PROCESS FOR CAPTURING PHYTOTOXINS IN A BIOLOGICAL REACTOR

Abstract

The present invention relates to a method for recovering phytotoxins from a fermentation juice obtained from the culture of microorganisms producing said phytotoxins. The method is characterized in that it comprises a first membrane filtration cycle comprising the following steps: Membrane filtration of the biomass fermentation juice so as to retain the phytotoxins in the retentate,Recovery of a retentate comprising said phytotoxins, andRecovery of a first permeate depleted in phytotoxins, said permeate having a phytotoxin content lower than that of the fermentation juice. The method may, if appropriate, be followed by at least one additional membrane filtration cycle of the retentate obtained in step (c) of the previous cycle.

Description

Title of the invention: METHOD FOR CAPTURING PHYTOTOXINS IN A BIOLOGICAL REACTOR FIELD OF THE INVENTION

[0001] The present invention relates to a method for recovering phytotoxins from a fermentation juice obtained from the culture of microorganisms producing said phytotoxins.

[0002] BACKGROUND OF THE INVENTION

[0003] The methods for recovering biomasses and compounds included in the biomasses and / or culture media are well known to those skilled in the art. These methods generally consist of centrifugation of the culture medium or (micro)filtration, also allowing purification of the biomass.

[0004] Microorganisms, and more particularly algae and microalgae, are today widely cultivated for their property of producing biomasses rich in lipids, proteins, carbohydrates, etc. In parallel with the multiplication of cells, microorganisms secrete molecules of interest including phytotoxins. These phytotoxins, present mainly in the culture medium, and more precisely in the fermentation juice, are generally eliminated during the recovery and purification of the biomass.

[0005] However, phytotoxins secreted by microorganisms have interesting biological properties depending on their nature, and can be used in various fields such as the agricultural or pharmaceutical industry. Isolated phytotoxins can also be used to adjust the concentrations of the batches of biomass produced. Indeed, the quality and concentration of compounds, including phytotoxins, present in biomass vary depending on the cultivation conditions. Finally, in the context of an environmental issue, it is interesting to be able to recover phytotoxins, in particular phytotoxins harmful to the environment, and thus limit their spread in nature.

[0006] The inventors have thus developed a process for recovering phytotoxins from a fermentation juice obtained from the culture of microorganisms.

[0007] BRIEF DESCRIPTION OF THE INVENTION

[0008] The present invention relates to a process for recovering phytotoxins from a fermentation juice obtained from the culture of microorganisms producing said phytotoxins, characterized in that it comprises a first membrane filtration cycle comprising the following steps: a. membrane filtration of the biomass fermentation juice so as to retain phytotoxins in the retentate, b. recovery of a retentate comprising said phytotoxins, and c. recovery of a first permeate depleted in phytotoxins, said permeate having a phytotoxin content lower than that of the fermentation juice, followed where appropriate by at least one additional cycle of membrane filtration of the retentate obtained in step (c) of the previous cycle, with the recovery of at least one second retentate comprising said phytotoxins and at least one second permeate depleted in phytotoxins having a phytotoxin content lower than that of the first permeate.

[0009] The method according to the invention can thus comprise m additional membrane filtration cycles, and the recovery of m+1 retentates comprising the phytotoxins and m permeates depleted in phytotoxins, m being an integer equal to or greater than 1.

[0010] The different membrane filtration cycles can be carried out on the same filtration membranes or on different membranes. The same applies to the filtration cycles, each of which can comprise similar or different membranes.

[0011] In the context of the present invention, the final concentration of phytotoxins obtained in the permeate recovered after the last filtration cycle can be determined by the equation: [00i2] cip] =

[0013] in which A is the number of cycles m+1, X is the concentration factor of the membrane and R is the molecular rejection rate of the membrane.

[0014] Phytotoxins are molecules with a molecular weight between 300 and 3000 Da.

[0015] Finally, the invention relates to the use of the phytotoxins included in the retentate or isolated as an additive in a biomass of microorganisms, and the use of the permeate resulting from the process as a food in a process for culturing microorganisms. Brief description of the drawings

[0016] [Fig.l]: Embodiment of the invention.

[0017] [Fig.2]: Representation of the number of filtration cycles n as a function of the concentration of phytotoxins (cylindrospermopsin) in the retentate.

[0018] [Fig.3]: Representation of the evolution of the concentration of phytotoxins (cylindrospermopsin) in the retentate as a function of the number of membrane filtration cycles.

[0019] [Fig.4]: Representation of the yield of phytotoxins recovered at each stage of membrane filtration depending on the type of membrane. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention relates to a process for recovering phytotoxins from a fermentation juice obtained from the culture of microorganisms producing said phytotoxins, characterized in that it comprises a first membrane filtration cycle comprising the following steps: a. membrane filtration of the biomass fermentation juice so as to retain phytotoxins in the retentate, b. recovery of a retentate comprising said phytotoxins, and c. recovery of a first permeate depleted in phytotoxins, said permeate having a phytotoxin content lower than that of the fermentation juice, followed where appropriate by at least one additional cycle of membrane filtration of the retentate obtained in step (c) of the previous cycle, with the recovery of at least one second retentate comprising said phytotoxins and at least one second permeate depleted in phytotoxins having a phytotoxin content lower than that of the first permeate.

[0021] In the context of the present invention, phytotoxins are understood to mean the molecules or chemical substances secreted by microorganisms during their cultivation, that is to say in parallel with the formation of a biomass of said microorganisms. The term "phytotoxin" thus includes the biotoxins produced by plants, fungi (also called mycotoxins), and algae (also called phycotoxins). In the context of the present invention, the phytotoxins are advantageously phycotoxins. Examples of phycotoxins are domoic acid, okadaic acid, saxitoxins, brevetoxins or ciguatoxins.

[0022] In the context of the present invention, the term "biomass" means a set of microorganism cells (i.e. several thousand cells or more) produced by cultivating said microorganisms on a culture medium and separated from this culture medium at the end of the culture after harvesting. As a result of their culture conditions, the microorganism cells in the biomass all have substantially the same composition. The microorganism biomass is a composition which comprises microorganism cells cultivated with water and possibly traces of nutrients and other elements present in the culture medium.

[0023] In the context of the present application, the biomass is a microalgal biomass. The microalgal biomass may be a raw biomass, a lysed biomass, a homogenized biomass or a dried biomass.

[0024] In the context of the present application, the term “raw biomass” designates a biomass obtained after harvesting, without other modification such as cell lysis or homogenization. Raw biomass comprises at least 70% water, and up to 90% water, preferably 80 to 85% water. Raw biomass also includes raw biomass washed after harvesting to remove certain nutrients and other elements present in the culture medium.

[0025] A “lysed biomass”, in the context of the present application, means a microalgal biomass in which at least 50% of the cells are lysed, preferably at least 70%, more preferably at least 80%, 85%, 90%, 95%, up to 100% of the cells are lysed. For this purpose, lysis is performed by a human using known methods, such as mechanical, chemical or enzymatic lysis, well known to those skilled in the art (WO2015 / 095688, WO2011 / 153246, US6750048 and WO2015 / 095694, WO 2018 / 178334, WO 2020 / 144330, WO 2020 / 053375, CN 106749633, CN102433015, CN1117973, Michelon & al. 2016). The raw biomass may comprise lysed cells due to the harvesting step. However, the ratio of lysed cells in the raw biomass is generally very low, less than 50%, with harvesting being performed to avoid significant damage to the biomass.Except for materials added for biomass lysis, lysed biomass contains only algal biomass in a transformed state.

[0026] A “dry biomass” in the context of the present application means that the biomass comprises less than 10% water, preferably from 1 to 10% water, more preferably from 2 to 7%. Dry biomass is a biomass obtained after a drying step carried out under conditions defined by a human being or a computer under the control of a human being.

[0027] In the context of the present application, the term "microalgae cultivation" means a human-controlled industrial cultivation method comprising seeding a culture medium chosen by said human with cells of said microalgae, allowing the cells to multiply and grow to a certain high cell density in the culture medium. For the avoidance of doubt, "microalgae cultivation" according to the invention does not mean harvesting microalgae found in nature.

[0028] In one embodiment, the microalgal biomass may be raw biomass, lysed biomass, homogenized biomass, or dried biomass.

[0029] The “microalgal cultures” comprise a step a) of cell growth and metabolite accumulation, and a step b) of biomass harvesting by separating all the microalgal cells forming the culture medium. The cell growth and accumulation may be a single step where the growth conditions allow the accumulation of metabolites. In another embodiment, the cell growth and accumulation may be carried out in two successive steps, where step a1) allows the growth of the cells to a certain density, and is followed by maturation step a2) where the metabolites are accumulated in the cells without significantly affecting the cell density in the culture medium. In some embodiments, growth step a1) also allows for some level of metabolite accumulation enhanced by maturation step a2). In some embodiments, maturation step a2) also allows for some level of improvement in cell density.

[0030] According to the invention, the phytotoxins are metabolites.

[0031] The term “culture medium” in the context of the present application designates an aqueous composition comprising the nutrients necessary for the growth of the microalgae. The culture medium comprises a nitrogen source, a phosphorus source, salts, and / or vitamins, and / or trace elements and other nutrients well known to those skilled in the art. For heterotrophic or mixotrophic cultures, the culture medium also comprises a source of organic carbon metabolized by the microalgae, while for autotrophic cultures, a source of mineral carbon such as CO2 is present in the culture medium.

[0032] The carbon source may be an organic carbon source for a culture in heterotrophic or mixotrophic mode. An “organic carbon source” in the context of the present application, designates an aliphatic molecule, in particular a polyol, an organic acid or a carbohydrate. Examples of such complex carbon sources are glycerol, glucose, fructose, xylose, sucrose, cellulose derivatives, lactic acid and salts, starch, starch derivatives, and mixtures thereof, and any complex product comprising at least one of these molecules.

[0033] The carbon source may be carbon dioxide for a culture in autotrophic mode where light provides energy for the cells to incorporate carbon during photosynthesis. This carbon dioxide is generally supplied to the cells throughout the culture in a gaseous or pre-dissolved form.

[0034] The methods for culturing microorganisms are well known to those skilled in the art, whether in heterotrophic, autotrophic or mixotrophic mode.

[0035] The microorganisms according to the invention are cultivated in biological reactors, reactors within which biological phenomena develop, such as growth of pure cultures or microorganisms or of a consortium of microorganisms.

[0036] The phytotoxins produced by microorganisms are mainly found in the fermentation juice obtained during cell culture. The term "fermentation" thus designates the operation which makes it possible to produce biomass and / or bioconversion products by culturing microorganisms.

[0037] In the context of the invention, the term “fermentation juice”, also called culture broth, refers to the liquid phase included in the biological reactor. The juice fermentation thus includes, among other things, water, nutrients and compounds secreted and / or produced by microorganisms.

[0038] The fermentation juice and the biomass are separated according to the usual separation methods. In particular, centrifugation (plate centrifuge or sedicant), and filtration (plate filter, press filter, ceramic or organic tangential filtration) will be mentioned. These techniques are well known to those skilled in the art who will be able to determine, depending on the nature and characteristics of the biomass and the fermentation juice, the most appropriate method.

[0039] In one embodiment, the method for recovering phytotoxins from a fermentation juice obtained from the culture of microorganisms producing said phytotoxins is thus preceded by a step of separating the biomass and the fermentation juice.

[0040] The fermentation juice can also be obtained from a fermentation must obtained by a process of fermentation of said microorganisms in a culture medium comprising an aqueous culture medium and nutrients necessary for the growth of said microorganisms.

[0041] The term "fermentation must" means the liquid / solid mixture produced by the microorganisms. In this case, the fermentation must is separated from the microorganism biomass according to methods well known to those skilled in the art, before extracting the fermentation juice.

[0042] Once the fermentation juice has been separated, the phytotoxins are extracted according to the method defined in the present application. The method for recovering phytotoxins from the fermentation juice of the biomass produced comprises at least one membrane filtration cycle, this filtration cycle comprising the following steps: a. membrane filtration of the fermentation juice of the biomass so as to retain the phytotoxins in the retentate, b. recovery of the retentate comprising said phytotoxins, and c. recovery of the permeate depleted in phytotoxins, said permeate having a lower phytotoxin content than fermentation juice.

[0043] The term "retentate" means all the molecules and / or particles that are retained during the membrane filtration process. On the contrary, the term "permeate" or "filtrate" means the molecules and / or particles that have passed through the membrane. In the context of the invention, the phytotoxins are mainly recovered in the retentate.

[0044] The filtration cycle may comprise one or more additional membrane filtration cycles of the retentate obtained in step (c).

[0045] In one embodiment, the method comprises at least one membrane filtration cycle of the retentate obtained in step (c), then leading to the recovery of at least at least a second retentate comprising said phytotoxins and a second permeate depleted in phytotoxins having a phytotoxin content lower than that of the first permeate.

[0046] In another embodiment, the method according to the invention comprises more than one additional membrane filtration cycle. Generally, the method may comprise m additional membrane filtration cycles, m being an integer between 1 and 10. In one embodiment, m is between 1 and 10, more particularly m is selected from the values ​​of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In a preferred embodiment, m has the value 1, 2 or 3. In another preferred embodiment, m has the value 1 or 2.

[0047] The process according to the invention comprising m additional membrane filtration cycles leads to the recovery of m+1 retentates comprising the phytotoxins and m permeates depleted in phytotoxins.

[0048] Membrane filtration cycles are carried out with a single membrane or on a new membrane, similar or different. The term "identical or different membrane" means membranes whose intrinsic characteristics are identical or different.

[0049] In one embodiment, each filtration cycle of the method according to the invention is carried out on the same filtration membrane. The permeate obtained from the membrane filtration is thus filtered again with this first membrane in the additional filtration cycle.

[0050] In another embodiment, each filtration cycle is carried out on a new membrane, it being understood that the latter may have intrinsic characteristics similar to or different from the membrane of the previous filtration cycle.

[0051] The number of additional membrane filtration cycles m or the total number of membrane filtration cycles n are determined based on several parameters, including the phytotoxin content of the fermentation juice, the concentration factor of the filtration membrane and the final phytotoxin concentration desired in the permeate recovered after the last filtration cycle.

[0052] Membranes are characterized by their intrinsic properties including pore size, cutoff threshold, selectivity and permeability. A person skilled in the art will be able to determine the type of membrane to be used according to the process parameters, such as the number of filtration stages, the concentration factor, the desired phytotoxin concentration in the final permeate, etc.

[0053] In the context of the present invention, the membranes are membranes whose cut-off threshold is lower than the molar mass of the filtered phytotoxins. Those skilled in the art will be able to determine the value of the cut-off thresholds, and consequently the appropriate membranes to use. In a preferred embodiment, the membranes thus have pores with a diameter between 0.6 and 0.7 nm. In parallel, the molecular weight cutoff threshold of the membranes is between approximately 100 and 300 Da. Particular examples of membranes are the membranes used in reverse osmosis. Such membranes are the SEPA CF II membranes marketed by Ge Osmonics and SW C4 marketed by Hydranautics.

[0054] The final phytotoxin concentration in the permeate recovered after the last membrane filtration cycle, as a function of the number of cycles m+1, can be determined by the following equation:

[0055] =

[0056] in which 2 is the number of cycles m+1, X is the concentration factor of the membrane and R is the molecular rejection rate of the membrane, and the values ​​of X and R being known.

[0057] It is also possible to determine the overall efficiency nk of a number of passages n in a membrane according to the following formula:

[0058] / n, = ----7~îT k \ ( j /

[0059] In the case where only one filtration cycle is used, the efficiency nk is calculated according to the formula:

[0060] “----L—-

[0061] Advantageously, the recovered permeate has a phytotoxin content lower than that of the fermentation juice. In one embodiment, the permeate has a phytotoxin content of less than 10% by weight relative to the total weight of the biomass, or even less than 8%, or even less than 7%, preferably less than 6%. In another embodiment, the phytotoxin content is between 0 and 5% by weight relative to the total weight of the biomass. Preferably, the phytotoxin content is between 0 and 4%, preferably between 0 and 3%, and more preferably between 0 and 2%. In another preferred embodiment, the phytotoxin content is less than 1% by weight relative to the total weight of the biomass. In the context of the present invention, the limit of 0% by weight relative to the total weight of the biomass corresponds to the detection limit of the phytotoxins present in the permeate.

[0062] The m+1 retentates comprising the phytotoxins recovered from the first membrane filtration cycle and the retentates from the m additional membrane filtration cycles are recovered and possibly combined.

[0063] An embodiment of the method for recovering phytotoxins from a fermentation juice obtained from microorganisms is shown in [Fig.l]. After culturing the microorganisms in the biological reactor (1), the fermentation juice (2) and the biomass (3) are separated. The biomass (3) is recovered, possibly treated and / or transformed according to its fate. In parallel, the fermentation juice (4) is purified by membrane filtration (5). The retentate (7) is isolated, while the permeate (6) can be isolated or undergo a new membrane filtration cycle m, leading to the production of the retentate m+1 and the permeate m. The permeate thus obtained is reintroduced into the biological reactor (1).

[0064] Microorganisms and cultured microorganisms are well known to those skilled in the art. These include bacteria, yeasts, or even protists, and more particularly algae and microalgae. According to a preferred embodiment, the cultured microorganisms are protists.

[0065] By protist is meant all eukaryotic unicellular microorganisms. Microalgae (Chlorophytes such as Chlorella, Senedesmus, Tetraselmis, Haematococcus; Charophytes, chrysophytes including diatoms; Nannochloropsis; Euglenophytes such as Euglena, Phacus; Rodophytes including Galdieria, etc.), unicellular fungi (Thrautochytrids such as Schizochytrium, Aurantiochytrium, etc.), cyanobacteria (Anabaena, Nostoc, Microcistis, Arthrospira, Spirulina, etc.) or heterotrophic flagellates (Crypthecodinium, etc.) are part of the protist group.

[0066] In one embodiment, the microorganisms according to the invention are algae and microalgae preferably belonging to the classes of Chlorophytes, Rodophytes, Thraustochytrids, Diatoms and Dinoflagellates.

[0067] When the microalgae are diatoms, they may be chosen from the following genera: Nitzschia, Navicula, Gyrosigma, Phaeodactylum, Thalassiosira, etc.

[0068] When the algae or microalgae are Dinoflagellates, they can be chosen from the species: Alexandrium, Fragilidinium, Coolia, Ostreopsis, Fukuyoa, Gambierdiscus, Goniodoma, Pyrocystis, Pyrodinium, Pyrophacus, Ceratium, Tripos, Lingulodinium, Amylax, Gonyaulax verior Gonyaulax, Ceratocorys, Protoceratium, Amphisolenia, Dinophysis, Histioneis Ornithocercus, Phalachroma, Triposolenia, Si-nophysis, Pseudophalacroma, Ansanella, Asulcocephalium, Baldinia, Biecheleria, Biecheleriopsis, Borghiella, Cystodinium, Leiocephalium, Pelagodinium, Phytodinium, Piscinoodinium, Polarella, Protodinium, Symbiodinium, Woloszynskia, Amyloodinium, Cryptoperidiniopsis, Paulsenella, Pfiesteria, Pemambugia, Dubosquodinium, Naiadinium, Scrippsiella, Theleodinium Apocalathium Crypthecodinium Stoeckeria, Chimonodinium, Thoracosphaera, Aduncodinium glandula, Tintinnophagus acutus, Azadinium, Amphidoma, Azadinium dexteroporum, Azadinium polongum, Azadinium concinnum, Azadinium caudatum,Durinskia, Galeidinium, Kryptoperidinium, Un-ruhdinium, Blixaea, Ensiculifera, Pentapharsodinium, Clade Monovela: Amphi-diniopsis, Archaeperidinium, Herdmania, Islandinium, Protoperidinium americanum, , P. fusiforme, P. fukuyoi, P. monovelum, P. parthenopes, Peridinium clade: Peridinium willei, P. volzii, P. cinctum, P. gatunense, P. bipes, P. limbatum, Protoperidinium sensu stricto: Protoperidinium abei, P. bipes, P. conicum, P. crassipes, P. divergens, P. denticulatum, P . elegans, P. excentricum, P. leonis, P. pallidum, P. pellucidum, P. pentagonum, P. punctulatum, P. thorianum, P. thulesense, Kolkwitziella, Diplopsa-lioideae III and Oceanica clade: Diplopsalopsis, Niea, Qia, Gotoius, Protoperidinium claudicans, Protoperidinium depressum, Diplopsalis caspica, D. lenticula, Prepe-ridinium meunieri, Heterocapsa, Blepharocysta, Podolampas, Roscojfia, Prorocentrum dentatum, P. donghaiense, P. emarginatum, P. fukuyoi, P. mexicanum, P. micans, P. minimum, P. rhathymum, P. shikoense, P. texanum, P. triestinum, P. tsaw-wassenense, Prorocentrum belizeanum, P. bimaculatum, P. concavum, P. consutum, P. foraminosum, P. maculosum, P. levis, P. lima, Prorocentrum glenanicum, P. panamense, P.pseudopanamense, Plagiodinium, Prorocentrum cassubicum, Akashiwo, Chytriodinium, Dissodinium, Erythropsidinium, Gymnodinium, Gym-noxanthella, Gyrodiniellum, Lepidodinium, Nematodinium, Nusuttodinium, Paragym-nodinium, Pellucidodinium, Pheopolykrikos, Polykrikos, Proterythropsis, Spinife-rodinium, Warnowia, Brachidinium, Karenia, Karlodinium, Takayama, Gyrodinium, Amphidinium, Amphidinium mootonorum, A. herdmanii, A. longum, A. carterae, To-rodinium, Kapelodinium, Esoptrodinium, Jadwigia, Tovellia, Blastodinium navicula, B. mangini, B. galatheanum, Blastodinium spinulosum, B. crassum, B. pruvoti, B.inornatum, Blastodinium contortum, Blastodinium oviforme, Ptychodiscus noctiluca, Ailadinium, Amphidiniella, Bysmatrum, Glenoaulax, Glenodiniopsis, Gloeodinium, Hemidinium, Heterodinium, Madanidinium, Oodinium, Palatinus, Parvodinium, Peridinium sociale, Peridiniopsis borgei, Pileidinium, Pseudadenoides, Rufusiella, Sa-bulodinium, Stylodinium, Thecadinium, Zooxanthella, Ankistrodinium, Apicoporus, Balechina, Bispinodinium, Ceratoperidinium, Margalefidinium, Cucumeridinium, Le-vanderina, Moestrupia, Testudodinium, and Togula. .

[0069] According to a particular embodiment of the invention, the protists are chosen from the genera Chlorella, Galdieria, Euglena, cyanobacteria, diatoms and Dinoflagellates.

[0070] The phytotoxins secreted by microorganisms are small molecules well known to those skilled in the art.

[0071] In the context of the present invention, phytotoxins are molecules secreted by cultivated microorganisms and whose molecular weight is between 300 and 3000 Da. Examples of phytotoxins, but not limited to, are compounds belonging to the families of microcystins, brevetoxins, saxitoxins, anatoxins a, okadaic acids, amphidinols, cylindrospermopsins, cyclic imines, and anabaenopeptins. Other examples of phytotoxins are karlotoxin, palytoxin, tetrodotoxin, yessotoxin, gambierol, amphidinolide, dinophysis, ovatoxins, ostreocins, gonyautoxins, ciguatoxins, spirolides, and cyclic imines.

[0072] The invention also relates to the use of the phytotoxins obtained according to the process of the invention. The phytotoxins present in the retentates may be purified by methods known to those skilled in the art. The phytotoxins thus isolated and purified may be used in the agricultural industry, as pesticides, or in the pharmaceutical or cosmetic industry as biologically active compounds. Another application of the phytotoxins, or even of the retentates rich in phytotoxins, is their use as an additive in a biomass of microorganisms. Indeed, the composition of the harvested biomasses varies according to the culture conditions, and the addition of phytotoxins is sometimes necessary to obtain a standardization of the composition of the batches of biomass.

[0073] The invention also relates to the use of the permeate(s) obtained according to the process of the invention. At the end of the process, the permeates comprising small quantities of phytotoxins are used as a culture medium, in particular in a biological reactor. It is understood that depending on the composition of these retentates, other elements may be added in order to obtain a culture medium suitable for the microorganisms. In one embodiment, the nth permeate obtained after n membrane filtration cycles is reintroduced into the biological reactor. This closed system thus makes it possible to recycle the permeates obtained. In another embodiment, the permeates are isolated, possibly combined, before being used as a culture medium. These permeates may also be stored for later use. EXAMPLES

[0074] The objective is to recycle the permeate still containing active molecules, the permeate will be used as feed during a number of loops n until the total extraction of the initial mass concentration of the feed.

[0075] Therefore the result of the concentration after a first membrane turn will depend on the concentration of the permeate, hence] the concentration of the feed after lambda turn:

[0076] ck w = CP[ ) ] *XR With A > 1 and If 2 = 1 Q = Co

[0077] where c^j = Cw* / j(l-XK-')

[0078]

[0079] To obtain batches that are as concentrated as possible between each turn in the membranes, the specificities of each of the phytotoxins must be taken into account.

[0080] In the case of cylindrospermopsin, a membrane size of less than 415 Da and with a high concentration factor should be preferred.

[0081] It is interesting to have membranes with different concentration factors in order to minimize the number of turns in the membrane but also to recover the most concentrated retentate, also ensuring optimization of the yield at each turn n in the membrane.

[0082] [Fig.2] represents the number of filtration turns n as a function of the concentration of phytotoxins (cylindrospermopsin) in the retentate. The concentration factors noted ck have the following values: ckl = 5, ck2=2.5, ck3=1.6 and ck4=1.25.

[0083] When the concentration factor is greater than or equal to 5 (ckl), each batch of retentates is more concentrated after each passage through the membranes, thus ensuring much more varied batch possibilities.

[0084] This method also ensures the increase in the concentration of each batch of retentate at each pass ([Fig.3]). The concentration factors noted ck have the following values: ckl = 5, ck2=2.5, ck3=1.6 and ck4=1.25.

[0085] The choice of the membrane concentration factor will also influence the overall efficiency of the system. Depending on the type of batch that one wishes to produce, it will be necessary to adapt the membrane concentration factor.

[0086] For example with the concentration factor of 1.6 (ck3) the evolution of the concen retentate concentrations remain constant. But with a higher concentration factor high such as 5 (ckl), the retentate concentration will only increase with each pass, thus allowing a greater variety of concentration for the establishment of several batches.

[0087] The yields are calculated with the following formula:

[0088] Canvas

[0089]

Claims

Claims

1. A process for preparing a composition comprising a biomass of microorganisms comprising the addition of phytotoxins as an additive to said biomass, characterized in that the phytotoxins are obtained by a process comprising the steps of: a. membrane filtration of a fermentation juice of the biomass so as to retain the phytotoxins in the retentate, said biomass being obtained from a culture of phytotoxin-producing microorganisms, b. recovery of a retentate comprising said phytotoxins, and c.recovery of a first permeate depleted in phytotoxins, said permeate having a phytotoxin content lower than that of the fermentation juice, followed where appropriate by at least one additional cycle of membrane filtration of the retentate obtained in step c of the previous cycle with the recovery of at least one second retentate comprising said phytotoxins and at least one second permeate depleted in phytotoxins having a phytotoxin content lower than that of the first permeate.

2. Method according to claim 1, characterized in that the method for obtaining the phytotoxins comprises m additional cycles of membrane filtration and the recovery of m+1 retentates comprising the phytotoxins and the same permeate depleted in phycotoxins, m being an integer of at least 1.

3. Method according to one of claims 1 or 2, characterized in that the membrane filtration cycles are carried out on the same membrane or each membrane filtration cycle is carried out on a new membrane, the membranes of each filtration cycle being identical or different.

4. Method according to one of claims 2 or 3, characterized in that the number of cycles m+1 is determined as a function of: - the phytotoxin content of the fermentation juice, - the concentration factor of the filtration membrane, and - the final concentration of phytotoxins in the permeate recovered after the last cycle.

5. Method according to claim 4, characterized in that the final concentration of phytotoxins in the permeate recovered after the last cycle as a function of the number of cycles m+1 is determined by the equation: CÀW =cw*i(i-xR1)*x* in which À is the number of cycles m+1, X is the concentration factor of the membrane and R is the molecular rejection rate of the membrane.

6. Method according to one of claims 2 to 5, characterized in that the m+1 retentates comprising the phytotoxins recovered from the first membrane filtration cycle and the m additional membrane filtration cycles are combined.

7. Method according to one of claims 1 to 6, characterized in that the phytotoxins are molecules with a molecular weight of between 300 and 3000 Da.

8. Method according to one of claims 1 to 7, characterized in that the phytotoxins belong to the families of microcystins, brevetoxins, saxitoxins, anatoxins a, okadaic acids, amphidinols, cylindrospermopsins, cyclic imines and ana-baenopeptins.

9. Method according to one of claims 1 to 8, characterized in that the fermentation juice in step (a) is obtained by separation of a biomass of microorganisms and a fermentation must itself obtained by a process of fermentation of said microorganisms in a culture medium comprising an aqueous medium and nutrients necessary for the growth of said microorganisms.

10. Method according to one of claims 2 to 9, characterized in that the same permeate obtained after m additional membrane filtration cycles is recycled into the culture medium of the microorganisms.

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