Processes for the biotechnological production of biological products

Abstract

The present invention relates to a process and an apparatus for the biotechnological production of a bioproduct. The process comprises a first and a second bioprocess carried out in a first and a second bioreactor (2, 3) and a tank (4). The first bioprocess is a carbon dioxide producing bioprocess resulting in the production of a bioproduct, and the second bioprocess is a chemoautotrophic bioprocess in which the CO2 produced in the first bioprocess is consumed by hydrogen bacteria oxidizing H2 with O2 as the electron acceptor. The electrolytically produced H2 and O2 are separated and O2 is fed directly to the second and / or first bioprocess, while H2 is initially dissolved in a medium contained in the tank (4). The H2 saturated medium is then fed to the second bioprocess. The biomass produced in the second bioprocess is fed to the first bioprocess as an additional C and N source.

Description

[Technical field]

[0001] The present invention relates to the field of biotechnology, in particular to processes for the biotechnological production of biological products. [Background technology]

[0002] Biotechnological production of bioproducts, e.g. bio-based chemicals, has been known for a long time and is expected to provide chemicals in a more sustainable and environmentally friendly manner for commercial use, e.g. in the chemical industry. As an example, so-called biofuels, such as bioethanol or biodiesel, have been produced in bioprocesses to replace fossil fuels derived from petroleum (see, e.g., U.S. Pat. No. 5,399,623 and U.S. Pat. No. 5,499,633). However, in view of global warming caused by the increasing amount of carbon dioxide (CO2) in the atmosphere, bioprocesses for large-scale production of chemicals must also minimize the emission of CO2 in order to be sustainable.

[0003] Both US Pat. No. 5,399,663 and US Pat. No. 5,493,313 describe the fixation of CO2 produced in a first bioprocess in a second bioprocess in order to reuse the produced CO2. US Pat. No. 5,493,663 discloses a method for converting a carbon source into lipids, in which lipid production is carried out in an aerobic fermenter with the oleaginous yeast Yarrowia lypolytica, and carbon dioxide generated during lipid production is converted into a carbon substrate by CO2 fixation carried out in an anaerobic fermenter by bacteria of the genus Clostridium sp. These clostridium use as a reducing agent H2 gas, which is produced by electrolysis of water and fed into the anaerobic fermenter. US Pat. No. 5,493,663 discloses the production of biofuels using photosynthetic plant cells that fix the produced CO2, for example by yeast cells producing biofuels.

[0004] US Pat. No. 5,399,433 describes, inter alia, a method for the capture and fixation of carbon dioxide, for example from flue gases, by oxyhydrogen ("Knallgas") bacteria, where oxygen and hydrogen can be provided, for example, by electrolysis of water. The biotechnological production of biofuels using "Knallgas" bacteria is also described in non-patent document 1. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2011 / 088364(A9) [Patent Document 2] International Publication No. 2009 / 133351(A2) [Patent Document 3] International Publication No. 2011 / 139804(A2) [Non-patent literature]

[0006] [Non-Patent Document 1] Brigham, C., "Perspectives for the biotechnological Production of biofuels from CO2 and H2 using Ralstonia eutropha and other 'Knallgas' bacteria," Applied Microbiology and Biotechnology, vol. 103 (2019), 10.1007 / s00253-019-09636-y Summary of the Invention [Problem to be solved by the invention]

[0007] It is an object of the present invention to improve the biotechnological production of bioproducts, in particular bio-based chemicals, with regard to sustainability, in particular minimizing the net production of carbon dioxide. [Means for solving the problem]

[0008] In a first aspect, the present invention relates to a process for the biotechnological production of a biological product, comprising: a) a first bioprocess comprising the cultivation of a first type of microorganism in a first medium at a first pressure p1 in a first bioreactor, the first bioprocess resulting in the production of said bioproduct and carbon dioxide CO2; b) a second bioprocess comprising the cultivation of a second type of microorganism in a second medium at a second pressure p2 in a second bioreactor, the second bioprocess producing biomass and consuming molecular oxygen O2, molecular hydrogen H2 and at least a portion of the CO2 produced in the first bioprocess of step a); c) electrolyzing water into O2 and H2, feeding at least a portion of the O2 produced by electrolysis to the second bioprocess and / or the first bioprocess, and feeding at least a portion of the H2 produced by electrolysis to the headspace and to a tank containing a third medium at a third pressure p3, the third medium being identical to the second medium in the second bioreactor; d) feeding at least a portion of the third medium containing H2 from the tank to a second bioprocess; wherein at least a portion of the biomass produced in the second bioprocess of step b) is used as a C source for the first bioprocess of step a), providing a process where p3 ≧ p2 ≧ p1.

[0009] The process combines two bioprocesses, the first being a CO2 producing process and the second being a CO2 consuming bioprocess, which involves non-photosynthetic biological fixation of the carbon dioxide produced in the first bioprocess by hydrogen oxidizing and oxygen reducing microorganisms, e.g. "Kunar gas" bacteria. The first and second bioprocesses are carried out spatially separated from each other in separate bioreactors. The CO2 produced in the first bioprocess is fed to the second bioprocess for at least partial, preferably at least mostly or completely consumed in the second bioprocess. The process of the invention is preferably a "CO2 neural" process, i.e. a process in which the produced carbon dioxide is at least essentially completely consumed, so that no CO2 is released to the environment. The biomass produced in the second bioprocess is used as an additional carbon source for the first bioprocess, together with at least a portion of the CO2 from the first bioprocess as a carbon source. The biomass may also be used as an additional nitrogen source. Furthermore, oxygen gas produced by electrolysis of water and used as an electron acceptor by the microorganisms of the second bioprocess is fed directly to the second bioprocess or, alternatively, to the first bioprocess, and hydrogen gas produced by electrolysis and used as an electron donor by the microorganisms of the second bioprocess is fed indirectly to the second bioprocess, not directly, but via a medium contained in a separate vessel. The advantage of this separate supply of oxygen and hydrogen to the second bioprocess is an increased safety, for example with respect to explosion protection. Furthermore, this serves as a countermeasure to the poor solubility of H2 in aqueous media.

[0010] The term "bioproduct" refers to a compound, particularly an organic compound, that is, for example, a component of a chemical reaction in a living cell, produced by a chemical reaction in a living cell, or participates in a chemical reaction in a living cell, in contrast to the product of a bioprocess, for example a chemical based on a fossil feedstock. A bio-based chemical derived from biomass may be structurally identical to or structurally different from an existing chemical derived from a fossil feedstock. Examples of bio-based chemicals include, for example, fatty acids and fatty acid derivatives, such as lipids, alcohols, such as diols, and acids, biopolymers or monomers for polymerization, peptides, etc. The term also encompasses the product of a bioprocess, for example biomass as a living, lyophilized, or hydrolyzed microorganism. As defined above, the term "bio-based chemical" relates to a compound, particularly an organic compound, that is, for example, a component of a chemical reaction in a living cell, produced by a chemical reaction in a living cell, or participates in a chemical reaction in a living cell. The term "biochemical" may also be used synonymously with "bio-based chemical". It should be noted that although the present invention may be described, and is primarily intended to be, in the context of the production of bio-based chemicals, this should not be construed to exclude the production of biomass, for example, as a bio-product.

[0011] The term "lipid" refers to organic compounds that are soluble in non-polar organic solvents (such as chloroform or ether) and normally insoluble in polar solvents such as water, and includes, for example, fats, oils, waxes, phospholipids and steroids.

[0012] The term "bioprocess" as used herein relates to any biological process, i.e. a process involving living cells, especially living microorganisms such as bacteria, or functional components of living cells, e.g. enzymes, cell-free systems or organelles, to obtain a desired product. The product may be a compound, e.g. an organic compound, a mixture of compounds, e.g. a mixture of organic compounds, or a biomass. An example is the production of lipids from substrates such as glucose, starch, glycerol or similar.

[0013] "Bioreactor" is understood to mean a reactor comprising a working volume capable of providing a suitable medium, for example an aqueous medium suitable for supporting the growth of a culture of microorganisms, for example bacterial cells, or capable of carrying out biological and / or biochemical processes and reactions or of creating conditions in which biological and / or biochemical processes and reactions can take place. These processes and reactions may be substance transformations, for example the synthesis, modification or degradation of substances by living cells or cell-free systems, or the growth of biomass, i.e. the growth of living cells, for example bacterial cells. Depending on the type of process to be carried out and / or the type of microorganism used to carry out the process, the bioreactor may comprise facilities for example for aeration / ventilation and / or stirring of the medium.

[0014] The term "closed" with respect to a bioreactor or vessel means that the photobioreactor or vessel is closed to the surrounding environment so that the photobioreactor or vessel can be pressurized. This does not exclude the possibility that the bioreactor or vessel may have openings, connections or the like through which, for example, a fluid, e.g., a gas, e.g., CO2, or a liquid, can be introduced or removed from the vessel.

[0015] The term "Knar gas bacteria", "hydrogen bacteria", "H2 bacteria" or "oxyhydrogen bacteria" as used herein refers to a physiologically defined group of bacteria that grow autotrophically, i.e., capable of oxidizing hydrogen (H2) and fixing carbon dioxide while using oxygen (O2) as the final electron acceptor. The abbreviation HOB is sometimes used in place of the term hydrogen bacteria or H2 bacteria. The term "oxyhydrogen" is sometimes used synonymously with "Knar gas". "Knar gas" is a mixture of gaseous hydrogen and gaseous oxygen. Knar gas bacteria are aerobic auxotrophic autotrophic bacteria. Examples of knargas bacteria are Hydrogenophilus thermoluteolus, Hydrogenobacter thermophilus, Hydrogenovibrio marinus, Cupriavidus metallidurans (formerly Alcaligenes eutropha or Ralstonia eutropha), Rhodococcus opacus, Xantobacter autotrophicus and Cupriavidus necator. The following simplified general reaction scheme describes aerobic CO2 fixation by knargas bacteria through H2 oxidation with O2 as the electron acceptor. H2+O2+CO2→Biomass+H2O

[0016] The term "chemotrophic" refers to organisms that use the oxidation of compounds as an energy source, in contrast to "phototrophic" organisms that derive their energy from light. The term "chemolithotrophic" refers to organisms that use inorganic compounds, e.g. hydrogen gas, as electron donors, in contrast to the term "chemoorganotrophic" which refers to organisms that use organic compounds as electron donors. The term "autotroph" or "heterotroph" refers to the carbon source, with CO2 being the carbon source in "autotrophic" organisms and organic carbon compounds being the carbon source in "heterotrophic" organisms. The term "mixotrophic" is sometimes used for organisms that can use both organic compounds and CO2 as carbon sources. The term "chemolithoautotrophic" refers to organisms that use inorganic compounds as energy sources and electron donors and CO2 as the carbon source. The term "facultative chemolithoautotrophic" refers to an organism that is also capable of growing chemoorganoheterotrophically. For example, the "Kunar gas" bacteria grow heterotrophically when H2 is at tropospheric concentrations and chemolithotrophically only when H2 is available at higher concentrations (Pumphrey GM, Ranchou-Peyruse A, Spain JC. Cultivation-independent detection of autotrophic hydrogen-oxidizing bacteria by DNA stable-isotope probing. Applied Environmental Microbiology, 77(14):4931-4938 (2011). doi:10.1128 / AEM.00285-11).

[0017] The term "yeast" refers to eukaryotic unicellular fungal microorganisms. Examples of yeasts are Saccharomyces cerevisiae, Candida albicans, and Yarrowia lipolytica. The term "oleaginous yeast" relates to yeast that are capable of storing 20% ​​or more of their cell dry weight in the form of lipids (e.g., fats, e.g. Abeln, F., Chuck, C.J., "The history, state of the art and future prospects for oleaginous yeast research", Microbial Cell Factories, Vol. 20, p. 221 (2021), doi:10.1186 / s12934-021-01712-1; Blomqvist, J., Pickova, J., Tilami, S.K. et al., "Oleaginous yeast as a component in fish feed", Scientific Reports, vol. 20, p. 221 (2021), doi:10.1186 / s12934-021-01712-1 Reports, vol. 8, p. 15945 (2018), doi:10.1038 / s41598-018-34232-x. Examples of oleaginous yeasts are Cutaneotrichosporon oleaginosus, Rhodotorula toruloides, Yarrowia lipolytica, Lipomyces starkeyi, Trichosporon oleaginosus (formerly Cryptococcus curvatus), and Rhodosporidium toruloides.

[0018] The term "electrolyzing water" refers to a process in which direct current is used to split water into hydrogen and oxygen. The term "electrolyzer" is used to denote a device capable of electrolyzing water.

[0019] The term "hydrogen", unless expressly stated or unmistakably clear from the context, relates to dihydrogen, i.e. the element molecule H2 consisting of two hydrogen atoms linked by a single bond. The terms "molecular hydrogen" or "gaseous hydrogen" may also be used interchangeably in place of H2.

[0020] The term "oxygen", unless expressly stated or unmistakably clear from the context, refers to the elemental molecule O2 consisting of two oxygen atoms linked by a single bond. The terms "molecular oxygen" or "gaseous oxygen" may also be used interchangeably in place of O2.

[0021] The term "C source" or "carbon source" refers to a compound that contains at least one carbon atom that can be incorporated or is incorporated into a compound that constitutes the cellular material. An example of a C source, particularly for autotrophic bacteria, is CO2.

[0022] The term "at least a portion of the biomass produced in the second bioprocess is used as a C source for the first bioprocess" should not be interpreted as meaning that the biomass is the only carbon source of the first bioprocess. Rather, the biomass is used as an additional carbon source used in the first bioprocess. Furthermore, this term does not exclude that the biomass can be used for other purposes, for example as an additional nitrogen source.

[0023] The term "CO2 fixation" or "carbon fixation" refers to the biological incorporation of inorganic carbon, especially in the form of carbon dioxide, into organic carbon compounds, such as carbohydrates. The term "non-photosynthetic biological fixation of carbon dioxide" refers to the fixation of carbon without photosynthesis, i.e. by non-photosynthetic organisms.

[0024] The terms "fermentation" or "fermentative" refer to any process in which microbial activity produces a desired change in a compound, food or beverage.

[0025] The phrase "a third medium is identical to a second medium" means that the third medium and the second medium have at least essentially the same composition, i.e., the same components in essentially the same concentrations. This term does not imply that the concentrations of dissolved gases, e.g., H2, O2, and CO2, are identical.

[0026] The expression "feeding at least a portion of the O2 produced by electrolysis to the second bioprocess and / or the first bioreactor" includes feeding all or a portion of the electrolytically produced O2 only to the first bioprocess, feeding all or a portion of the electrolytically produced O2 only to the first bioprocess, or feeding at least a portion of the electrolytically produced O2 partially to the first bioprocess and partially to the second bioprocess, either simultaneously or intermittently. The terms "part" or "partially" may refer to a partial stream of a continuous total gas stream, or a portion of the O2 gas stream separated in time. Thus, a portion of the continuous stream of oxygen led to the second bioprocess may be separated from the total oxygen stream and used for the oxygen supply of the first bioprocess (see below), and / or the total oxygen gas stream may be fed, for example, to the second bioprocess during a first period and to the first bioprocess during a second period. The formulation that oxygen is supplied "directly" to the second bioprocess does not exclude that the oxygen is intermediately stored in a storage tank, for example a gas cylinder.

[0027] The phrase "carbon dioxide produced in a first bioprocess is recovered from the first bioprocess and fed to a second bioprocess" should not be interpreted as meaning that CO2 is selectively recovered from the first bioprocess and fed to the second bioprocess, but rather that air containing CO2 is recovered from the first bioprocess, for example from the headspace of a first bioreactor, and the CO2-containing air, optionally enriched or concentrated in CO2, is fed to the second bioreactor.

[0028] In the process of the invention, the electrolytically produced oxygen may in one embodiment be fed only to the second bioprocess for the oxygen supply of the second type of microorganism. In an alternative embodiment, the electrolytically produced oxygen may be fed only to the first bioprocess, which in this embodiment is preferably an aerobic or at least microaerobic bioprocess. In this embodiment, the oxygen supply of the aerobic second bioprocess is provided by a gas stream containing CO2 produced in the first bioprocess, which gas stream is recovered from the first bioprocess and fed to the second bioprocess. The second bioprocess is thus fed with residual oxygen from the first bioprocess. In another embodiment, the electrolytically produced oxygen is fed partly to the first bioprocess and partly to the second bioprocess. The oxygen may be divided in terms of amount or time, i.e. into two partial streams fed continuously to the first and second bioprocesses or fed intermittently to these bioprocesses.

[0029] In a preferred embodiment of the process of the invention, the third pressure p3 is equal to or greater than the second pressure p2, which is equal to or greater than the first pressure p1. Particularly preferred, the third pressure p3 is greater than the second pressure p2, which is greater than the first pressure p1, i.e. p3>p2>p1. More preferably, p1 is greater than atmospheric pressure. The greater the pressure p3 in the vessel with the third medium to which H2 gas is supplied, the greater the amount of H2 that can be dissolved in the third medium due to the pressure dependence of the solubility of the gas. The lower pressure p2 in the second bioreactor compared to the pressure p3 facilitates the release of H2 into the second medium. The vessel may be configured as a third bioreactor.

[0030] The first bioprocess may be any bioprocess that results in the production of a desired bioproduct and carbon dioxide CO2. Preferably, the first bioprocess is a bioprocess that does not involve the production of strongly reducing gases such as H2 or CO. The first bioprocess may be an aerobic, anaerobic or microaerobic process. Preferably, the first bioprocess is an aerobic or at least microaerobic process. The bioproduct may be recovered from the first bioprocess by any method appropriate for the respective bioproduct, for example by recovering at least a portion of the cells cultivated in the first bioprocess, if the bioproduct is stored in cells. The cells recovered from the first bioprocess may then be further processed to obtain the bioproduct. The first bioprocess may also be a bioprocess for the production of biomass, for example cell mass of living microorganisms grown in the first bioprocess. In a preferred embodiment of the process according to the invention, the first bioprocess is an aerobic bioprocess. More preferably, the first bioprocess is a fermentation process for the production of lipids. Preferably, the fermentation process involves the cultivation of an oleaginous yeast, preferably Rhodosporidium toruloides.Alternatively or additionally, the first bioprocess includes the production of carotenoids, preferably using Rhodosporidium toruloides (see, e.g., Igreja, WS; Maia, FDA; Lopes, AS; Christe, RC, "Biotechnological Production of Carotenoids Using Low Cost-Substrates Is Influenced by Cultivation Parameters: A Review," International Journal of Molecular Sciences, 2009, 111-115, 2011). Sciences, 22, 8819 (2021), doi:10.3390 / ijms22168819; Yaegashi, J., Kirby, J., Ito, M. et al., "Rhodosporidium toruloides: a new platform organism for conversion of lignocellulose into terpene biofuels and bioproducts," Biotechnology For Biofuels And Bioproducts, 10, 241 (2017), doi:10.1186 / s13068-017-0927-5). The microorganisms used in the first bioprocess, such as yeast cells, may be genetically engineered.

[0031] In the case of an aerobic first bioprocess, the electrolytically produced oxygen may be fed only to the first bioprocess or may also be partially fed to the first bioprocess to support the aerobic process. The latter may be carried out, for example, by a) splitting the O2 gas stream into two gas streams, one of which is fed to the second bioprocess and the other to the first bioprocess, or b) feeding the entire O2 gas stream intermittently to the first or to the second bioprocess. A combination of both options a) and b) is of course also possible. As mentioned above, it is also possible that oxygen is fed only to the first bioprocess, the second bioprocess being oxygenated by a gas stream containing CO2 recovered from the first bioprocess.

[0032] In a preferred embodiment of the process of the invention, the second bioprocess is a non-photosynthetic bioprocess and involves chemolithoautotrophic consumption of O2, H2 and CO2. In this embodiment, the second bioprocess is a bioprocess in which knargas bacteria are grown which oxidize H2 with O2 as the electron acceptor and fix carbon dioxide. Preferably, the carbon dioxide originates at least in large part, and more preferably entirely, from the first process. The microorganisms used in the second bioprocess may be genetically engineered.

[0033] Particularly preferred, the first bioprocess is an aerobic process for the fermentative production of lipids and / or carotenoids, comprising the cultivation of yeast cells, preferably of the genus Rhodosporidium, particularly preferred of Rhodosporidium toruloides, and the second bioprocess comprises the chemolithoautotrophic cultivation of knargas bacteria, preferably of the genus Hydrogenophilus, particularly preferred of Hydrogenophilus thermoluteolus, with H2 and O2. The O2 produced by electrolysis is fed into the second bioreactor, while the H2 from the electrolysis is fed into a tank where it is dissolved in a third medium, a part of which is fed into the second bioreactor to feed the hydrogen bacteria with H2. The main carbon source for the first process may be, for example, carbonaceous waste and by-products (e.g. biomass hydrolysate, molasses, etc.). However, other carbon sources are also possible, for example glycerol or glucose. In this embodiment, the carbon dioxide produced by the yeast cells is fed to a second bioprocess comprising the production of a biomass of knargas bacteria, which grows chemolithoautotrophically. Thus, in a preferred embodiment of the process of the invention, the first type of microorganism is a microorganism of the genus Rhodosporidium, preferably Rhodosporidium toruloides, and the second type of microorganism is a microorganism of the genus Hydrogenophilus, preferably Hydrogenophilus thermoluteolus. Other combinations of knargas bacteria for the second bioprocess and yeast or bacteria for the first bioprocess are of course also possible.

[0034] In a preferred embodiment of the process according to the invention, the second bioprocess is carried out at a temperature between 40 and 55° C., preferably between 45 and 55° C., 50 and 55° C. or 50 and 53° C. This is particularly preferred in the case of the cultivation of thermophilic knargas bacteria, such as Hydrogenophilus thermorteolus.

[0035] In a preferred embodiment of the process of the present invention, the third medium in the tank is maintained anaerobic. This may be achieved, for example, by not aerating the medium and as a result of the activity of knargas bacteria in the medium, which consume any oxygen with H2 introduced into the medium.

[0036] The first, second and third media are in each case aqueous media suitable for the intended purpose, e.g., to support the growth of the microorganism to be cultured.

[0037] Fresh medium for the second bioprocess is preferably fed into the tank, but may alternatively or additionally also be fed into the second bioreactor.

[0038] In a preferred embodiment of the process of the present invention, a portion of the second medium of the second bioreactor containing the second type of microorganism is withdrawn from the second bioreactor, the biomass contained in the second medium is at least partially separated from the second medium, hydrolyzed and fed to the first bioprocess, and the second medium separated from the biomass is recycled to the second bioprocess. Alternatively, or in addition, the medium separated from the biomass may be returned to the tank. In this embodiment, the biomass produced in the second bioprocess is used as an additional carbon and nitrogen source for the first bioprocess, i.e., as a substrate for growing the microorganisms cultivated in the first bioreactor. Furthermore, at least a portion of the biomass produced in the second bioprocess and separated from the second medium may be withdrawn from the overall process and used as a separate bioproduct, for example, fish feed.

[0039] In a further preferred embodiment of the process of the invention, a portion of the second medium from the second bioreactor is fed into the tank and sprayed into the headspace of the tank containing the third medium, and an amount of the third medium, preferably essentially the same as the amount of the second medium fed into the tank, is fed from the tank into the second bioreactor. Since the medium in the second bioreactor and the medium in the tank are the same, a portion of the medium in the second bioreactor and the medium in the tank are preferably continuously circulated from the second bioreactor to the tank and back to the second bioreactor. The medium is sprayed into the headspace of the tank, for example with the help of a pump and a nozzle. The pump increases the pressure of the portion of the second medium fed into the tank to a pressure > p3, atomizing the medium in the headspace of the tank. At the same time, an amount of the third medium in the tank, preferably the same amount, is returned to the second bioreactor. In this embodiment, a portion of the second and third media, including the bacteria therein, circulates between the second and third bioprocesses. The fine distribution of the second media in the headspace of the tank promotes the transfer of H2 from the gas phase to the liquid phase. In this way, the electrolytically generated H2 gas supplied to the tank is efficiently dissolved in the medium in the tank and supplied to the second bioprocess.

[0040] The process of the present invention may be configured as a batch process, a semi-batch process or a continuous process. Preferably, the process of the present invention is configured as a continuous process, i.e. a continuous process.

[0041] Carbon dioxide produced in the first bioprocess is withdrawn from the first bioreactor, for example from the headspace of the first bioreactor, and fed into the second bioreactor. After recovery from the first bioreactor and prior to introduction into the second bioreactor, the carbon dioxide is preferably separated through a suitable separation membrane, such as a hollow fiber membrane for separating N from CO (see, for example, Tong Z, Sekizkardes AK. Recent Developments in High-Performance Membranes for CO Separation, Membranes (Basel), Vol. 11, No. 2, p. 156 (2021), doi:10.3390 / Membranes11020156; Khalilpour R., Mumford K., Zhai H., Abbas A., Stevens G., Rubin ES, Membrane-based carbon capture from flue gases: a review). The effluent is concentrated by using a hollow fiber membrane such as "SEPURAN® Green" (Evonik Industrie, Essen, Germany), for example.

[0042] The CO2 recovered from the first bioreactor may optionally be intermediately stored in a storage tank, e.g., a gas cylinder, for example in case the first and second bioprocesses are not running in sync with respect to carbon dioxide production and consumption, and for better control of the CO2 supply to the second bioprocess.

[0043] In a preferred embodiment, the electrolytically produced oxygen is at least partially fed directly to the second bioprocess to support the metabolic activity of the knargas bacteria. The oxygen may be fed continuously or intermittently to the second bioprocess. The oxygen may be intermediately stored in a storage tank, e.g. a gas cylinder. The oxygen may be fed to the second bioprocess depending on the physical parameters measured in the second bioprocess, e.g. pH, pO2, pH2, temperature, cell mass, etc. Flow regulation may be applied to control the flow rate of oxygen fed into the second bioreactor. In this embodiment, the electrolytically produced oxygen may be additionally fed to the first bioprocess, particularly in the case of aerobic bioprocesses, continuously or intermittently. The oxygen flow rate to the first bioprocess may also be subject to regulation.

[0044] In a second aspect, the present invention relates to an apparatus for the biotechnological production of a biological product by the process of the invention, comprising: a) a first bioreactor configured for carrying out a first bioprocess, the first bioprocess comprising the cultivation of a first type of microorganism in a first medium at a first pressure p1, the first bioprocess resulting in the production of a bioproduct and carbon dioxide CO2; b) a second bioreactor configured for carrying out a second bioprocess comprising the cultivation of a second type of microorganism in a second medium at a second pressure p2, the second bioprocess being a process which produces biomass and consumes molecular oxygen O2, molecular hydrogen H2 and at least a portion of the CO2 produced in the bioprocess of step a); c) a vessel configured to contain the headspace and a third medium at a third pressure p3; d) an electrolyser for electrolyzing water into O2 and H2; The apparatus further comprises: - a first fluid connection fluidly connecting a first outlet of the first bioreactor to a first inlet of the second bioreactor for the transfer of CO2 produced in the first bioreactor to the second bioreactor; - a second fluid connection fluidly connecting a first outlet of the second bioreactor to a first inlet of the first bioreactor for the transfer of biomass produced in the second bioreactor to the first bioreactor; a third fluid connection fluidly connecting the second outlet of the second bioreactor to the first inlet of the vessel for the transfer of a second medium to the vessel; a fourth fluid connection fluidly connecting the first outlet of the vessel to a second inlet of a second bioreactor for the transfer of a third medium to the second bioreactor; a fifth fluid connection fluidly connecting the first outlet of the electrolyser to a third inlet of the second bioreactor for the transfer of oxygen O2 to the second bioreactor; a sixth fluid connection fluidly connecting the first outlet of the electrolyser to the third inlet of the first bioreactor for the transfer of oxygen O2 to the first bioreactor; a seventh fluid connection fluidly connecting a second outlet of the electrolyser to a second inlet of the vessel for the transfer of hydrogen H2 to the vessel; The present invention relates to an apparatus comprising:

[0045] The apparatus of the present invention is configured to carry out the process of the present invention and includes a first bioreactor, a second bioreactor, and a tank, which may be configured as a bioreactor. The bioreactors and the tank are fluidly connected to each other to carry out the process of the present invention. Thus, the apparatus includes fluid connections, such as pipes or tubes or the like, that fluidly interconnect the bioreactors and the tanks to allow the transfer of CO2 produced in the first bioreactor to the second bioreactor, to transfer biomass from the second bioreactor to the first bioreactor, to supply oxygen generated by the electrolyzer to the second bioreactor and / or the first bioreactor, hydrogen into the tank, and to circulate the medium between the second bioreactor and the tank.

[0046] In a preferred embodiment of the device of the invention, the second fluid connection comprises, between the first outlet of the second bioreactor and the first inlet of the first bioreactor, a separation device for separating the second type of microorganism from the second medium and a hydrolysis device for hydrolyzing the second type of microorganism separated from the second medium by the separation device. The separation device may be a centrifuge device or a hollow fiber membrane module operating in batch or continuous mode. The medium separated from the second type of microorganism may be returned to the second bioreactor, fed into the tank or withdrawn from the process.

[0047] In a further preferred embodiment of the apparatus of the invention, the third fluid connection fluidly connecting the second outlet of the second bioreactor to the first inlet of the vessel extends into an upper portion of the vessel including the headspace, the extension comprising a nozzle for atomizing the second medium provided in the vessel.

[0048] More preferably, the first fluid connection fluidly connecting the first outlet of the first bioreactor to the first inlet of the second bioreactor comprises a CO2 concentrator, preferably a membrane-based CO2 concentrator, for concentrating the CO2 produced in and recovered from the first bioreactor before it is transferred to the second bioreactor. Even more preferably, the first fluid connection comprises a CO2 storage tank, e.g. a gas cylinder, for storing CO2. Thus, CO2 can be stored intermediately and fed to the second bioprocess when needed. This is advantageous, as it allows decoupling of CO2 production in the first bioprocess from CO2 consumption in the second bioprocess, e.g. in case of process upsets or to compensate for unequal CO2 production and consumption rates.

[0049] A sixth fluid connection fluidly connecting the first outlet of the electrolyser to the third inlet of the first bioreactor for the transfer of oxygen O2 to the first bioreactor shares a portion of the fifth fluid connection. In a preferred embodiment of the device of the invention, a fifth fluid connection fluidly connecting the first outlet of the electrolyser to the third inlet of the second bioreactor extends into the first bioreactor and fluidly connects the first outlet of the electrolyser to the third inlet of the first bioreactor. Preferably, a two-way valve is inserted into the fifth fluid connection so that the oxygen flow can be split and directed to both the first and second bioreactors simultaneously or intermittently to either the first or second bioreactor.

[0050] The vessels may be configured as bioreactors, such that the apparatus includes a first, second and third bioreactor.

[0051] In the following, the invention will be described in more detail, by way of example only, with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0052] [Figure 1]1 is a schematic diagram showing an embodiment of a device according to the invention, with gas flow shown in dashed lines and liquid flow shown in solid lines. [Diagram 2] 3 is a schematic diagram showing the gas and liquid flows of the embodiment of the device according to the invention shown in Figure 2, with the gas flows shown in dashed lines and the liquid flows shown in solid lines. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] Figure 1 shows a simplified schematic diagram of the device 1 of the invention. The device comprises three compartments A, B and C. Compartment A comprises a first bioreactor 2, compartment B comprises a second bioreactor 3 and compartment C comprises a tank 4 which may be configured as a third bioreactor. The first bioreactor 2 and the second bioreactor 3 comprise a first stirring device 21 and a second stirring device 31, respectively, for stirring a first medium 23 in the first bioreactor 2 or a second medium 33 in the second bioreactor 3. A first pressure p1 prevails in the first bioreactor 2, a second pressure p2 in the second bioreactor 3 and a third pressure p3 in the tank 4.

[0054] In compartment A, which contains the first bioreactor 2, a first bioprocess is carried out, which is a bioprocess in which a desired biochemical is produced by a first type of microorganism cultivated in the first bioreactor 2, with the production of carbon dioxide CO2. An example of such a first bioprocess is the aerated yeast fermentation with Rhodosporidium toruloides for variable lipid production, where e.g. carbon-containing waste materials and by-products (biomass hydrolysates, molasses, etc.) are used as the main C source. In addition to lipids, R. toruloides is used as a feed in aquaculture (shrimp farms, salmon farms, see e.g. Blomqvist, J., Pickova, J., Tilami, SK, et al., Oleaginous yeast as a component in fish feed, Scientific Reports, 8, 15945 (2018), doi:10.1038 / s41598-018-34232-x) or as a colorant in the food industry (see e.g. Igreja, WS, Maia, FA, Lopes, AS, Christe, RC, Biotechnological Production of Carotenoids Using Low Cost Substrates Affected by Culture Parameters, Review). (See “Cost-Substrates Is Influenced by Cultivation Parameters: A Review,” International Journal of Molecular Sciences, Vol. 22, p. 8819 (2021), doi:10.3390 / ijms22168819) to produce carotenoid-containing biomass that can be used for bioprocessing.The lipid profile can be tailored by genetic modification of R. toruloides (Wen Z., Zhang S., Odoh CK, Jin M., Zhao ZK, Rhodosporidium toruloides-A potential red yeast chassis for lipids and beyond, FEMS Yeast Research, vol. 20, no. 5, p. foaa038 (2020), doi:10.1093 / femsyr / foaa038). The lipid profile produced can be adapted to change the lipids for the cosmetic / food industry, e.g., from palm oil-like to cocoa butter-like classification.

[0055] Air 100 is bubbled through the first bioreactor 2, which contains a first medium 23 and cells of a first type of microorganism, here for example the oleaginous yeast Rhodosporidium toruloides. Alternatively or additionally, oxygen generated in an electrolyser 5 for electrolyzing water may be fed to the first bioreactor 2. The exhaust air containing CO2 produced in the first bioreactor 2 is withdrawn from the headspace 32 of the first bioreactor 2 by means of a compressor 9 and fed as a C source to the bioprocess carried out in the second bioreactor 3 of compartment B via a first fluid connection 61. The first fluid connection 61, which may for example be a pipe or tube, fluidly connects the first outlet 25 of the first bioreactor 2 to the first inlet 34 of the second bioreactor 3. On the way to the second bioreactor 3, the CO2 is concentrated using a CO2 concentrator 8, for example a membrane CO2 concentrator 8, so that CO2-depleted air 102, which mainly contains N2, is removed from the exhaust air stream. The airflow rate vg2 (see FIG. 2) at which CO2, or more precisely air enriched with CO2, is fed to the second bioreactor 3 is therefore set by the aeration rate, i.e. the rate at which air 100 is fed to the first bioreactor 2 and the compressor 9. In the illustrated embodiment, the first fluid connection 61 now further comprises a storage tank 13 for intermediate storage of CO2 after the CO2 concentrator 8 in the direction of the flow of CO2. Bioproducts 101, e.g. bio-based chemicals produced in compartment A, e.g. chemicals discharged in the first medium 23 or accumulated in the grown cells in the first bioprocess, can be recovered from the bioprocess in compartment A.

[0056] In compartment B, a second type of microorganism, the chemolithotrophic so-called "Knar gas bacteria", grows dependent on CO2, H2 and O2 in a second bioreactor 3. The second type of microorganism cultivated in compartment B can be any microorganism with specified gas requirements. An example of a suitable microorganism is Hydrogenophilus thermoluteolus, which has a particularly high growth rate (Arai H, Shomura Y, Higuchi Y, Ishii M. Complete Genome Sequence of a Moderately Thermophilic Facultative Chemolithoautotrophic Hydrogen-Oxidizing Bacterium, Hydrogenophilus thermoluteolus TH-1, Microbiology Resource Announcements, Vol. 7, No. 6, p. e00857-18 (2018), doi:10.1128 / MRA.00857-18). The bioprocess in compartment B receives CO2 and residual O2 from the associated bioprocess in compartment A as well as pure H2 and O2 from electrolyser 5, which is described in more detail below.

[0057] The O2 and H2 used by the hydrogen bacteria growing in the second bioreactor 3 are generated by the electrolyser 5. O2 is supplied directly to the second bioreactor 3 via a fifth fluid connection 65, which fluidly connects the first outlet 55 of the electrolyser 5 with the third inlet 38 of the second bioreactor 3. In the embodiment of the device 1 of the invention shown in FIG. 1, an extension 651 of the fluid connection 65 fluidly connects the third inlet 28 of the first bioreactor 2 with the fluid connection 65 and thus with the first outlet 55 of the electrolyser 5, so that the O2 generated by the electrolyser 5 can also be supplied to the first bioreactor 2 via a sixth fluid connection 66, which shares a part of the fifth fluid connection 65. For example, a two-way valve 86 may be used to split the O2 gas flow into a first portion vg4 (see FIG. 2) that is fed to the second bioreactor 3 and a second portion vg5 that is fed to the first bioreactor 2 via the extension 651. The valve 86 may alternatively be used to direct the O2 flow only to the first bioreactor 2 or to direct the O2 flow intermittently to the second bioreactor or to the first bioreactor 2. The O2 gas may be intermediately stored in a separate storage tank (not shown).

[0058] Due to the significantly lower solubility of H2 in the aqueous medium used and in order to separate the gas phase containing O2 or H2 to avoid flammable spaces and thus create explosion protection, H2 is not fed directly to the second bioreactor 3. Instead, the H2 inlet is relocated to compartment C, which contains the tank 4 containing the third medium 43, which corresponds to the second medium 33 in the second bioreactor 3. H2 gas is fed to the tank 4 via a seventh fluid connection 67, which fluidly connects the second outlet 57 of the electrolyzer 5 with the second inlet 46 of the tank 4. The H2 gas may also be stored intermediately in a separate storage tank (not shown). The electrolyzer 5 builds up an overpressure in the tank 4. In this embodiment, the pressure p3 prevailing in the tank 4 is the highest pressure compared to the pressures p1 and p2 in the other compartments, i.e. compartments A and B. The increased pressure provides a higher solubility coefficient of H2. The pressure is regulated by the current I (proportional to the H2 gas feed rate vg3, see FIG. 2) passed through the electrolyzer 5 and by the position (closedness) of the second valve 82. The medium circulating between compartments B and C has an additional positive effect on the H2 gas introduction. For this medium cycle operation, third and fourth fluid connections 63, 64 are arranged between the second bioreactor 3 and the tank 4. The third fluid connection 63 fluidly connects the second outlet 37 of the second bioreactor 3 with the first inlet 44 of the tank, which is arranged in the upper part 41 of the tank 4 at the height of the headspace 42 of the tank 4. A first pump 10 is arranged in the third fluid connection 63 to pump the second medium 33 from the second bioreactor 3 to the tank 4. A fourth fluid connection 64 fluidly connects the first outlet 45 of the tank 4 with the second inlet 36 of the second bioreactor 3 and allows a return flow of the third medium 43 from the tank 4 to the second bioreactor 3. The flow of the third medium 43 to the second bioreactor 3 can be controlled, for example, with a second valve 82. A part of the second medium 33 is withdrawn from compartment B and brought to a pressure >p3 using the first pump 10 in order to finely spray it through the nozzle 12 into the headspace 42 of the tank 4 in compartment C. This fine distribution in the headspace 42 of the tank 4 with H2 atmosphere ensures a further enrichment of H2 in the liquid phase.The inflow of H2 saturated medium to the second bioprocess in compartment B can be regulated by means of vl1 and vl2 (see FIG. 2) in a circuit with a first pump 10 and a second valve 82. There may also be a second type of microorganism in this cycle, namely the knar gas bacteria, which can benefit from different dissolved gas concentrations.

[0059] In compartment B there is a pressure p2, preferably lower than pressure p3, which allows the dissolved H2 to be better released into the surrounding medium, where the inflow of CO2 and O2 is increased by stirring with the second stirring device 31 and an optional gas cycle between compartments A and B via a valve 81 arranged in a ninth fluid connection 69 fluidly connecting the third outlet 39 of the second bioreactor 3, arranged at the level of the headspace 32 of the second bioreactor 3, with the second inlet 26 of the first bioreactor 2, arranged at the level of the headspace 22 of the first bioreactor 2.

[0060] The pressure p1 in compartment A is preferably lower than in B and C, but is preferably slightly above atmospheric pressure. In compartments B and C, excess pressure can be reduced or controlled, if necessary, by means of valves 83 and 84.

[0061] The biomass generated in compartment B is preferably continuously withdrawn via the second pump 11 and the separation device 6 once a sufficient cell density of the second type of microorganism is reached. In this process, the second medium 33 containing the second type of microorganism is withdrawn from the second bioreactor 3, the cells are separated from the second medium 33 using a separation device 6, for example a continuous centrifuge device, for example a disc stack centrifuge device or a hollow fiber membrane module, hydrolyzed in the hydrolysis device 7 and fed to the first bioreactor 2. For this purpose, a second fluid connection 62, for example a pipe or tube, fluidly connects the first outlet 35 of the second bioreactor 3 with the first inlet 24 of the first bioreactor 2. The separation device 6 is arranged after the pump 11 and the hydrolysis device 7 is arranged after the separation device 6 with respect to the flow direction of the second medium 33. In this embodiment, the second medium 33 separated from the cells is returned to the bioprocess in compartment B via an eighth fluid connection 68, which fluidly connects the part of the separation device 6 with the separated second medium 33 to the fourth inlet 30 of the second bioreactor 3. Optionally, the second medium may be removed from the circuit via a fifth valve 85, for example for reprocessing, and reintroduced into compartment C. The withdrawn biomass is hydrolyzed using a hydrolysis device 7 and fed to the bioprocess in compartment A as an additional C and N source. At least a part of the biomass may also be recovered and used as a separate bioproduct, for example as fish feed. Depending on the operating mode of the bioprocess in compartment A (for example batch or continuous), the biomass post-processing may be performed continuously or at intervals. In this way, an estimated 20-60% of the expensive glucose in the lipid fermentation step can be saved, significantly shifting the C balance towards higher lipid yields.

[0062] In compartment C, fresh medium 103 may be introduced into the process, for example at the third inlet 48 of vessel 4. The first type of microorganisms and / or the first medium 23 may be withdrawn from compartment A, for example via the third outlet 29 of the first bioreactor 2.

[0063] FIG. 2 shows a simplified scheme of the apparatus shown in FIG. 1, illustrating gas (dashed lines) and liquid (solid lines) flows into and between compartments A, B or C (see description of FIG. 1). [Explanation of symbols]

[0064] 1. Apparatus of the Present Invention 2. First bioreactor 21 First stirring device 22 Headspace 23 First Medium 24 First Entrance 25 Exit 1 26 Second Entrance 28 The Third Entrance 29 Third Exit 3. Second bioreactor 30 The Fourth Entrance 31 Second stirring device 32 Headspace 33 Second Medium 34 First Entrance 35 Exit 1 36 Second Entrance 37 2nd Exit 38 The Third Entrance 39 Third Exit 4 tanks 41 Top of the tank 42 Headspace 43 The Third Medium 44 First Entrance 45 Exit 1 46 Second Entrance 48 Third Entrance 5. Electrolysis Apparatus Exit 55 (1st) Exit 57 (2nd) 6 Separation device 61 first fluid connection 62 second fluid connection 63 Third fluid connection 64 Fourth fluid connection 65 Fifth fluid connection 651 Extension part 66 Sixth fluid connection 67 Seventh fluid connection 68 Eighth fluid connection 69 9th fluid connection 7 Hydrolysis equipment 8 CO2 concentrator 81 Valve 82 Second Valve 83 Valve 84 Valves 85 The Fifth Valve 86 Two-way valve 9. Compressor 10 First Pump 11 Second Pump 12 Nozzles 13 CO2 Storage Tank 100 Air 101 Biological products 102 CO2 deficient air 103 Fresh medium p1 First pressure p2 Second pressure p3 The third pressure

Claims

1. 1. A process for the biotechnological production of a biological product, comprising: a) a first bioprocess comprising the cultivation of a first type of microorganism in a first medium at a first pressure p1 in a first bioreactor, said bioproduct and carbon dioxide CO ２ and a first bioprocess resulting in the production of b) a second bioprocess comprising the cultivation of a second type of microorganism in a second medium at a second pressure p2 in a second bioreactor, producing biomass and molecular oxygen O ２ and molecular hydrogen H ２ and the CO produced in the first bioprocess of step a). ２ and a second bioprocess that consumes at least a portion of c) Water O ２ and H ２ The O produced by electrolysis was ２ to the second bioprocess and / or the first bioprocess, and H produced by electrolysis is ２ to a headspace and a vessel containing a third medium at a third pressure p3, said third medium being identical to the second medium in said second bioreactor; d) H ２ feeding at least a portion of the third medium from the vessel to the second bioprocess; wherein at least a portion of the biomass produced in the second bioprocess of step b) is used as a C source for the first bioprocess of step a).

2. 2. The process of claim 1, wherein p3 > p2 > p1, preferably p3 > p2 > p1.

3. 3. The process according to claim 1 or 2, wherein the first bioprocess is an aerobic bioprocess, preferably a fermentation process, more preferably a fermentation process for the production of lipids and / or carotenoids.

4. The second bioprocess is ２ , H ２ and CO ２ The process according to any one of claims 1 to 3, comprising the chemoautotrophic consumption of

5. 5. The process according to any one of claims 1 to 4, wherein the second bioprocess is carried out at a temperature between 40 and 55°C, preferably 45 to 55°C, 50 to 55°C or 50 to 53°C.

6. 6. The process according to claim 1, wherein a portion of the second medium of the second bioreactor containing the second type of microorganism is withdrawn from the second bioreactor, the biomass contained in the second medium is at least partially separated from the second medium, hydrolyzed and fed to the first bioprocess, and the second medium separated from the biomass is recycled to the second bioprocess.

7. 7. The process of any one of claims 1 to 6, wherein a portion of a second medium from the second bioreactor is fed into the tank and sprayed into the headspace of the tank, and an equal amount of a third medium is fed from the tank to the second bioreactor.

8. O produced by electrolysis ２ The process according to any one of claims 1 to 7, wherein a portion of said first bioprocess is fed to said first bioprocess.

9. 9. The process according to any one of claims 1 to 8, wherein the first type of microorganism is a microorganism of the genus Rhodosporidium, preferably Rhodosporidium toruloides, and the second type of microorganism is a microorganism of the genus Hydrogenophilus, preferably Hydrogenophilus thermoluteolus.

10. The process according to any one of claims 1 to 9, wherein p1 is greater than atmospheric pressure.

11. The process according to any one of claims 1 to 10, wherein the process is a continuous process.

12. An apparatus (1) for the biotechnological production of a bioproduct by the process according to any one of claims 1 to 11, comprising: a) a first bioreactor (2) configured for carrying out a first bioprocess, said first bioprocess comprising the cultivation of a first type of microorganism in a first medium (23) at a first pressure p1 ... ２ and a first bioreactor (2) resulting in the production of b) a second bioreactor (3) configured for carrying out a second bioprocess comprising the cultivation of a second type of microorganism in a second medium (33) at a second pressure p2, said second bioprocess producing biomass and molecular oxygen O ２ and molecular hydrogen H ２ and the CO produced in the bioprocess of step a). ２ and a second bioreactor (3) which is a process consuming at least a portion of c) a vessel (4) configured to contain the headspace (42) and the third medium (43) at a third pressure p3; d) Water ２ and H ２ an electrolysis device (5) for electrolyzing the The device (1) further comprises: the CO produced in the first bioreactor (2) ２ a first fluid connection (61) fluidly connecting a first outlet (25) of the first bioreactor (2) to a first inlet (34) of the second bioreactor (3) for the transfer of - a second fluid connection (62) fluidly connecting a first outlet (35) of the second bioreactor (3) to a first inlet (24) of the first bioreactor (2) for the transfer of biomass produced in the second bioreactor (3) to the first bioreactor (2); a third fluid connection (63) fluidly connecting a second outlet (37) of said second bioreactor (3) to a first inlet (44) of said tank (4) for the transfer of a second medium (33) to said tank (4); a fourth fluid connection (64) fluidly connecting a first outlet (45) of said vessel (4) to a second inlet (36) of said second bioreactor (3) for the transfer of a third medium (43) to said second bioreactor (3); -Oxygen ２ a fifth fluid connection (65) fluidly connecting the first outlet (55) of the electrolyser (5) to the third inlet (38) of the second bioreactor (3) for the transfer of -Oxygen ２ a sixth fluid connection (66) fluidly connecting the first outlet (55) of the electrolyzer (5) to the third inlet (28) of the first bioreactor (2) for the transfer of - Hydrogen H ２ a seventh fluid connection (67) fluidly connecting a second outlet (57) of the electrolyser (5) to a second inlet (46) of the tank (4) for the transfer of An apparatus (1) comprising:

13. 13. The apparatus (1) according to claim 12, wherein the second fluid connection (62) comprises, between the first outlet (35) of the second bioreactor (3) and the first inlet (24) of the first bioreactor (2), a separation device (6) for separating the second type of microorganisms from the second medium (33) and a hydrolysis device (7) for hydrolyzing the second type of microorganisms separated by the separation device (6) from the second medium (33).

14. 14. The apparatus (1) according to claim 12 or 13, wherein the third fluid connection (63) fluidly connecting the second outlet (37) of the second bioreactor (3) to the first inlet (44) of the tank (4) extends into an upper part (41) of the tank (4) including the headspace (42), the extension including a nozzle (12) for atomizing the second medium (33) supplied to the tank (4).

15. A first fluid connection (61) fluidly connects the first outlet (25) of the first bioreactor (2) to the first inlet (34) of the second bioreactor (3). ２ prior to its transfer to said second bioreactor (3). ２ An apparatus (1) according to any one of claims 12 to 14, comprising a concentrator (8).

16. The first fluid connection (61) fluidly connects a first outlet (25) of the first bioreactor (2) to a first inlet (34) of the second bioreactor (3). ２ CO for intermediate storage ２ An apparatus (1) according to any one of claims 12 to 15, comprising a storage tank (13).

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