0079] In a particular embodiment, the enzymes are immobilized in the form of an enzyme precipitate on a fiber-type support, and more precisely on nanofibers.

[0080] 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-vinylacetate).

[0081] 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 even helical helix. The support is possibly a rectangular plate or glass tube. Here, a glass plate or tube is understood to mean a plate or tube of macroscopic size. For example, a glass plate with a surface area of ​​at least 1 cm 2 , preferably at least 2 cm 2, with a length of at least 1 cm, preferably at least 1.25 cm, a width of at least 0.5 cm, preferably at least 0.8 cm and a height of less than 0.5 mm, preferably less than 0.13 mm may be used.

[0082] In the case of a glass support, such as a glass plate, and as shown in Figure 3 A, the enzymes 311 can be immobilized using a coating 312 provided on the plate 31. For example, the enzymes can thus be immobilized by disulfide bonds, in particular via cysteine ​​molecules using a silanization process. Trimethoxysilane is then deposited on the surface of the glass and silver nanoparticles capped with cysteine ​​are added to form a layer of cysteine ​​capable of binding to the enzymes. The enzymatic conversion module can then comprise one or more plates, in particular made of glass, on which the enzymes are immobilized. The plates 31 are preferably arranged parallel to each other and placed inside the structure 3, as shown in Figure 3B. It is noted that, in accordance with the representation of Figure 3B, the structure 3 can have a tubular structure.

[0083] When the support comprises a plurality of glass plates, the distance between the latter is at least 2 mm, preferably 4 mm. In a preferred configuration, the plates are preferably spaced from 2 mm to 10 mm, preferably from 4 mm to 6 mm. Enzymes are then advantageously immobilized on each surface of these glass plates, and the culture medium can then circulate between the plates, thus increasing the CCh / enzyme contact. Thus, the conversion of CO2 into HCO3' is improved.

[0084] Furthermore, the use of a glass plate limits the deposition of biomass on the support. Consequently, this limits the fouling of the culture system. In addition, the use of a glass plate allows an optimal surface coverage rate and therefore easier access to enzymes. Indeed, it is known to use hollow nanofibers as a support for enzymes, but this type of system experiences rapid fouling, and the surface coverage rate of enzymes is not satisfactory. This disadvantageously results in reduced efficiency of the system.

[0085] The enclosure may comprise one or more supports comprising said immobilized enzymes. With reference to Figure 3B, the glass plates may in particular be arranged in parallel to each other within the conversion module.

[0086] CO2 supply device

[0087] The CO2 supply device allows the supply of CO2 into the culture medium.

[0088] The CO2 supply device can be arranged in the reactor, or outside the reactor in the culture medium circulation circuit, upstream or downstream of the conversion module in the circulation direction.

[0089] According to a preferred embodiment, 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 (figure 1).

[0090] There are many CO2 supply devices including membranes, membrane bubblers or bubble columns, ceramic bubblers, or even bubble diffuser stones.

[0091] According to a particular embodiment, the CO2 supply device is a membrane contactor.

[0092] 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.

[0093] The implementation parameters of the CO2 supply device suitable for obtaining a CO2-enriched culture medium are adapted according to the process parameters including the CO2 content of the culture medium before enrichment, the kinetics of conversion of CO2 into HCO3' and the circulation speed of the culture medium.

[0094] Membrane contactor

[0095] 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 circulation fluid(s). The membrane contactor thus comprises a first circulation circuit of a first circulation fluid for supplying carbon dioxide (CO2), called the CCL supply fluid, in contact with a first face of the transfer membrane, and a second circulation circuit of a second circulation fluid for receiving CO2, called the CO2 receiving fluid, in contact with the second face of the transfer membrane. Each fluid circulation circuit is independent of one another and can be closed or open.

[0096] 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 conditions for growth of the microorganisms.

[0097] In one embodiment, the CO2 supply fluid is in the form of gas, preferably an air / CCh mixture comprising at least 5% CO2, or at least 25% CO2, more preferably at least 50% CO2 and still more preferably at least 75% CCh. Advantageously, the supply fluid is gaseous CO2 with a content of 100% CO2.

[0098] In another embodiment, the CO2 supply fluid is in the form of a liquid comprising a CO2 absorbing solvent in which CO2 is dissolved.

[0099] CO2 absorbing solvents are solvents whose chemical composition allows the absorption of CO2 molecules. These solvents are generally solutions containing carbonate ions (CO3 2 ), bicarbonate ions (HCO3), primary, secondary or tertiary amines or amino acids. Such solvents are, for example, solutions comprising P-alanine, sarcosine, taurine, 6-aminohexanoic acid, monoethanolamine (MEA), diethanolamine (DEA), methyl diethanolamine (MDEA), or carbonate compounds such as potassium carbonate (K2CO3), sodium carbonate (NaaCCh) or ammonium bicarbonate (NH4HCO3). These compounds are preferentially present in CO2 absorbing solutions at concentrations between 0.1 and 1.0 M.

[0100] The CO2 receiving fluid is the culture medium. Said CO2 receiving fluid may also include the cultivated photosynthetic microorganisms and the nutrients necessary for their growth.

[0101] 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 the form of gas, and the CO2 receiving fluid, which is the culture medium, through a membrane, also called a transfer membrane.

[0102] Within the membrane contactor, the two fluids, the CO2 supply fluid and the CO2 receiving fluid, are in circulation.

[0103] The circulation of the CO2 supply fluid can be ensured by different means, in particular by using a source of CCh already under pressure or by 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 enabled by a stirring 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 a pump. A vacuum pump will then preferably be used.

[0104] Advantageously, the use of a membrane contactor according to the invention allows a CO2 injection process which does not generate deleterious shear stresses in the reactor, which avoids the formation of foam in the culture.

[0105] In one embodiment, the flows of the CO2 supply fluid and the CO2 receiving fluid are parallel. In another embodiment, the flows are perpendicular. In yet another embodiment, the flows are crossed. The flows of the CO2 supply fluid and the CO2 receiving fluid may be crossed, i.e., the CO2 supply fluid and the CO2 receiving fluid flow in opposite directions.

[0106] With reference to Figure 2, the membrane contactor thus comprises an inlet (210) or (211) and an outlet (210) or (211) for a first circulation fluid, as well as an inlet (220) or (221) and an outlet (220) or (221) for a second circulation 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 circulation fluid in contact with a first face of the membrane and a second circulation circuit for the second circulation fluid in contact with a second face of the membrane.

[0107] The membrane thus promotes contact between the two circulating fluids without having direct physical contact. The properties of the membrane determine the nature and characteristics of the exchanges between the two circulating fluids.

[0108] 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.

[0109] The membrane can be a porous or non-porous membrane, 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.

[0110] Hydrophilic membranes allow the passage of water and therefore, for example, the dehydration of a solvent. Typically, these membranes are made of 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 in essentially flat modules, with the "permeate" compartment connected to a partially vacuum chamber, so as to maintain the acting force and evacuate the separation products. [OR I] 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 compounds in the mixture that allows selective transfer through the membrane. Very high selectivities are thus obtained, with transfer fluxes generally remaining low.

[0112] Membrane contactors are characterized by the transfer membranes that compose them, more particularly by the geometric configuration and structure of these membranes.

[0113] The membranes can be of flat, spiral, tubular or hollow fiber geometric configuration (pipes or tubes).

[0114] According to a preferred embodiment, the membrane(s) have a geometric configuration of hollow fibers. Advantageously, the membrane contactor comprises several fibers dispersed in bundles of tubes. The external diameter of the membranes is greater than 50 μm, preferably greater than 60 μm, even more preferably greater than 70 μm. In another embodiment, the membranes with a hollow fiber configuration have an external diameter of at least less than 100 μm. In another embodiment, the external diameter of the membranes is less than 2 mm, preferably less than 1.5 mm, more preferably less than 1 mm. Advantageously, the external diameter of the membranes with a hollow fiber configuration is between 100 μm and 1 mm. The internal diameter of the membranes according to the invention is preferably less than 900 μm, or even less than 850 μm. In a preferred embodiment, the external diameter of the membranes with a hollow fiber configuration is between 100 and 800 μm.

[0115] The internal diameter of the 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 the 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, even more preferably between 10 and 100 pm. Advantageously, the internal diameter of the membranes with a hollow fiber configuration according to the invention is between 20 and 90 pm.

[0116] With reference to Figure 2B and according to one embodiment, the membrane contactor comprises a transfer membrane in a hollow fiber configuration, the latter 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. It is thus possible to define an internal face and an external face of the membrane corresponding respectively to the internal face of the fiber (or tube) (21) and to the external face of the fiber (or tube) (22).

[0117] 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.

[0118] For the purposes of this application, transfer membranes with a hollow fiber configuration are microporous membranes or non-porous membranes.

[0119] In one embodiment, the transfer membrane of the membrane contactor is a porous membrane, and comprises pores whose diameter is less than 10 μm. Advantageously, the porous membrane is microporous, and has pores with a diameter of less than 8 μm, preferably less than 5 μm, or even less than 3 μm, more preferably less than 2 μm. In another embodiment, the microporous membrane comprises pores whose diameter is greater than 0.001 μm, 0.002 μm, 0.003 μm, 0.004 μm, or even greater than 0.005 μm. Advantageously, the diameter of the pores of the membrane is thus between 0.005 and 2 μm. In one embodiment, the diameter of the pores is less than 1 μm, or even less than 0.5 μm and preferably less than 0.2 μm or even less than 0.1 μm. In another embodiment, the pore diameter of the membrane is between 0.005 and 0.1 μm.Yet, 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 pore diameter of the membrane is approximately 0.01 pm, 0.02 pm, 0.03 pm, 0.04 pm or 0.05 pm.

[0120] In a preferred embodiment, the transfer membrane of the membrane contactor is a dense membrane. The diffusion of the molecules is then characterized by a phenomenon of diffusion through the membrane via the adsorption of molecules having a strong affinity with the membrane. This embodiment has the advantage of reducing or even preventing the clogging of the membrane of the membrane contactor with the various elements transported in the liquid medium (such as, for example, cellular debris) compared to a porous membrane which has potential attachment zones on its structure and thus makes it possible to maintain the same gas exchange performance across the membrane throughout the duration of the culture process.

[0121] According to one embodiment, the transfer membrane is a non-porous membrane with an internal diameter of 150 to 450 μm and an external diameter of 250 to 550 μm.

[0122] According to one embodiment, the transfer membrane is a porous membrane with an internal diameter of 150 to 250 μm and an external diameter of 250 to 350 μm and comprising pores with a diameter of 20 to 40 μm.

[0123] According to another embodiment, the transfer membrane is a dense membrane with an internal diameter of 350 to 450 μm and an external diameter of 5 to 100 mm.

[0124] Advantageously, the transfer membrane, porous or dense, in particular dense, has a thickness of less than 1000 μm, preferably from 100 μm to 1000 μm.

[0125] The use of a membrane contactor according to the invention makes it possible in particular to ensure all or part of the exchanges 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 between the two circulating fluids.

[0126] According to one embodiment of the invention, the membrane contactor has an exchange surface between the two circulation fluids of at least 0.003 m 2 / L of CO2 receiving circulating fluid, preferably at least 0.006 m 2 / L. In parallel, the exchange surface between the two circulating fluids is less than or equal to 0.020 m 2 / L per liter of CO2 receiving circulation 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 m 2 / L of CO2 receiving circulation fluid. In another embodiment, the membrane contactor has an exchange surface area of ​​between 0.003 and 0.012 m 2 / L of CO2 receiving circulation fluid, preferably between 0.006 and 0.012 m 2 / CO2 receiving circulation fluid.

[0127] The materials used for the production of transfer membranes are generally polymers, such as polyethylene (PE), polypropylene (PP), polysulfone (PSu), polyether sulfone (PES), polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE), polyimide (PI), polyamide (PA), polycarbonate (PC), polyacrylonitrile (PAN), and cellulose acetate (CA). The transfer membranes can also be made from silicone or silicone derivatives. In the context of the present application, the term silicone includes inorganic compounds formed from a chain of silicon and oxygen atoms and whose silicon atoms can carry groups. Mention will be made in particular of polymers belonging to the siloxane family, and more particularly polydimethylsiloxane (PDMS) or compositions comprising polydimethylsiloxane (PDMS), in particular a ternary system composed of PDMS, water and tetrahydrofuran (THF). Advantageously, the transfer membranes of the present application are made from silicone, preferably PDMS, in particular said membranes are thin PDMS membranes with a thickness of between 0.01 and 1.00 cm in sheet or roll form.Examples of commercial transfer membranes are thin silicone membranes such as SSP-M823 from SSP® or PDMS from Saint-Gobain. Such membrane contactors and their operating principles are known per se. They are commercially available, notably under the references pervélys and 3M™ liqui-Cel.

[0128] The combination of the use of a conversion enzyme and a membrane contactor makes it possible to reduce the CO2 concentration, or even lead to the absence of CO2 molecules, in the culture medium, thus accentuating the differences in CO2 concentrations and pressure between the culture medium constituting the CO2 receiving fluid and the CO2 supply fluid. In this way, the diffusion of CO2 molecules from the CO2 supply fluid to the culture medium is promoted.

[0129] In an alternative embodiment, the conversion enzymes are advantageously immobilized within the membrane contactor. In this embodiment, the membrane contactor also fulfills the role of the enzyme conversion module. This therefore represents a saving of space and cost.

[0130] According to this alternative embodiment, the enzymes can be immobilized on plates provided inside the membrane contactor, as previously described, or directly deposited on the surfaces of the membrane contactor.

[0131] Oxygen (O2) evacuation device

[0132] In a preferred embodiment of the invention, the reactor is included in a closed circuit so as to isolate the culture from environmental hazards external to the reaction, such as temperature variations or variations in the gas composition of the ambient atmosphere.

[0133] In this case, it is preferable to evacuate the oxygen (O2) produced during photosynthesis by means of an oxygen evacuation device to promote the growth of photosynthetic microorganisms and avoid possible overpressures in the reactor enclosure. This may be one or more valves associated with the reactor to allow the evacuation of overpressure gases.

[0134] 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 membrane of the membrane contactor, by a path opposite to that of the CO2.

[0135] In the case where the CO2 supply means is a membrane contactor, and in particular when the CO2 supply fluid is a liquid, the evacuation of the dioxygen will be carried out by a suitable means, independent of said CO2 supply membrane contactor, for example using one or more valve(s) or with another membrane contactor adapted to the evacuation of dioxygen from the culture medium. A membrane contactor adapted to the evacuation of O2 comprises the same structure as a CO2 supply membrane contactor as described above but with a FO2 evacuation circulation 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 liquid.

[0136] Conversion enzyme regeneration device

[0137] In a particular embodiment, the culture system further comprises an enzyme regeneration device (531). This regeneration device, placed in bypass of the main circuit of the culture medium, consists of a module for supplying a proton acceptor, said proton acceptor is preferentially chosen from asparagine, glutamine, histidine, serine, threonine, tyrosine, aspartic acid and glutamic acid, even more preferentially, 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 solution proton acceptor (531), is activated. The solution then passes through the enzymatic conversion module (53) in order to regenerate the conversion enzymes. The proton acceptor solution circulation cycle can be repeated several times in the enzymatic conversion module instead of the culture medium. The number of regeneration cycles to be applied is determined according to the different culture parameters and the characteristics of the enzymes used. The circulation of the proton acceptor solution is enabled by a pump (54) placed, in the direction of circulation of the solution, between the enzymatic conversion module (53) and the proton acceptor solution supply module (531) or between the proton acceptor solution supply module (531) and the enzymatic conversion module (53).

[0138] Cleaning device

[0139] In another particular embodiment, the culture system further comprises a cleaning device (56) for the culture medium circulation circuit. This device is placed in bypass of the main culture medium circuit and consists of a set of cleaning operations comprising one or more washes with water, a wash with an alkaline solution and a wash with an acid solution. The alkaline solution is preferably a sodium hydroxide-based solution, comprising from 0.5 to 5% sodium hydroxide. The acid solution is an aqueous solution based on nitric acid for removing limescale and sodium hydroxide deposits in 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 acid solution and rinsing the device again with water.It is understood that the operations can be repeated several times as required individually or sequentially. The cleaning solutions circulate through the various elements of the culture system, such as the biological reactor (51), the CO2 supply device (52) or the enzymatic conversion module (53), in particular by means of pumps (54) and valves (55). A suitable arrangement of valves and pumps within the culture system can thus allow the cleaning of one or more elements independently or in combination. The elements of the system are optimally arranged depending on the objectives.

[0140] Cultivation system

[0141] The invention also relates to a system for culturing photosynthetic microorganisms comprising a culture system comprising a biological reactor, a carbon dioxide (CO2) supply device and a module for enzymatic conversion of CO2 into HCO3' ions coupled to the reactor, said conversion module comprising enzymes for converting CO2 into HCO3' immobilized on a support.

[0142] In one embodiment, the culture medium is first supplied with CO2 at the CO2 supply device before passing through the enzymatic conversion module where catalysis of CO2 into HCO3' ions occurs.

[0143] In particular, with reference to Figure 1, the culture system according to the invention comprises a biological reactor (1), a CO2 supply device (2), said CO2 supply device preferably being a membrane contactor, and an enzymatic conversion module (3) in which the enzymes are immobilized on a support.

[0144] The CO2 supply device is advantageously a membrane contactor. With reference to Figure 2 and according to one embodiment, the membrane contactor comprises a transfer membrane (20) in hollow fiber configuration, these being dispersed in the form of a bundle of tubes in the membrane contactor. According to one embodiment, the CO2 supply fluid circulates in a first space (21) corresponding to the interior of the hollow fibers, the fluid circulating according to a flow between the inlet (210) or (211) and the outlet (210) or (211) of the bundle of hollow fibers. 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 according to a flow between the inlet (220) or (221) and the outlet (220) or (221) of the membrane contactor.

[0145] In another embodiment, the CO2 supply fluid circulates in the membrane contactor, i.e. in a space (22) delimited by the watertight walls of the membrane contactor, the CO2 supply fluid then circulating 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 circulates inside the hollow fibers (21) from the inlet (210) or (211) to the outlet (210) or (211).

[0146] With reference to the embodiment of the invention illustrated in Figure 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 enzymatic conversion of CO2 into HCO3' ions (53). The system presented is preferably a closed circuit.

[0147] Preferably, the different elements of the device are arranged as shown in Figure 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 circulates from the biological reactor to the membrane contactor (52) where the CO2 supply is made, then the culture medium enriched with CO2 passes through the enzymatic conversion module (53) where the catalysis of CO2 molecules into HCO3' ions occurs, before the culture medium returns to the biological reactor (51).

[0148] Preferably, the system may further comprise one or more pumps. (54) to ensure the continuous circulation of fluids as well as valves (55). It is understood that probes, sensors and other elements for measuring culture parameters or safety systems can be added to the system.

[0149] 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 be associated with different CO2 supply devices, in particular but not only a membrane contactor.

[0150] In particular, Figure 4 shows a specific embodiment comprising as CO2 supply device a membrane contactor, a cleaning device and an enzyme regeneration device.

[0151] It is understood that the culture system according to the invention comprising a biological reactor (1), a CO2 supply device (2) and an enzymatic conversion module (3) is used for the culture of photosynthetic microorganisms comprising at least one step under autotrophic culture conditions, preferably only steps under autotrophic culture conditions.

[0152] EXAMPLES

[0153] 5 separate cultures of Chlorella sorokiniana are grown in a 2.5 L photobioreactor, under artificial illumination with wavelengths between 400 and 460 nm and between 620 and 700 nm and an intensity of 350 pmol / m2 / s.

[0154] These cultures differ from each other 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 conversion enzyme from Sigma, CAS: 9001-03-0 with a dosage of 0.1 g / l). The carbonic anhydrase is free in the culture medium or immobilized on a support in an enzymatic conversion module coupled to the biological reactor. The different conditions are summarized in Table 1 below.

[0155] [Table 1] 0156] When a device coupled to the reactor is used (conversion module or membrane contactor, i.e. experiments B, D and E) and there is therefore a circuit in which the culture medium circulates, the flow of the culture medium is induced by a paddle stirrer with a speed of 500 revolutions per minute (rpm). Note that the homogenization of the medium is ensured using the same stirring blade at 500 rpm.

[0157] Optical density (OD) measurements were made in duplicate at 750 nm with a spectrophotometer (7315, JENWAY).

[0158] In addition, daily productivity is calculated from the DO and a correlation coefficient (CC) specific to the cultivated microorganism according to the following formula, particularly when the biomass concentration is too low to have an accurate value of the dry mass.

[0159] Productivity (DO) (gL^.day 1 ) = ((DO(day D)-DO (day Dl))x 0.6) / (time (day D)-time (day Dl))x24

[0160] For the present culture of Chlor ella sorokinina:

[0161] Productivity (DO) (gL^.day 1 ) = ((DO(day D)-DO (day Dl))x 0.6) / (time (day D)-time (day Dl))x24d

Claims

Claims 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 a module for enzymatically converting CO2 into HCO3' ions coupled to the reactor, said method comprising the steps of enriching the culture medium with CO2 via the CO2 supply device, to obtain a culture medium enriched in CO2, and contacting the culture medium enriched in CO2 obtained with enzymes for converting CO2 into HCO3' in the conversion module to obtain a culture medium enriched in HCO3' ions, in which conversion module the enzymes for converting CO2 into HCO3' are immobilized on a support, the method being characterized in that the support is a glass plate.

2. Method according to claim 1, characterized in that the conversion enzymes are carbonic anhydrases.

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

4. Method according to one of claims 1 to 3, characterized in that the culture medium meets the conditions of autotrophic culture.

5. System for culturing photosynthetic microorganisms comprising a biological reactor, a carbon dioxide (CO2) supply device and a module for enzymatic conversion of CO2 into HCO3' ions coupled to the reactor, said conversion module comprising enzymes for converting CO2 into HCO3- 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 one of claims 5 or 6, characterized in that the flow of culture medium is directed from the biological reactor to the CO2 supply device, then from the CO2 supply device to the enzymatic conversion module.

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

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

Images