BR112021018944B1 - IMPROVED FERMENTATION OF GREENHOUSE GAS - Google Patents

Abstract

IMPROVED FERMENTATION OF GREENHOUSE GAS. The present invention relates to a method for cultivating a microorganism capable of using an organic raw material, comprising the steps of: (i) cultivating the microorganism in one or more bioreactors (1); (ii) capture CO2 from one or more bioreactors (1) and reduce the CO2 to an organic feedstock in a reduction unit (3); and (iii) feed at least part of the organic raw material from the reduction unit (3) into one or more bioreactors (1).

Description

Field

[0001] The present invention relates to a method for cultivating a microorganism capable of using an organic raw material. According to another aspect, the present invention relates to a system for cultivating a microorganism capable of using an organic raw material.

Fundamentals

[0002] Microorganisms are used on a large scale for the industrial production of the microorganisms themselves or metabolites produced by the microorganisms. These industrial fermentation processes and the flanking operations built into a factory or biorefinery are energy intensive and CO2 is emitted due to energy use and also the metabolism of the microorganisms themselves. In the face of climate change, there is an urgent need for industrial fermentation processes with reduced CO2 emissions, or without CO2 emissions, or even with negative CO2 emissions.

Detailed Description

[0003] According to a first aspect, the present invention relates to a method for cultivating a microorganism capable of using an organic raw material, comprising the steps of: (i) cultivating the microorganism in one or more bioreactors (1); (ii) capturing CO2 from one or more bioreactors (1), preferably in a capture unit (2), and reducing the CO2 to an organic feedstock, preferably in a reduction unit (3) ; and (iii) feeding at least a part of the organic raw material, preferably from the reduction unit (3) into one or more bioreactors (1), preferably feeding at least a part of the organic raw material from the reduction unit (3) in one or more bioreactors.

[0004] The present inventors found that the invention provides for the cultivation of microorganisms with reduced CO2 emissions, or even without CO2 emissions. Furthermore, when feeding organic feedstock into one or more bioreactors, the carbon substrate used for microorganism growth, i.e. sugar, ethanol, glycerol, oligosaccharides, polysaccharides, oil or sugar alcohol, can be reduced. This means that the biomass yield per sugar used is increased, which provides an improved production process.

[0005] A "bioreactor", as used in the present context, is a fermentation vessel suitable for the controlled cultivation of a microorganism in the presence or absence of oxygen.

[0006] A 'CO2 capture unit' is a physical means suitable for capturing CO2 from the exhaust gas of one or more bioreactors (1).

[0007] A 'CO2 reduction unit' is a physical means, such as an apparatus or equipment, that is suitable for reducing CO2 to an organic raw material.

[0008] Microorganisms capable of utilizing an organic raw material, as used in the present context, means microorganisms that can assimilate the organic raw material.

[0009] ‘Organic raw materials’, as used in the present context, are carbon-containing raw materials resulting from CO2 reduction. Preferably, the present organic raw material is derived from CO2. More preferably, the present organic raw material is derivable from CO2 using electrochemical CO2 reduction, microbial CO2 reduction, enzymatic CO2 conversion process or an organic synthesis process. Alternatively, the organic raw materials used in the present context can be defined as CO2-derived energy carriers. Preferably, the amount of organic feedstock introduced into one or more bioreactors is less than the amount of substrate in one or more bioreactors.

[00010] In a preferred embodiment, the present organic feedstock, or CO2-derived energy carrier, is chosen from the group consisting of formic acid (HCOOH), methanol (CH3OH), ethylene (C2H4), ethanol ( C2H5OH), 1-propanol (CH3CH2CH2OH), methane (CH4), acetic acid (CH3COOH) and carbon monoxide (CO). More preferably, the present organic raw material is formic acid. The advantage of using formic acid is that it can be efficiently produced from CO2, and microorganisms may be able to utilize formic acid. Microorganisms may inherently utilize formic acid, or microorganisms may be engineered to be capable of utilizing formic acid. Therefore, the present microorganism is preferably capable of utilizing formic acid.

[00011] In a preferred embodiment, the present organic raw material is not formatted. The disadvantage of formate is that formate is an anion that needs to be balanced with a cation, which needs to be supplied through titration. Later in fermentation, the absorption of formic acid results in the need for back-titration to balance the cation. As a result, a salt will be produced as a co-product, which is highly undesirable for economic and environmental reasons.

[00012] 'Cultivating the microorganism', as used in the present context, comprises the production of biomass and/or the production of a compound of interest, such as extracellular metabolites and/or intracellular metabolites, including complex compounds such as enzymes, lipids, polysaccharides, vitamins and antibiotics.

[00013] Preferably, the fermentation is on an industrial scale. More preferably, one or more bioreactors (1) each have a volume of more than 10 liters, more preferably more than 100 liters, even more preferably more than 1,000 liters, more preferably more than 10,000 liters.

[00014] Step (i) of cultivating the microorganism in one or more bioreactors (1) can be anaerobic fermentation and/or aerobic fermentation.

[00015] Step (ii) of capturing CO2 from one or more bioreactors (1) and reducing the CO2 to an organic feedstock in a reduction unit (3) means capturing CO2 from the exhaust gas of one or more bioreactors ( 1) and reduce the CO2 captured from organic raw materials. Capturing and reducing CO2 to an organic feedstock in a reduction unit (3) can be accomplished by known methods and equipment. Preferably, the reduction of CO2 is the electrochemical reduction of CO2. Alternatively, reducing CO2 to an organic feedstock in one reduction unit (3) is photoelectrochemical CO2 reduction, an enzymatic CO2 reduction, or a microbial CO2 reduction. An example of an electrochemical process for reducing CO2 in formic acid is disclosed in US 2012/0228147.

[00016] In a preferred embodiment, the exhaust gas from one or more bioreactors comprises a concentration of CO2 that is more than 50%, preferably more than 55%, preferably more than 60%, preferably more than preferably more than 65%, preferably more than 70%, preferably more than 75%, preferably more than 80%, preferably more than 85%, preferably more than 90% , preferably more than 95%. Alternatively, the exhaust gas comprises a CO2 concentration that is less than 50%, preferably less than 40%, preferably less than 30%, preferably less than 20%, preferably less to 10%, preferably less than 5%. Preferably, the exhaust gas from one or more bioreactors comprises a CO2 concentration of more than 90% and the exhaust gas from one or more other bioreactors comprises a CO2 concentration of less than 10%. For example, anaerobic processes provide an exhaust gas with a very high CO2 concentration, while aerobic processes provide an exhaust gas with a low CO2 concentration.

[00017] Step (iii) of feeding at least part of the organic raw material from the reduction unit into one or more bioreactors (1) comprising feeding at least 10% (w/w) of the organic raw material of the reduction unit (3). More preferably, the feed is at least 20% (w/w), at least 30% (w/w), at least 40% (w/w), at least 50% (w/w), at least at least 60% (w/w), at least 70% (w/w), at least 80% (w/w), at least 90% (w/w), at least 95% (w/w) or at least minus 99% (w/w) of the organic raw material from the reduction unit (3) to the bioreactors (1). More preferably, 100% of the organic feedstock formed is fed into one or more bioreactors (1). Advantageously, the present method can be optimized in that the amount of organic raw material that is reduced from CO2 corresponds to the amount of organic raw material that can be used for co-feeding in the bioreactors. This provides an improved method that reduces CO2 emissions and can be implemented at lower costs because the equipment required for CO2 reduction can be smaller. On the other hand, in view of carbon pricing, it may be advantageous to reduce all CO2 formed to organic raw material. Any surplus organic feedstock, i.e. feedstock that cannot be co-fed to the bioreactors (1), can be used for other commercial purposes. Therefore, the present method may comprise a step of collecting formed organic raw material and packaging and/or transportation.

[00018] In a preferred embodiment, the present method uses two or at least two bioreactors, more preferably three or at least three bioreactors, more preferably four or at least four bioreactors. Bioreactors can ferment the same microorganism, or each bioreactor can ferment a different type of microorganism. For example, in case two bioreactors are used, the same microorganisms can be fermented, but also two different types of microorganisms can be fermented. The advantage of combining different types of microorganisms is that a combination can be chosen that makes efficient use of each other's waste streams. Or different types of CO2-reduced organic feedstocks may be used by different microorganisms because one microorganism may be better at utilizing one type of organic feedstock than another. This flexibility increases the applicability of the present invention.

[00019] In a preferred embodiment, the present microorganism is chosen from the group consisting of yeast, filamentous fungi, bacteria and algae. In another preferred embodiment, the microorganism comprises two different microorganisms from the group consisting of yeast, filamentous fungi, bacteria and algae. More preferably, the microorganism comprises three or even four different microorganisms from the group consisting of yeast, filamentous fungi, bacteria and algae. For example, the present microorganism comprises yeasts and filamentous fungi. More preferably, the present method comprises cultivating yeast in a bioreactor and cultivating a filamentous fungus in a second bioreactor.

[00020] A yeast cell is preferably a yeast belonging to the genus Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces or Yarrowia. More preferably, the present yeast is Kluyveromyces lactis, Saccharomyces cerevisiae, Hansenula polymorpha, Yarrowia lipolytica or Pichia pastoris.

[00021] Filamentous fungi include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK) . Filamentous fungi are characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan and other complex polysaccharides. Vegetative growth occurs through hyphae lengthening and carbon catabolism is necessarily aerobic. Strains of filamentous fungi include, but are not limited to, strains of Acremonium, Agaricus, Aspergillus, Aureobasidium, Chrysosporium, Coprinus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces , Panerochaete, Pleurotus, Schizophyllum, Talaromyces, Rasamsonia, Thermoascus, Thielavia, Tolypocladium, and Trichoderma.

[00022] The preferred filamentous fungi belong to a species of the genus Acremonium, Aspergillus, Chrysosporium, Myceliophthora, Penicillium, Talaromyces, Rasamsonia, Thielavia, Fusarium or Trichoderma and, more preferably, a species of Aspergillus niger, Acremonium alabamense, Aspergillus awamori, Aspergillus foetidus, Aspergillus lojae, Aspergillus fumigatus, Talaromyces emersonii, Rasamsonia emersonii, Aspergillus oryzae, Chrysosporium lucknowense, Fusarium oxysporum, Myceliophthora thermophila, Trichoderma reesei, Thielavia terrestris or Penicillium chrysogenum. A more preferred filamentous fungus belongs to the genus Aspergillus, more preferably the filamentous fungus belongs to the species Aspergillus niger or is Aspergillus niger.

[00023] The term “bacteria” includes Gram-negative and Gram-positive microorganisms. Suitable bacteria can be selected from, for example, Escherichia, Anabaena, Caulobactert, Gluconobacter, Rhodobacter, Pseudomonas, Paracoccus, Bacillus, Brevibacterium, Corynebacterium, Rhizobium (Sinorhizobium), Flavobacterium, Klebsiella, Enterobacter, Lactobacillus, Lactococcus, Methylobacterium, Staphylococcus , Streptomyces, Actinomycetes, Xanthomonas or Sphingomonas. Preferably, the bacterial cell is selected from the group consisting of B. subtilis, B. amyloliquefaciens, B. licheniformis, B. puntis, B. megaterium, B. halodurans, B. pumilus, G. oxydans, Caulobactert crescentus CB 15, Methylobacterium extorquens, Rhodobacter sphaeroides, Pseudomonas zeaxanthinifaciens, Paracoccus denitrificans, E. coli, C. glutamicum, Staphylococcus carnosus, Streptomyces lividans, Sinorhizobium melioti and Rhizobium radiobacter.

[00024] The present algae are preferably chosen from the group consisting of glaucophytes, rhodoplasts and chloroplasts. More preferably, the present algae are heterotrophic algae, more preferably heterotrophic algae such as Chlorella, Nannochloropsys, Nitzschia, Thraustochytrium or Schizochyttrium.

[00025] In a preferred embodiment, the microorganisms according to the invention comprise at least one polynucleotide encoding a compound of interest or at least one polynucleotide encoding a compound involved in the production of a compound of interest by the cell.

[00026] The compound of interest can be any biological compound. The biological compound can be biomass or a biopolymer or metabolite. The biological compound may be encoded by a single polynucleotide or a series of polynucleotides that make up a biosynthetic or metabolic pathway or may be the direct result of the product of a single polynucleotide or products of a series of polynucleotides. The biological compound may be native to the host cell or heterologous.

[00027] The term "heterologous biological compound" is defined herein as a biological compound that is not native to the cell; or a native biological compound in which structural modifications have been made to alter the native biological compound.

[00028] The term "biopolymer" is defined herein as a chain (or polymer) of identical, similar or different subunits (monomers). The biopolymer can be any biopolymer. The biopolymer may be, for example, but is not limited to, a nucleic acid, polyamine, polyol, polypeptide (or polyamide) or polysaccharide.

[00029] The biopolymer can be a polypeptide. The polypeptide can be any polypeptide with a biological activity of interest. The term "polypeptide" is not intended herein to refer to a specific length of the encoded product and therefore encompasses peptides, oligopeptides, and proteins. Polypeptides further include naturally occurring allelic and engineered variations of the above-mentioned polypeptides and hybrid polypeptides. The polypeptide may be native or may be heterologous to the host cell. The polypeptide may be a collagen or gelatin, or a variant or hybrid thereof. The polypeptide may be an antibody or parts thereof, an antigen, a clotting factor, an enzyme, a hormone or a hormone variant, a receptor or parts thereof, a regulatory protein, a structural protein, a reporter or a protein transport, protein involved in the secretion process, protein involved in the folding process, chaperone, peptide amino acid transporter, glycosylation factor, transcription factor, synthetic peptide or oligopeptide, intracellular protein. The intracellular protein can be an enzyme such as a protease, ceramidases, epoxide hydrolase, aminopeptidase, acylases, aldolase, hydroxylase, aminopeptidase, lipase. The polypeptide may also be an extracellularly secreted enzyme. These enzymes can belong to the groups of oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, catalase, cellulase, chitinase, cutinase, deoxyribonuclease, dextranase, esterase. The enzyme may be a carbohydrase, for example, cellulases, such as endoglucanases, ß-glucanases, cellobiohydrolases or ß-glucosidases, hemicellulases or pectinolytic enzymes, such as xylanases, xylosidases, mannanases, galactanases, galactosidases, pectin methyl esterases, pectin lyases, pectate lyases, endo polygalacturonases, exopolygalacturonases, rhamnogalacturonases, arabanases, arabinofuranosidases, arabinoxylan hydrolases, galacturonases, lyases, or amylolytic enzymes; hydrolase, isomerase or ligase, phosphatases such as phytases, esterases such as lipases, proteolytic enzymes, oxidoreductases such as oxidases, transferases or isomerases. The enzyme may be a phytase. The enzyme may be an aminopeptidase, asparaginase, amylase, a maltogenic amylase, carbohydrase, carboxypeptidase, endoprotease, metalloprotease, serine protease catalase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase , glucoamylase, alpha-glucosidase, beta-glucosidase, haloperoxidase, protein deaminase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phospholipase, galactolipase, chlorophylase, polyphenoloxidase, ribonuclease, transglutaminase, or glucose oxidase, hexose oxidase or mono-oxygenase.

[00030] Preferably, the compound of interest is a heterologous product. Preferably, the compound of interest is a glucose oxidase. More preferably, the compound of interest is a heterologous glucose oxidase. In another preferred embodiment, the compound of interest is a lipolytic enzyme, e.g., a lipolytic enzyme having one or more of the activities selected from the group consisting of: lipase (triacylglycerol lipase), phospholipase (e.g., phospholipase A1 and/or or phospholipase A2 and/or phospholipase B and/or phospholipase C), galactolipase.

[00031] According to the present invention, a polypeptide or enzyme can also be a product as described in WO 2010/102982. According to the present invention, a polypeptide can also be a fused or hybrid polypeptide to which another polypeptide is fused at the N-terminus or at the C-terminus of the polypeptide or fragment thereof. A fused polypeptide is produced by fusing a nucleic acid sequence (or a portion thereof) encoding one polypeptide with a nucleic acid sequence (or a portion thereof) encoding another polypeptide.

[00032] Techniques for producing fusion polypeptides are known in the art and include ligation of the coding sequences encoding the polypeptides so that they are in frame and expression of the fused polypeptide is under the control of the same(s). ) promoter(s) and terminator(s). Hybrid polypeptides may comprise a combination of partial or complete polypeptide sequences obtained from at least two different polypeptides, one or more of which may be heterologous to the host cell. Examples of fusion polypeptides and signal sequence fusions are, for example, as described in WO 2010/121933.

[00033] The biopolymer can be a polysaccharide. The polysaccharide can be any polysaccharide, including, but not limited to, a mucopolysaccharide (e.g., heparin and hyaluronic acid) and nitrogen-containing polysaccharide (e.g., chitin). In a more preferred option, the polysaccharide is hyaluronic acid. In another preferred option, the polysaccharide is a hydrocolloid, for example, xanthan, gellan, pectin, Welan or other polysaccharide.

[00034] The polynucleotide encoding the compound of interest or encoding a compound involved in the production of the compound of interest according to the invention may encode an enzyme involved in the synthesis of a primary or secondary metabolite, such as organic acids, alcohols, lipids, carotenoids, beta-lactams, antibiotics and vitamins. Such a metabolite can be considered as a biological compound according to the present invention. Preferably, the present compound of interest is beta-lactam. Preferably, the present compound of interest is ethanol.

[00035] The term "metabolite" encompasses both primary and secondary metabolites; the metabolite can be any metabolite. Preferred metabolites are citric acid, gluconic acid, adipic acid, fumaric acid, itaconic acid and succinic acid.

[00036] The metabolite can be encoded by one or more genes, as in a biosynthetic or metabolic pathway. Primary metabolites are products of the primary or general metabolism of a cell, which relate to energy metabolism, growth and structure. Secondary metabolites are products of secondary metabolism (see, for example, R. B. Herbert, The Biosynthesis of Secondary Metabolites, Chapman and Hall, New York, 1981).

[00037] The primary metabolite may be, but is not limited to, an amino acid, fatty acid, nucleoside, nucleotide, sugar, triglyceride or vitamin. For example, vitamin A, B2, C, D or E.

[00038] The secondary metabolite may be, but is not limited to, an alkaloid, coumarin, flavonoid, polyketide, quinine, steroid, peptide or terpene. The secondary metabolite can be an antibiotic, appetite suppressant, attractant, bactericide, fungicide, hormone, insecticide or rodenticide. The preferred antibiotics are cephalosporins and beta-lactams. Other preferred metabolites are exo-metabolites. Examples of exo-metabolites are Aurasperone B, Funalenone, Kotanin, Nigragilin, Orlandin, other naphtho-g-pyrones, Pyranonigrin A, Tensidol B, Fumonisin B2 and Ochratoxin A.

[00039] The biological compound can also be the product of a selectable marker. A selectable marker is a product of a polynucleotide of interest whose product provides biocidal or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Selectable markers include, but are not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferase), bar (phosphinotricinacetyltransferase), hygB (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (adenyl sulfate transferase), trpC (anthranilate synthase), ble (phleomycin resistant protein), hyg (hygromycin), NAT or NTC (Nourseothricin), as well as their equivalents.

[00040] According to the invention, the compound of interest is preferably a polypeptide as described in the list of compounds of interest.

[00041] Preferably, the polypeptide is an enzyme as described in the list of compounds of interest. Preferably, a glucose oxidase. In another embodiment, the enzyme is a lipolytic enzyme.

[00042] According to another embodiment of the invention, the compound of interest is preferably a metabolite.

[00043] According to yet another embodiment, the compound of interest is biogas.

[00044] The present microorganism may already be capable of producing the compound of interest. The microorganism may also be provided with a homologous or heterologous nucleic acid construct encoding a polypeptide wherein the polypeptide may be the compound of interest or a polypeptide involved in the production of the compound of interest. The person skilled in the art knows how to modify a microorganism so that it is capable of producing the compound of interest.

[00045] In another preferred embodiment, the present method further comprises the step of: (iv) electrolyzing water into H2 and O2 in an electrolysis unit (4) and feeding at least part of the H2 into the reduction unit (3) to reduce CO2 to organic raw material.

[00046] The advantage of electrolyzing water into H2 and O2 is that no H2 needs to be obtained from external sources. The electricity required for the electrolysis unit (4) can advantageously be obtained from surplus electricity from other equipment on site, to provide a sustainable solution for the electricity required. Alternatively, electricity is generated from renewable energies such as solar, wind or hydro.

[00047] In another preferred embodiment, the electrolysis unit (4) is combined with the reduction unit (3). Preferably, step (iv) of water electrolysis is carried out simultaneously and in the presence of the CO2 reduction step. Preferably, the following reaction is carried out during CO2 reduction and water electrolysis:

[00048] In other words, it is preferable that the water electrolysis step is carried out in the presence of CO2. The advantage is that an improved process or system is provided. Therefore, it is preferred that the present step of (ii) capturing CO2 from one or more bioreactors (1), preferably in a capture unit (2), and reducing the CO2 to an organic feedstock, in a manner preferred in a reduction unit (3) further comprises electrolysis of water in the presence of CO2. In another preferred embodiment, the present method further comprises the step of: (v) feeding at least part of the O2 into one or more bioreactors (1). O2 is the byproduct of water electrolysis. It can be advantageously used in one or more bioreactors, for example for aerobic fermentation. Furthermore, it was found that all aeration of one or more bioreactors can be carried out using O2 from the electrolysis unit (4). Therefore, O2 from a different source is no longer required, which provides an additional sustainability improvement. For example, no air compressor is needed anymore, or a smaller compressor can be used, because the electrolysis unit (4) can supply the O2 under pressure comparable to the pressure of a conventional compressor. Preferably, step (iv) of electrolyzing water into H2 and O2 in an electrolysis unit (4) is carried out at a pressure within the range of 0.5 to 10 bar (50 kPa to 1000 kPa), of preferably 1 to 8 bar (100 kPa to 800 kPa), more preferably 1.5 to 6 bar (150 kPa to 600 kPa), more preferably 2 to 4 bar (200 kPa to 400 kPa) , preferably bar of absolute pressure.

[00049] In another preferred embodiment, the present method further comprises the step of: (vi) feeding at least part of the H2 into one or more bioreactors (1). Any excess H2, that is, H2 that is not required for CO2 reduction, can advantageously be co-fed into one or more bioreactors. Microorganisms that can utilize H2 can be fermented by co-feeding with H2. By doing so, the amount of CO2 resulting from the microorganism's metabolism can be reduced. This provides a further sustainability advantage. Furthermore, by H2 co-feeding, the amount of sugar or other carbon-containing raw material required for fermentation can be reduced, which provides a significant cost improvement. Therefore, the present microorganism, or one of the present microorganisms, is preferably capable of utilizing H2. More preferably, the present microorganism is capable of utilizing the present organic raw material and H2. Alternatively, one microorganism is capable of utilizing the present organic raw material and another type of microorganism is capable of utilizing H2. For example, the present method comprises a fermentation step of a yeast capable of using formic acid in a bioreactor and the fermentation of a filamentous fungus capable of using H2 in another bioreactor. More specifically, the present method comprises a step of cultivating Saccharomyces cerevisiae capable of utilizing formic acid in a bioreactor and cultivating Penicillium chrysogenum capable of utilizing H2, or cultivating Saccharomyces cerevisiae capable of utilizing H2 in a bioreactor and cultivating Penicillium chrysogenum capable of utilizing acid formic in another bioreactor.

[00050] In another preferred embodiment, the cultivation of a yeast produces yeast biomass, while the cultivation of a filamentous fungus produces a compound of interest. Therefore, the present method preferably comprises a step of harvesting biomass or yeast biomass and/or comprises a step of harvesting a compound of interest.

[00051] The present method can be improved by monitoring the fermentation process. In a preferred embodiment, the present method further comprises the steps of: (vii) selecting at least one process parameter of the fermentation process, for which current measured values are determined during the course of the fermentation process; (viii) comparing a respective current measured value of at least one selected process parameter with a corresponding estimated value of a model parameter estimated by a process model for this at least one process parameter; (ix) i) compare a variation between the respective current measured value and the corresponding estimated value for at least one selected process parameter with a predetermined limit value; and (x) ) changing at least one defined model parameter employed in the process model when the predetermined threshold value is exceeded by the variation: wherein steps (vii) to (ix) with a respective changed model parameter are executed until the variation is placed below a predetermined threshold value; and, optionally, where, after a predetermined number of repetitions of steps (vii) to (ix), the threshold value is reached by the variation, the method is discontinued and a warning is generated as output.

[00052] In one or more bioreactors (1), in the CO2 capture unit (2), in the CO2 reduction unit (3) and/or in the electrolysis unit (4), various environmental and/or process parameters can be regulated and controlled for the respective fermentation processes, such as pH value, temperature, CO2 supply, H2 supply, oxygen supply, nitrogen supply, sugar content, organic raw material content and/or temperature settings. mixer. However, fermentation processes are biologically complex and very sensitive. Constant and close monitoring of the fermentation process is therefore necessary to maintain the corresponding environmental conditions in one or more bioreactors for a consistent and optimal course of the process and for the biomass or bioactive cells used in the nutrient solution to grow and be able to produce the desired biomass and/or compound of interest.

[00053] It is favorable if a process parameter of the fermentation process, for which during the course of the fermentation process measured values can be determined approximately in real time, is selected as the process parameter to be measured. For this reason, the variation between the process model and the course of the fermentation process can be determined approximately in real time, especially if the process model is calculated in parallel with the ongoing fermentation process. A direct comparison between the measured value of the selected process parameter and the estimated value calculated or predicted by the process model can thus be carried out for the process parameter. An almost instantaneous intervention is also activated if there is an error in the fermentation process.

[00054] Conveniently, repetitions of steps (vii) to (ix) of the present method are carried out, that is, the comparison between the measured value and the estimated value of the selected process parameter. Here, comparisons of the variance with the predetermined threshold value and the modification of the process model model parameter when the threshold value is exceeded are performed with the respective modified model parameter at short intervals in time, such as every 10 seconds. The process model can thus be adjusted quickly, for example, with the presence of biological variability or variability in process control (e.g. differences in raw materials, or fluctuations in substrate composition) so that a prediction correspondingly Validity of the fermentation process can be carried out for the subsequent course of the process. In case of errors in the bioprocess, an almost instantaneous response can occur. An almost instantaneous intervention allows damage to the bioprocess and/or equipment, for example, to be avoided or prevented.

[00055] In a preferred embodiment of the invention, what is known as the respiratory quotient, also abbreviated to RQ, is selected as a process parameter that is measured during the course of the fermentation process. The respiratory quotient is a process parameter in fermentation processes, which represents an indicator of the processes within a bioactive cell. The respiratory quotient describes a ratio between the CO2 produced at a given time and the O2 consumed at the same time. During the course of a fermentation process, the respiratory quotient can be measured very easily in real time, for example using what is known as exhaust gas analysis.

[00056] Alternatively or additionally, a biomass concentration and/or a substrate concentration can be selected as process parameters, which are measured during the course of the fermentation process. The determination of the current measured values of the biomass concentration throughout the fermentation process occurs, for example, based on the electrical properties of the biomass. The actual measured values for the substrate concentration (e.g. sugar, etc.) can also be determined in the course of the fermentation process, such as based on spectroscopic properties using spectroscopy, in particular using reflection spectroscopy.

[00057] Furthermore, it is advantageous if what is known as a deterministic process model is employed to estimate the parameters of the fermentation process. With a deterministic approach to an illustration of a fermentation process as a model, knowledge of the respective process-specific biochemical processes inside and outside the biomass or cells employed is converted into mathematical equations during the fermentation process.

[00058] In another preferred embodiment, steps (vi) to (ix) are performed by a computer-implemented method, more preferably a computer-implemented method using algorithms. The assistance of a computer and algorithms is beneficial because variations in process parameters can be easily compared with historical fermentation process data and this historical data can be used for correct interpretation of process parameter variation.

[00059] Given the beneficial sustainability achievements of the present invention, the present invention relates, according to another aspect, to a system for cultivating a microorganism capable of utilizing an organic raw material, said system comprising one or more bioreactors (1) for cultivating said microorganism, a CO2 capture unit (2) for capturing CO2 from one or more bioreactors (1), a CO2 reduction unit (3) for reducing CO2 to formic acid and /or one more conduits (10) for introducing the organic raw material into the one or more bioreactors (1).

[00060] In a preferred embodiment, the present system further comprises an electrolysis unit (4) for water electrolysis and/or a conduit (11) for introducing H2 from the electrolysis unit (4) into the CO2 reduction unit (3 ). Preferably, the present electrolysis unit (4) comprises means for pressurizing the O2 and/or the electrolysis reaction. This is advantageous because pressurized O2 can be introduced into one or more bioreactors (1).

[00061] In yet another preferred embodiment, the present system further comprises one or more conduits (12) for the introduction of H2 and/or O2 into one or more bioreactors (1). This is advantageous in that optimal use can be made of both products, between the CO2 reduction unit (3) and one or more bioreactors (1). Depending on the course of the fermentation process, the amount of H2 feeding the CO2 reduction unit (3) and/or the amount of H2 and/or O2 feeding one or more bioreactors (1) can be adjusted to achieve an optimal CO2 reduction. CO2, an optimal output and/or a reduced substrate intake.

[00062] In a preferred embodiment, the present system further comprises one or more inlets (13) for introducing substrate into one or more bioreactors (1) and one or more outlets (14) for the product formed in one or more bioreactors (1).

[00063] In yet another preferred embodiment, the present system further comprises a computer-implemented system for a fermentation monitoring tool to simulate and/or monitor the fermentation method as defined in any of the preceding claims, said system implemented by The computer comprises at least one processor, a user interface, a control system interface configured to adjust one or more process parameters of the fermentation method, a memory comprising computer-readable medium storing instructions for simulating and/or monitoring the fermentation method. fermentation, wherein the instructions for simulating and/or monitoring the fermentation method configure the processor to: (vi) select at least one process parameter of the fermentation method, for which the current measured values are determined during the course of the method fermentation; (vii) comparing a respective current measured value of at least one selected process parameter of the fermentation method with a corresponding estimated value of a model parameter estimated by a process model for this at least one process parameter; (viii) comparing a variation between the respective current measured value and the corresponding estimated value for at least one selected process parameter with a predetermined limit value; and (ix) changing at least one defined model parameter employed in the process model when the predetermined threshold value is exceeded by the variation: wherein steps (vii) to (ix) with a respective changed model parameter are executed until the variation is placed below a predetermined threshold value; and, optionally, wherein, in cases where, after a predetermined number of repetitions of steps (vii) to (ix), the threshold value is reached by the variation, the method is discontinued and a warning is generated as output in the interface of the user.

Description of the figures

[00064] In the embodiment of figure 1, the substrate sugar is introduced through conduits 13 into bioreactor 1. Bioreactor 1 provides the conditions that allow the fermentation of microorganisms capable of using organic raw material. The product produced, such as the microorganism or a compound of interest produced by microorganisms, leaves the bioreactors 1 through conduits 14. The CO2 formed in the bioreactors 1 is introduced into a CO2 capture unit 2 through conduits 15. In the reduction unit 3, CO2 is reduced to organic raw material using H2. The organic raw material is introduced into the bioreactors 1 through the conduits 10, allowing the fermentation of microorganisms that are capable of using the organic raw material. Preferably, the amount of organic feedstock introduced into the bioreactors is less than the amount of sugar in the substrate. The advantage is that CO2 is not released into the environment and the amount of sugar required for the fermentation process can be reduced. The H2 required for CO2 reduction in organic feedstock can be introduced in reduction 3 and can be obtained from a supplier. Preferably, the electrolysis unit 4 electrolyzes water into H2 and O2. H2 can be introduced into the reduction unit 3 via conduit 11. O2 can be introduced into the bioreactor 1. Alternatively, air can be introduced into the bioreactor 1 for aerobic processes.

[00065] In the embodiment of figure 2, in addition to the embodiment of figure 1, a second bioreactor 1' is present. The electrolysis unit 4 electrolyzes water into H2 and O2. H2 can be introduced into the reduction unit 3 through conduit 11. Advantageously, H2 can be introduced into a bioreactor 1' for fermentation of microorganisms capable of using H2. This is advantageous because the H2 co-feed reduces the amount of sugar needed and can reduce the amount of CO2 formed by the microorganisms in bioreactor 1'.

[00066] Figure 3 shows a calculation of the required substrate and O2, the ethanol and yeast produced and the CO2 emitted, in a conventional system.

[00067] In the embodiment of figure 4, the aerobic bioreactor 1 produces yeast through outlet 14, using sugar through inlet 13 and O2 through inlet 12. O2 is produced by the electrolysis unit 4. The electrolysis unit 4 produces H2 which is introduced into the reduction unit 3 via conduit 11. The CO2 from the aerobic bioreactor 1 is captured in unit 2, via conduit 15. The CO2 is reduced to formic acid in the reduction unit 3 and introduced into the yeast bioreactor 1 via the conduit 10.

[00068] In operation, the embodiment of figure 4 allows the production of the same amount of yeast as in figure 3. However, no CO2 is released into the environment and the amount of sugar and O2 required for yeast fermentation is significantly reduced .

[00069] In the embodiment of figure 5, an aerobic fermentation for the production of yeast in bioreactor 1 is combined with anaerobic fermentation of ethanol for the production of ethanol in bioreactor 1'. O2 produced by the electrolysis unit 4 is introduced into the aerobic yeast bioreactor 1 through conduit 12. H2 produced by the electrolysis unit 4 is introduced into the anaerobic ethanol bioreactor 1' through conduit 12' and into the reduction unit 3 through the conduit 11. CO2 is only captured from the aerobic yeast bioreactor 1 through conduit 15. Formic acid is introduced into the aerobic yeast bioreactor 1 only through conduit 10, and any excess formic acid is collected for external use.

[00070] In operation, the modality in figure 5 allows the production of ethanol without the production of CO2. Furthermore, the amount of sugar required for ethanol production is reduced due to the H2 feed. Aerobic yeast fermentation produces the same amount of yeast using a reduced amount of sugar. No CO2 is released into the environment. The O2 required for fermentation is entirely derived from the electrolysis unit. Therefore, figure 5 shows a system that allows a CO2 neutral fermentation process.

[00071] In the embodiment of figure 6, yeast production in the aerobic bioreactor 1 is combined with the production of ethanol in the anaerobic bioreactor 1'. O2 produced by the electrolysis unit 4 is introduced into the aerobic yeast bioreactor 1 through conduit 12. H2 produced by the electrolysis unit 4 is introduced into the reduction unit 3 through conduit 11. CO2 is only captured from the anaerobic ethanol bioreactor 1 'through conduit 15. Formic acid is introduced into the aerobic yeast bioreactor 1 only through conduit 10.

[00072] In operation, the embodiment of figure 6 produces only the amount of formic acid necessary for aerobic yeast fermentation in bioreactor 1. Any excess CO2 from both bioreactors 1 that is not necessary for the production of formic acid is released . This modality allows a reduction in the sugar necessary for yeast fermentation, while minimizing the energy required for the electrolysis of water in the electrolysis unit 4.

[00073] In the embodiment of figure 7, the aerobic bioreactor 1 produces yeast through outlet 14, using sugar through inlet 13 and O2 through inlet 12. The anaerobic bioreactor 1' produces ethanol through outlet 14' using sugar through inlet 13'. CO2 from both aerobic yeast bioreactor 1 and anaerobic ethanol fermentation bioreactor 1' is captured in unit 2, via conduits 15. CO2 is reduced to formic acid in reduction unit 3 and introduced into both aerobic yeast bioreactor 1 and anaerobic ethanol fermentation bioreactor 1' using conduits 10. Any excess formic acid is collected through an outlet in conduit 10. The electrolysis unit 4 electrolyzes water into H2 and O2. H2 is introduced into the reduction unit 3 through conduit 11. O2 is introduced into the aerobic yeast fermentation bioreactor 1. Any excess O2 leaves the system through conduit 16.

[00074] In operation, the embodiment of figure 7 achieves the production of ethanol and yeast using minimal amounts of sugar. All CO2 is captured and reduced to formic acid. Microorganisms grown in bioreactors are capable of using formic acid.

[00075] In the embodiment of figure 8, the CO2 from the aerobic yeast bioreactor 1 and the anaerobic bioreactor 1' is captured in unit 2, via conduits 15. The CO2 is reduced to formic acid in the reduction unit 3 and introduced into both aerobic yeast bioreactors 1 and anaerobic bioreactor 1' using conduits 10. The electrolysis unit 4 electrolyzes water into H2 and O2. H2 is introduced into the reduction unit 3 through conduit 11. O2 is introduced into the aerobic yeast fermentation bioreactor 1. Any excess O2 leaves the system through conduit 16.

[00076] In operation, the embodiment of figure 8 achieves the production of ethanol and yeast using minimal amounts of sugar. The amount of CO2 that is captured and reduced to formic acid is adapted to the amount needed to produce formic acid. Any excess CO2 is emitted. Microorganisms grown in bioreactors only need to be able to use formic acid. In this embodiment, electricity is reduced to that required for minimal electrolysis of water to form formic acid.

[00077] Figure 9 shows a calculation of the required substrate and O2, the penicillin and yeast produced and the CO2 emitted, in a conventional system.

[00078] Figure 10 shows an embodiment of the invention in which CO2 from the aerobic yeast bioreactor 1 and the aerobic penicillin bioreactor 1' is captured in unit 2 via conduits 15. The CO2 is reduced to formic acid in the reduction unit 3 and introduced into both the aerobic yeast bioreactor 1 and the aerobic penicillin bioreactor 1' using conduits 10. The electrolysis unit 4 electrolyzes water into H2 and O2. H2 is introduced into the reduction unit 3 through conduit 11. O2 is introduced into the aerobic yeast fermentation bioreactor 1 and the aerobic penicillin bioreactor 1'. The yeast in bioreactor 1 is capable of utilizing formic acid. The penicillin in bioreactor 1' is capable of utilizing formic acid.

[00079] In operation, the embodiment of figure 10 produces the same amount of penicillin and yeast as in comparative figure 9, while the amount of sugar required to produce the same yield is reduced from 63.3 kt to 48.6 kt, and the amount of externally supplied O2 is reduced from 31.8 kt to 16.1 kt.

Claims (14)

1. Method for cultivating a microorganism capable of using an organic raw material, characterized by the fact that it comprises the steps of: (i) cultivating the microorganism in one or more bioreactors (1); (ii) capture CO2 from one or more bioreactors (1) and reduce the CO2 to an organic feedstock in a reduction unit (3); and (iii) feed at least part of the organic raw material from the reduction unit (3) into one or more bioreactors (1). 2. Method according to claim 1, characterized by the fact that the organic raw material is chosen from the group consisting of formic acid, methanol, ethylene, ethanol, 1-propanol, methane, acetic acid and carbon monoxide. 3. Method according to claim 1 or 2, characterized by the fact that the organic raw material is formic acid. 4. Method according to any of the preceding claims, characterized by the fact that the microorganism is chosen from the group consisting of yeast, filamentous fungi, bacteria and heterotrophic algae. 5. Method, according to any of the previous claims, characterized by the fact that it additionally comprises the step of: (iv) electrolyzing water into H2 and O2 in an electrolysis unit (4) and feeding at least part of the H2 into the reduction (3) to reduce CO2 to organic raw material. 6. Method, according to claim 5, characterized by the fact that it additionally comprises the step of: (v) feeding at least a part of the O2 into one or more bioreactors (1). 7. Method, according to claim 5 or 6, characterized by the fact that it additionally comprises the step of: (v) feeding at least a part of the H2 into one or more bioreactors (1). 8. Method according to any of the preceding claims, characterized by the fact that the microorganism capable of using an organic raw material comprises at least one polynucleotide encoding a compound of interest or at least one polynucleotide encoding a compound involved in the production of a compound of interest by the cell. 9. Method according to any one of the preceding claims, characterized by the fact that it comprises monitoring the fermentation process comprising the steps of: (vi) selecting at least one process parameter of the fermentation process, for which the current measured values are determined during the course of the fermentation process; (vii) comparing a respective current measured value of at least one selected process parameter with a corresponding estimated value of a model parameter estimated by a process model for this at least one process parameter; (viii) comparing a variation between the respective current measured value and the corresponding estimated value for at least one selected process parameter with a predetermined limit value; and (ix) changing at least one defined model parameter employed in the process model when the predetermined threshold value is exceeded by the variation: wherein steps (vii) to (ix) with a respective changed model parameter are executed until the variation is placed below a predetermined threshold value; and, optionally, where, after a predetermined number of repetitions of steps (vii) to (ix), the threshold value is reached by the variation, the method is discontinued and a warning is generated as output. 10. System for cultivating a microorganism capable of using an organic raw material, said system characterized by the fact that it comprises one or more bioreactors (1) for cultivating said microorganism, a CO2 capture unit (2) for capture CO2 from one or more bioreactors (1), a CO2 reduction unit (3) to reduce CO2 to the organic feedstock and one more conduit (10) to introduce the organic feedstock into one more bioreactor (1) . 11. System, according to the previous claim, characterized by the fact that it additionally comprises an electrolysis unit (4) for water electrolysis and a conduit (11) for introducing H2 from the electrolysis unit (4) to the CO2 reduction unit (3). 12. System, according to any of the previous claims, characterized by the fact that it additionally comprises one or more conduits (12) for the introduction of H2 and/or O2 into one or more bioreactors (1). 13. System, according to any of the previous claims, characterized by the fact that it additionally comprises one or more inlets (13) for introducing substrate into one or more bioreactors (1) and one or more outlets (14) for the product formed in one or more bioreactors (1). 14. System according to any one of the preceding claims, characterized by the fact that it further comprises a computer implemented system for a fermentation simulation tool for simulating the fermentation method as defined in any one of the previous claims, said system implemented by The computer comprises at least one processor, a user interface, a control system interface configured to adjust one or more process parameters of the fermentation method, a memory comprising computer-readable medium storing instructions for simulating the fermentation method, wherein the instructions for simulating the fermentation method configure the processor to: (vi) select at least one fermentation method process parameter, for which current measured values are determined during the course of the fermentation method; (vii) comparing a respective current measured value of at least one selected process parameter of the fermentation method with a corresponding estimated value of a model parameter estimated by a process model for this at least one process parameter; (viii) comparing a variation between the respective current measured value and the corresponding estimated value for at least one selected process parameter with a predetermined limit value; and (ix) changing at least one defined model parameter employed in the process model when the predetermined threshold value is exceeded by the variation: wherein steps (vii) to (ix) with a respective changed model parameter are executed until the variation is placed below a predetermined threshold value; and, optionally, wherein, in cases where, after a predetermined number of repetitions of steps (vii) to (ix), the threshold value is reached by the variation, the method is discontinued and a warning is generated as output in the interface of the user.