A fermentor and method of performing a fermentation process

Abstract

The present disclosure provides a fermentor comprising a fluid containment space forming a closed loop flow path for a fermentation fluid, at least one substrate inlet for inlet of a substrate liquid, and at least one gas injection point for injection of gas. The fermentor further comprises a fermentation outlet for withdrawing fermentation fluid from the space, and at least one circulation member for circulation of the fermentation fluid. At least a part of the flow path forms a meandering section comprising serially connected sections of a first group of sections and sections of a second group of sections, wherein the first group of sections has a predominantly vertical extend and the second group of sections has a predominantly horizontal extend, the sections of the first group of sections being arranged alternately sections of the second group of sections. The sections of the first group of sections have a total length exceeding 70% of the total flow length.

Description

[0001] A FERMENTOR AND METHOD OF PERFORMING A FERMENTATION PROCESS

[0002] FIELD OF THE DISCLOSURE

[0003] The present disclosure relates to a fermentor and to a method of performing a fermentation process in a fermentor.

[0004] BACKGROUND OF THE DISCLOSURE

[0005] Typically, in biotech processes, microorganisms are cultivated in a tank, most often referred to as a bioreactor, into which the substrates necessary for the microorganisms to grow and make the desired product are added .

[0006] Such cultivation processes typically occur in aqueous solutions, often referred to as fermentation liquids / fermentation broths, containing a variety of substrates such as carbon sources as well as nitrogen sources, phosphates, sulphates, and a variety of other components such as minerals, trace metals, and vitamins, depending on the fermenting microorganism and the products to be produced in the cultivation process.

[0007] In many fermentation processes, the microorganisms are depended on oxygen for growth. Oxygen is most often added as a gas by pumping compressed atmospheric air, pure oxygen, or oxygen enriched air, into the fermentation liquid . All living cells requires at least one carbon source for growth. In many traditional fermentation processes, the carbon source(s) is added in a fluid form, such as sugars (glucose, sucrose, maltose, starch etc), organic acids, such as acetic acid, citric acid and succinic acid, or alcohols, such as methanol or ethanol. In some cases, depending on the requirement of the organism(s) used in the cultivation process the carbon source(s) is added as gases to the fermentation liquid such as methane, natural gas, biogas, ethane, propane, butane, or carbon dioxide, and also non-carbon source such as hydrogen gas (H2) and ammonia gas is required by some organisms.

[0008] A large amount of energy is typically used in conventional fermentation tanks to ensure that enough of the gaseous substrate(s) is dissolved in the fermentation fluid.

[0009] DESCRIPTION OF THE DISCLOSURE

[0010] It is an object of embodiments of the disclosure to provide an improved fermentor. It is an object of embodiments of the disclosure to provide an improved method of performing a fermentation process.

[0011] According to a first aspect, the invention provides a fermentor comprising :

[0012] - a fluid containment space forming a closed loop flow path for a fermentation fluid;

[0013] - at least one substrate inlet for inlet of a substrate liquid to the space;

[0014] - at least one gas injection point for injection of gas into the fermentation fluid;

[0015] - a fermentation outlet for withdrawing fermentation fluid from the space;

[0016] - at least one circulation member for circulation of the fermentation fluid in the flow path; wherein the flow path has a total flow length being a distance along the flow path from a starting point to an end point, wherein at least a part of the flow path forms a meandering section comprising serially connected sections of a first group of sections and sections of a second group of sections, wherein the first group of sections has a predominantly vertical extend and the second group of sections has a predominantly horizontal extend, the first group of sections comprising at least four sections, and the second group of section comprising at least three sections, and wherein sections of the first group of sections are arranged alternately sections of the second group of sections, wherein the predominantly vertical extend is in the range of 50-130 degrees from horizontal, and wherein the predominantly horizontal extend is in the range of + / -40 degrees from horizontal, and wherein the sections of the first group of sections have a total length exceeding 50% of the total flow length.

[0017] The fermentor is a reactor suitable for conducting a fermentation process, where the fermentation process may be defined as the growth or maintenance of living cells under aerobic, anaerobic, or partially aerobic conditions such that a desired product can be obtained. The desired product may be the cells themselves, or substances produced by the cells, or converted by the cells. The fermenter comprises a fluid containment space forming a flow path for a fermentation fluid. The fermentation fluid comprises at least one fermenting microorganism and may be a fermentation liquid or a fermentation gas. Fermentation of the microorganisms may be carried out using pure cultures of only one fermenting microorganism species or using blends / a consortium of different microorganisms. The flow path may form a closed loop to allow circulation of the fermentation fluid in the closed loop, such as continuous circulation. During the circulation of the fermentation fluid, gaseous and / or liquid substances may be added to the fluid containment space, and / or portions of the fermentation fluid may be withdrawn from the fluid containment space.

[0018] As an example, the least one fermenting microorganism may be a yeast, a filamentous fungus, or a bacteria. The at least one fermenting microorganism may be selected from a group comprising at least Saccharomyces cerevisiae, Saccharomyces boulardii, Candida tropicalis, Candida utilis, Kluyveromyces lactis, Pichia pastoris, Brettanomyces, Yarrowia lipolytica, Schizosaccharomyces pombe, Hansenula poiymorpha, Torulaspora delbrueckii, Zygosaccharomyces rouxii, Aspergillus niger, Aspergillus oryzae, Aspergillus nidulans, Aspergillus sojae, Trichoderma reesei, Penicillium species, Fusarium species, Rhizopus species, Neurospora crassa, Mucor species, Acremonium chrysogenum (Penicillium chrysogenum), Myceliophthora thermophila (Sporotrichum thermophile), Beauveria bassiana, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus bulgaricus, Lactobacillus easel, Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sake!, Lactococcus lactis, Streptococcus thermophilus, Methylococcus capsulatus, Methylosinus trichosporium, Methylocystis species, Methylocella Silvestris, Methylocystis hirsute, Methylomicrobium album, Methylomonas species, Methylosinus sporium, Methylothermus thermalis, Methylobacillus flagellates, Methylobacterium extorquens, Methylophilus methylotrophus, Paracoccus denitrificans, Pseudomonas putida, Xanthobacter autotrophicus, Bacillus species, Escherichia coli, Desulfovibrio species, Methanogenic Archaea, Thermoplasma species, Acetobacterium woodie, Aquifex aeolicus, Hydrogenophilus thermoluteolus, Alcaligenes acidovorans, Aneurinibacillus danicus, Acetobacter aceti, Gluconobacter species, Clostridium species, Propionibacterium species, Mycoplasma species, Pediococcus species, Shewanella putrefaciens, Campylobacter species, Clostridium butyricum, Geobacillus species, Enterococcus species. Other fermenting microorganisms may also be selected.

[0019] A product of the fermentation process may be at least one of biomass, single cell protein, gasses, enzymes, peptides, hormones, vitamins, organic acids, chemical precursors, alcohols, and other metabolites or organic chemicals. The fermenter comprises at least one substrate inlet for inlet of a substrate liquid to the space. The substrate liquid is added as a source for growth for the fermenting microorganism(s). In one embodiment, the substrate liquid may comprise substrates, such as sugars (glucose, sucrose, maltose, starch etc.), organic acids, such as acetic acid, citric acid and succinic acid, or alcohols, such as methanol or ethanol. Other substrates may additionally or alternatively be added. The substrate liquid may further comprise inorganic salts and trace metals. The substrate inlet may comprise a closing mechanism for opening and closing the substrate inlet. In one embodiment, the closing mechanism may be configured for manual operation. The closing mechanism may alternatively or additionally be configured for automatic operation. Adjustment, such as closing and / or opening of the substrate inlet may be carried out in response to a computer implemented setpoint and / or a computer generated setpoint.

[0020] The at least one substrate inlet may further be used to add e.g., pH adjusting chemicals to improve the fermentation conditions for the microorganism(s). To monitor the conditions of the fermentation fluid provided in the space, the fermentor may further comprise at least on pH sensor and / or at least one temperature sensor arranged in the fluid containment space.

[0021] The fermentor further comprises at least one gas injection point for injection of gas into the fermentation fluid. The gas may be a substrate gas for growth for the microorganism(s). In one embodiment, the gas may comprise at least one of atmospheric air, pure oxygen, oxygen enriched atmospheric air, methane, natural gas, biogas, ethane, propane, butane, carbon dioxide, carbon monoxide, hydrogen (H2), and ammonia gas. Dependent on the microorganism(s) and / or the product, other gasses may alternatively be used, or may be used in addition hereto.

[0022] At least one of the at least one gas injection points may be configured for injection of a pressurized gas. This may facilitate increase of pressure in the tubular part of the flow path.

[0023] The fermentor comprises a fermentation outlet for withdrawing fermentation fluid from the space. The withdrawn fermentation fluid may comprise the production of the fermentation product. In one embodiment, the fermentor may comprise a recirculation inlet which may be configured for inlet of a part of the withdrawn fermentation fluid back to the fluid containment space. In another embodiment, the fermentation outlet may be in fluid communication with the substrate inlet whereby a part of the withdrawn fermentation liquid may enter the fluid containment space with the substrate inlet.

[0024] In one embodiment, the withdrawn fermentation liquid with its content of biomass, dissolved gasses, dissolved metabolites and other extracellular chemicals and enzymes may be pumped to a reservoir tank from the fermentation outlet. After the tank, the fermentation liquid may be passed to a separator, such as a centrifuge, a decanter, or a filtration unit, e.g., a filter drum for separation into a thick fraction rich in biomass and a thin fraction (supernatant) which may contain most of the extracellular and dissolved components and minerals. Depended on the final value-added product, the thick fraction may be further processed to obtain a final protein product or fractionated further into nucleic acids, vitamins, fatty acids or other value-added cellular components. The thin supernatant fraction which may be rich in minerals and nutrients may either be recirculated into the fermentor, or the content of extracellular enzymes, peptides, vitamins, hormones, organic acids, and other dissolved molecules may be further purified and up-concentrated.

[0025] The fermentor comprises at least one circulation member for circulation of the fermentation fluid in the flow path. As an example, the at least one circulation member may be an in-line pump arranged in the flow path. At least one of the at least one circulation member may comprise a pump, such as a propeller pump, a lobe pump, or a turbine pump, which may also act as a pressure controlling means in the fermentor.

[0026] The fermentor may further comprise at least one mixing element for dispersion of gas injected in the fermentation fluid. The mixing element may as an example be static mixer. In an alternative embodiment, the mixer may be a dynamic mixer. It should be understood, that a fermentor may comprise one or more static mixer and / or one or more dynamic mixers. The dispersion of gas may facilitate microbial growth, as gas may be made accessible for the microorganisms when dissolved in the fermentation fluid. The fermentation process may be enhanced by providing a plurality of mixing elements along the flow path, as the transfer of gaseous substrates to the fermentation fluid may often be a limiting step for rate of the metabolism and growth of the organisms. Thus, the limiting step may be counteracted or partly counteracted by the application of a plurality of mixing elements along the flow path. This may solve or at least partly solve a problem of solubilise enough of the gaseous nutrients in the fermentation fluid at a sufficiently high rate to satisfy the demands of the microorganisms.

[0027] The transfer rate of gaseous substances from the gas phase into the liquid phase may be improved if very small bubbles are used for the gas injection or if the pressure inside the fermentor is above atmospheric pressure, or if the temperature in the fermentation fluid is reduced. Reducing the temperature significantly may however often lead to a slower growth and thereby a lower productivity of the fermentation process. Gas bubbles in a liquid tend to fuse together to larger bubbles leading to a decrease of the transfer rate of gaseous substances from the gas phase into the liquid phase. This may be counteracted by the application of a plurality of mixing elements along the flow path.

[0028] The flow path has a total flow length being a distance along the flow path from a starting point to an end point. As the flow path may form a closed loop, the starting point and the end point of the flow path may in one embodiment be at the same position along the flow path.

[0029] At least a part of the flow path forms a meandering section comprising serially connected sections of a first group of sections and sections of a second group of sections, wherein the first group of sections has a predominantly vertical extend and the second group of sections has a predominantly horizontal extend. The predominantly vertical extend is in the range of 50-130 degrees from horizontal, and the predominantly horizontal extend is in the range of + / -40 degrees from horizontal.

[0030] By a predominantly vertical extend being in the range of 50-130 degrees from horizontal should be understood, that a tangent to a centre line through the respective section is in the range of 50-130 degrees from horizontal. Likewise, by a predominantly horizontal extend being in the range of + / -40 degrees from horizontal should be understood, that a tangent to a centre line through the respective section is in the range of + / -40 degrees from horizontal.

[0031] The first group of sections comprises at least four sections, and the second group of section comprises at least three sections, where sections of the first group of sections are arranged alternately sections of the second group of sections.

[0032] As an example, the fermentor may comprise four substantially vertical sections and three substantially horizontal section being alternatingly arranged. The first substantially vertical section may at one end be attached to the last (fourth) substantially vertical section by an additional substantially horizontal section. In an alternative embodiment, the first substantially vertical section may at one end be attached to the last (fourth) substantially vertical section by a tank. In a further example, the fermentor may comprise four sections being arranged at an angle of 50 degrees from horizontal and three substantially horizontal section being alternatingly arranged.

[0033] It should be understood that that the at least four sections having predominantly vertical extend being in the range of 50-130 degrees from horizontal may be arranged at different angles from horizontal; i.e., the angle of each of the at least four sections need not be the same. It should further be understood that that the at least three sections having predominantly horizontal extend being in the range of + / -40 degrees from horizontal may be arranged at different angles from horizontal; i.e., the angle of each of the at least three sections need not be the same.

[0034] The sections of the first group of sections have a total length exceeding 70% of the total flow length, such as exceeding 75%, such as exceeding 80%, such as exceeding 85%, or even exceeding 90% of the total flow length.

[0035] As the risk of gas-liquid phase separation may be predominant in the second group of sections; i.e. sections having a horizontal extend, or an extend closer to horizontal, this risk may be considerably reduced when the sections of the first group of sections have a total length exceeding 70% of the total flow length. The higher hydrostatic pressure in the first group of sections and the lower tendency of phase separation in the first group of sections may result in a gas to liquid mass transfer being 3-5 times more efficient in the first group of sections than in the second group of sections.

[0036] To further reduce the risk of gas-liquid phase separation, the length of each of the sections in the second group may be less than six times the diameter of the sections in the second group, such as less than five times the diameter, such as less than four times the diameter, such as less than three times the diameter, such as less than twice the diameter. In the context of the present disclosure, the term 'diameter' should be understood as the largest dimension of the tubular part transverse to the flow path.

[0037] Furthermore, by providing a fermentor of a first group of sections comprising at least four sections, and a second group of section comprising at least three sections, the total height of the fermentor may be reduced compared a U-shaped fermentor having the same volume, same diameter of the tubular sections, and comprising two substantially vertical sections connected by a single horizontal section, where height is the dimension of the fermentor from ground level to the highest point above ground level along a vertical line.

[0038] Additionally, by maintaining the diameter of the tubular part and maintaining the velocity of the fluid flow along the flow path, it may be possible to e.g., double the volume of the flow path without the need for one or more considerably larger circulation pumps, and without requiring a considerably larger material thickness for tubes / pipes which may be used to form the flow path.

[0039] The volume may be increased by adding one or more sections to the first and second groups of sections being alternately arranged. This may increase the length of the flow path and thereby the contact time between gas and fermentation fluid which may increase utilization of gaseous nutrients. This flexible design of alternating sections may further facilitate expansion of an existing fermentor as one or more sections may be added to the first and second groups of sections. When increasing the length of the flow path by adding more alternating sections, the average pressure in the flow path may be increased (see also Example 1), increasing the gas-liquid mass transfer and thereby the efficiency of the fermentor.

[0040] In one embodiment, the fermentor may comprise four substantially vertical sections and three substantially horizontal section being alternatingly arranged. A substantially vertical sections may be attached to a first end of a substantially horizontal section via a bend. However, a first part of the bend may belong to the first group of sections whereas a second part of the bend may belong to the second group of sections, as a tangent to a centre line through the first part may be in the range of 50 degrees from horizontal, and a tangent to a centre line through the second part may be in the range of + / -40 degrees from horizontal. A second bend may connect the second end of the substantially horizontal section to a second substantially vertical section. To minimise the length of the second group of sections, in this embodiment, the substantially horizontal sections, two substantially vertical sections may be attached to each other via two bends, where the substantially horizontal section; i.e. a section of the second group, is formed by parts of the two bends forming the transition from one section of the first group of sections to a second section of the first group of sections. In this embodiment, the section having a substantially horizontal extend may be formed by the short transition part form one section having a substantially vertical extend to the next section having a substantially vertical extend.

[0041] In one embodiment, the fluid containment space may be constituted by a tank and a tubular part being in fluid connection, where a first outlet connects the tank to the tubular part to provide an outlet flow path for the fermentation fluid from the tank to the tubular part, and a first inlet connects the tubular part to the tank to provide an inlet flow path for the fermentation fluid from the tubular part to the tank. The part of the flow path forming a meandering section may be formed by the tubular part or a part of the tubular part. The tubular part may be formed by a plurality of tubes / pipes being attached to each other to form a hollow path for the fermentation fluid. In the tubular part, a long contact time between gas and the fermentation fluid may be achieved, as gas may be present in both the first group and the second group of sections. This may increase the utilization of gas in the fermentation fluid.

[0042] The tank may be arranged above the tubular part relative to ground level upon which the fermentor may be erected. In one embodiment, the first inlet and the first outlet may be arranged at a bottom part of the tank, where the bottom part faces the ground. This may facilitate a flow out of the tank into the tubular part and a flow from the tubular part into the tank. In the tank, excess waste gasses, such as metabolically produced CO2 may be released from the liquid.

[0043] The fermentor comprising a fluid containment space comprising a tank and a tubular part may have a horizontal extend corresponding substantially to the horizontal extend of the tank, as all the meandering sections or at least a considerably part of the meandering sections may be located below the tank.

[0044] A first circulation member may be arranged in the flow path. The first circulation member may be arranged in a first section of the first group of sections, the first section being the section immediately after the first outlet. By arranging the first circulation member at an upper end of the first section, the first circulation member may secure a flow from the tank to the tubular part.

[0045] A first gas injection point may additionally be arranged at an upper end of the first section of the first group of sections having a predominantly vertical extend. Thereby, it may be possible to obtain a nearly non-compressed injection of the gas into the fermentation fluid with less need for compressors as the gas only has to overcome a hydrostatic pressure of e.g., a few meters.

[0046] The first circulation member may as an example be arranged between the first outlet and the first gas injection point.

[0047] The fermentor may further comprise a first pressure controlling device for controlling a pressure in the flow path. The first pressure controlling device may be arranged in a last section of the first group of sections, the last section being the section immediately before the first inlet to the tank. By arranging the first pressure controlling device close to the first inlet into the tank, the first pressure controlling device may assist in maintaining a pressure in the tubular part higher than atmospheric pressure and higher than in the tank. The first pressure controlling device may further assist in maintaining a pressure in the tank being lower than the pressure in the tubular part to allow efficient degassing of the fermentation fluid in the tank.

[0048] The pressure in the tank may be higher than atmospheric pressure, such as 0.5 - 5 bar, thereby adding to the pressure in the tubular parts of the fermentor to further increase the transfer of gaseous nutrients from the gas phase to the liquid phase.

[0049] The first pressure controlling device may as an example comprise at least one of a valve, such as pressure-controlled valve, a static mixer, a hydrocyclone, a pump, such as a propeller pump, a plate with holes, a narrowing of the diameter or the cross-section of the tubular part in which the first pressure controlling device is placed.

[0050] The fermentor may further comprise ion sensor(s) or analyser(s) for sensing at least one of the ions phosphate, sulphate, ammonium, nitrate, and hydrogen ions (pH), oxygen sensor(s) for measuring the dissolved oxygen concentration, and at least one thermo-sensor for sensing the temperature. The sensor(s) may be provided in the flow path in which the fermentation fluid is circulated. In a preferred embodiment, the sensor(s) may be arranged in the area of the substrate inlet and / or the gas injection point(s) or in by-pass arrangements attached thereto.

[0051] The sensor(s) and analyser(s) may deliverer signals to a data processing system, where the received signals optionally are processed. The flow of injected gas(es), water, minerals, and pH controlling means supplied via the substrate inlet and / or the gas injection point(s) may be calculated and may be optimised via pre-programmed amounts. Additionally, or alternatively, the temperature may be adjusted by a temperature regulating device, such as a heating / cooling jacket which as an example may be mounted close to the first inlet into the tank. The jacket may have fittings for circulating heating or cooling media through the jacket. In a further alternative, the temperature may be adjusted by application of a by-pass stream to thereby provide the possibility of cooling the fermentation fluid.

[0052] The fermentation outlet for withdrawing fermentation fluid from the fluid containment space may preferably be arranged in the tank. However, in an alternative embodiment, the fermentation outlet may be arranged in the tubular part, such as at a lower part of one of the sections.

[0053] A cross-sectional shape of the tubular part transverse to the flow path may be substantially circular or oval to increase the volume of the flow path relative to the flow direction. In an alternative embodiment, the cross-sectional shape may be polygonal, rectangular, hexagonal, or of another shape.

[0054] A cross-sectional shape of the flow path may be substantially uniform along sections of the flow path, such as along the meandering section. As an example, a flow path having a cross- sectional shape being circular may have a constant diameter along most of the sections. However, at least one section of the flow path may additionally comprise one or more sections having a conical shape or have one or more sections with shorter or longer zones being narrowed or broadened, as this may be used to regulate the pressure along the flow path. In one embodiment, the inner volume of the flow path may be in the range of 1 litre to 1000 m3, preferably in the range of 0.2-500 m3, such as in the range of 25-250 m3.

[0055] In one embodiment, at least a part of the meandering section may form a helical shape.

[0056] The fermentor may further comprise an additional opening. The additional opening may be provided in an upper part of the flow path for adding and / or removal of gas, e.g., for degassing of CO2 and / or excess CH4 and / or other gases such as CO, H2, etc. As an example, the additional opening may be placed above the liquid surface in the tank which may create a gas flow for flushing a volume in the tank above the liquid surface (the headspace) co- currently, con-currently, or cross-currently to the liquid flow in the tank.

[0057] The fermentor may further comprise a plurality of flow modification elements arranged in the flow path. The flow modification elements may be configured for changing a flow of the fermentation fluid. The flow modification elements may preferably be arranged in the tubular part. As an example, the flow modification element may comprise static or dynamic mixers for efficient dispersion of the gases in the liquid. The static or dynamic mixers may be placed in the area of the gas injectors, such as immediate below. The static or dynamic mixers may be arranged at regular intervals along the first sections of the flow path. The flow rate of the fermentation fluid in the flow path may be adjusted to be sufficiently high to carry the injected gas through the static mixer and through the flow path. The gas bubbles may thus be carried with the fermentation fluid through a downstream first section to the lower end and further through a second section to an upstream first section so that the gas bubbles are redispersed by means of the regularly spaced static mixing elements several times in the liquid. Alternatively or additionally, the flow modification element(s) may be one or more sections of the tubular part having a smaller diameter, as narrowing the diameter of the tubular part of the flow path may also modify the flow through the tubular part.

[0058] According to a second aspect, the invention provides a method of performing a fermentation process in a fermentor comprising :

[0059] - a fluid containment space forming a closed loop flow path for a fermentation fluid;

[0060] - at least one substrate inlet for inlet of a substrate liquid to the space;

[0061] - at least one gas injection point for injection of gas into the fermentation fluid;

[0062] - a fermentation outlet for withdrawing fermentation fluid from the space; - at least one circulation member for circulation of the fermentation fluid in the flow path; wherein the flow path has a total flow length being a distance along the flow path from a starting point to an end point, wherein at least a part of the flow path forms a meandering section comprising serially connected sections of a first group of sections and sections of a second group of sections, wherein the first group of sections has a predominantly vertical extend and the second group of sections has a predominantly horizontal extend, the first group of sections comprising at least four sections, and the second group of section comprising at least three sections, and wherein sections of the first group of sections are arranged alternately sections of the second group of sections, wherein the predominantly vertical extend is in the range of 50-130 degrees from horizontal, and wherein the predominantly horizontal extend is in the range of + / -40 degrees from horizontal, and wherein the sections of the first group of sections have a total length exceeding 50% of the total flow length, the fermentation fluid comprising at least one fermenting microorganism, the method comprising the steps of:

[0063] - adding a substate liquid to the fermentation fluid in the space;

[0064] - injecting gas into the fermentation fluid;

[0065] - circulating the fermentation fluid along the flow path;

[0066] - withdrawing a portion of the fermentation fluid via the fermentation outlet, wherein the method further comprises a first step of increasing pressure in the space in a first zone relative to a pressure in a second zone in the space to increase mass transfer of the gas.

[0067] The substrate liquid may be added via the at least one substrate inlet. The substrate liquid may comprise substrates, such as sugars, and may comprise nutrient salts, pH adjusting chemicals. Furthermore, the temperature of the fermentation fluid may be regulated during circulation in the flow path. And cultivating may occur while the fermentation fluid is circulated. The second zone may be a tank forming part of the fluid containment space. The first zone may be a part of the tubular part.

[0068] It should be understood, that a skilled person would readily recognise that any feature described in combination with the first aspect of the disclosure could also be combined with the second aspect of the disclosure, and vice versa.

[0069] The fermentor according to the first aspect of the disclosure is very suitable for performing the method steps according to the second aspect of the disclosure. The remarks set forth above in relation to the fermentor are therefore equally applicable in relation to the method.

[0070] The method may further comprise a second subsequent step of decreasing pressure in the second zone relative to the pressure in the first zone to initiate release of trapped gas from the fermentation fluid in the tank. The released gas may be removed from the tank via a valve.

[0071] The method may comprise an additional step of recycling a part of withdrawn fermentation fluid to the space. The fermentation fluid may be withdrawn trough the fermentation outlet. The withdrawn fermentation liquid with its content of biomass, dissolved gasses, dissolved metabolites and other extracellular chemicals and enzymes may be pumped to a reservoir tank from the fermentation outlet. After the tank, the fermentation liquid may be passed to a separator for separation into a thick fraction rich in biomass and a thin fraction (supernatant) which may contain most of the extracellular and dissolved components and minerals. In an alternative embodiment, the withdrawn fermentation liquid with its content may be pumped directly to a separator. In one embodiment, the thin supernatant fraction which may be rich in minerals and nutrients may be recirculated into the fermentor.

[0072] The method may comprise a step of controlling pressure in the flow path at a level above atmospheric pressure by increasing pressure and by closing the fermentation outlet. The increased pressure in the flow path; i.e., in the tubular part and / or in the tank may increase mass transfer of the at least one injected gas from the gas phase to the liquid phase in the flow path. By closing the fermentation outlet, e.g., by a valve removal of gaseous components from the flow path may be avoided.

[0073] The gas injected via the at least one gas injection points may comprise at least one of atmospheric air, pure oxygen, oxygen enriched atmospheric air, methane, natural gas, biogas, ethane, propane, butane, carbon dioxide, carbon monoxide, hydrogen (H2), and ammonia gas. The at least one microorganism may belong to a group of organisms capable of metabolising carbon dioxide and / or hydrogen.

[0074] BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Embodiments of the disclosure will now be further described with reference to the drawings, in which:

[0076] Fig. 1 illustrates an embodiment of a fermentor;

[0077] Fig. 2 illustrates an alternative embodiment of a fermentor;

[0078] Fig. 3 illustrates a U-shaped fermentor;

[0079] Fig. 4 illustrates an embodiment of a fermentor;

[0080] Fig. 5 illustrates gas-liquid phase separation in a U-shaped fermentor as illustrated in Fig. 3;

[0081] Fig. 6 illustrates gas-liquid phase separation in a fermentor as illustrated in Fig. 4;

[0082] Fig. 7 illustrates a comparison of non-uniformity in a U-shaped fermentor as illustrated in Fig. 3 and a fermentor as illustrated in Fig. 4;

[0083] Figs. 8A and 8B illustrate comparisons of pressure in a U-shaped fermentor as illustrated in Fig. 3 and a fermentor as illustrated in Fig. 4;

[0084] Fig. 9 illustrates process data from a U-shaped fermentor as illustrated in Fig. 3; and

[0085] Fig. 10 illustrates process data from a fermentor as illustrated in Fig. 4.

[0086] DETAILED DESCRIPTION OF THE DRAWINGS

[0087] It should be understood that the detailed description and specific examples, while indicating embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the scope of the disclosure will become apparent to those skilled in the art from this detailed description. Fig. 1 illustrates an embodiment of a fermentor 1, and Fig. 2 illustrates an alternative embodiment of a fermentor 1.

[0088] The fermentor 1 comprises a fluid containment space 2 forming a closed loop flow path for a fermentation fluid. The flow direction in the flow path is indicated by the arrows. In both illustrated embodiments, the fluid containment space 2 is constituted by a tank 4 and a tubular part 6 being in fluid connection and forming a flow path for the fermentation fluid from the tank 4 to the tubular part 6 and back to the tank 4.

[0089] The fermentation fluid comprises at least one fermenting microorganism which may be a yeast, a filamentous fungus, or a bacteria. The at least one fermenting microorganism may be selected from a group comprising at least Saccharomyces cerevisiae, Saccharomyces boulardii, Candida tropicalis, Candida utilis, Kluyveromyces lactis, Pichia pastoris, Brettanomyces, Yarrowia lipolytica, Schizosaccharomyces pombe, Hansenula poiymorpha, Torulaspora delbrueckii, Zygosaccharomyces rouxii, Aspergillus niger, Aspergillus oryzae, Aspergillus nidulans, Aspergillus sojae, Trichoderma reesei, Penicillium species, Fusarium species, Rhizopus species, Neurospora crassa, Mucor species, Acremonium chrysogenum (Penicillium chrysogenum), Myceliophthora thermophila (Sporotrichum thermophile), Beauveria bassiana, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus bulgaricus, Lactobacillus easel, Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sake!, Lactococcus lactis, Streptococcus thermophilus, Methylococcus capsulatus, Methylosinus trichosporium, Methylocystis species, Methylocella Silvestris, Methylocystis hirsute, Methylomicrobium album, Methylomonas species, Methylosinus sporium, Methylothermus thermalis, Methylobacillus flagellates, Methylobacterium extorquens, Methylophilus methylotrophus, Paracoccus denitrificans, Pseudomonas putida, Xanthobacter autotrophicus, Bacillus species, Escherichia coli, Desulfovibrio species, Methanogenic Archaea, Thermoplasma species, Aceto bacterium woodie, Aquifex aeolicus, Hydrogenophilus thermoluteolus, Alcaligenes acidovorans, Aneurinibacillus danicus, Acetobacter aceti, Gluconobacter species, Clostridium species, Propionibacterium species, Mycoplasma species, Pediococcus species, Shewanella putrefaciens, Campylobacter species, Clostridium butyricum, Geobacillus species, Enterococcus species. Other fermenting microorganisms may also be selected.

[0090] The fermentor 1 further comprises at least one substrate inlet 8 for inlet of a substrate liquid to the space 2 and at least one gas injection point 10 for injection of gas into the fermentation fluid.

[0091] The substrate liquid is added as a source for growth for the fermenting microorganism(s).

[0092] The substrate liquid may comprise substrates, such as sugars (glucose, sucrose, maltose, starch etc.), organic acids, such as acetic acid, citric acid and succinic acid, or alcohols, such as methanol or ethanol. Other substrates may additionally or alternatively be added. The substrate liquid may further comprise inorganic salts and trace metals.

[0093] The substrate inlet 8 may further be used to add e.g., pH adjusting chemicals to improve the fermentation conditions for the microorganism(s).

[0094] The at least one gas injection point 10 is used for injection of gas into the fermentation fluid. The gas may be a substrate gas for growth for the microorganism(s). The gas may comprise at least one of atmospheric air, pure oxygen, oxygen enriched atmospheric air, methane, natural gas, biogas, ethane, propane, butane, carbon dioxide, carbon monoxide, hydrogen (H2), and ammonia gas.

[0095] The fermentor 1 further comprises a fermentation outlet 12 for withdrawing fermentation fluid from the space 2. In the illustrated embodiments, the fermentation outlet 12 is placed in the bottom of the tank 2. The product of the fermentation process may be at least one of biomass, single cell protein, gasses, enzymes, peptides, hormones, vitamins, organic acids, chemical precursors, alcohols, and other metabolites or organic chemicals, which product may be withdrawn from the fermentor 1 with the withdrawn fermentation fluid.

[0096] The fermentor 1 comprises a circulation member 14 for circulation of the fermentation fluid in the flow path. In the illustrated embodiments, the circulation member 14 is an in-line pump arranged in the flow path. The circulation member 14 further acts as a pressure controlling means in the fermentor 1.

[0097] The fermentor 1 further comprises a plurality of mixing elements 16 for dispersion of gas injected in the fermentation fluid. In the illustrated embodiments, the plurality of mixing elements is in the form of static mixers.

[0098] A first outlet 18 connects the tank 4 to the tubular part 6 to provide an outlet flow path for the fermentation fluid from the tank 4 to the tubular part 6, and a first inlet 20 connecting the tubular part 6 to the tank 4 to provide an inlet flow path for the fermentation fluid from the tubular part 6 to the tank 4.

[0099] At least a part of the flow path forms a meandering section comprising serially connected sections 22 of a first group of sections and sections 24 of a second group of sections, where the first group of sections has a predominantly vertical extend and the second group of sections has a predominantly horizontal extend, where the predominantly vertical extend is in the range of 50-130 degrees from horizontal, and where the predominantly horizontal extend is in the range of + / -40 degrees from horizontal. The first group of sections comprises at least four sections 22, and the second group of sections comprises at least three sections 24. The sections 22 of the first group of sections are arranged alternately sections 24 of the second group of sections.

[0100] In the illustrated embodiment in Fig. 1, the fermentor 1 comprises four substantially vertical sections 22 belonging to the first group of sections and three substantially horizontal sections 24 belonging to the second group of sections, the sections 22,24 of the first and section group being alternatingly arranged. In the illustrated embodiment in Fig.2, the fermentor 1 comprises six substantially vertical sections 22 belonging to the first group of sections and five substantially horizontal sections 24 belonging to the second group of sections, the sections 22,24 of the first and section group being alternatingly arranged.

[0101] In both embodiments, a substantially vertical section 22 is attached to a first end of a substantially horizontal section 24 via a bend 26. A first part of the bend 26 may belong to the first group of sections whereas a second part of the bend 26 may belong to the second group of sections, as a tangent to a centre line through the first part may be in the range of 50-130 degrees from horizontal, and a tangent to a centre line through the second part may be in the range of + / -40 degrees from horizontal. A second bend 26 connects the second end of the substantially horizontal section 24 to a second substantially vertical section 22.

[0102] The flow path has a total flow length being a distance along the flow path from a starting point to an end point, where the starting point may be at the outlet 18, and the end point may be at the inlet 20. The sections 22 of the first group of sections have a total length exceeding 70% of the total flow length.

[0103] To monitor the conditions of the fermentation fluid provided int the space 2, the fermentor 1 further comprises at least on pH sensor 28 and at least one temperature sensor 30 arranged in the fluid containment space 2.

[0104] A temperature regulating device 32 in the form of a heating / cooling jacket is mounted at a substantially vertical section 24 close to the inlet 20 to the tank 4.

[0105] A pressure controlling device 34 is arranged in the last of the substantially vertical section 24 close to the inlet 20. In the illustrated embodiments, the pressure controlling device 34 is in the form of narrowing the diameter of a part of the section 24. In addition to the circulation member 14 and the pressure controlling device 34, the pressure in the flow path may be increased by injection of pressurized air or another gas via the gas injection point 10. Gas separation in the tank 4 may be improved by flushing the volume above the liquid surface in the tank 4 using a flushing gas via a flushing valve 36 arranged in the upper part of the tank 4.

[0106] A degassing valve 38 in the tank 4 may be used to release gas(es) from the tank, which gas(es) may have been released from the fermentation liquid.

[0107] EXAMPLES

[0108] The following examples describes three different uses and applications of the present disclosure.

[0109] Example 1

[0110] Example 1 describes Single Cell Protein (SCP) production by fermentation of Methylococcus capsulatus optionally together with Alcaligenes acidovorans, Aneurinibacillus danicus, and Bacillus firmus, using methane, natural gas, or biogas as carbon and energy source. Ammonia gas is used as nitrogen source. In addition to these gaseous substrates, the cultivation of M. capsulatus requires water, phosphate, sulphate, and several minerals including calcium, potassium, iron, copper, and others (added as sulphates, phosphates, nitrates, or chlorides in a mineral mixture). Sodium hydroxide and sulphuric acid are used for pH adjustment.

[0111] The overall biological reaction to produce biomass looks like the below stoichiometry: a x CH4+ b x O2 + c x NH3 CHi.6sOo.36No.22 (biomass) + d x CO2 + e x H2O

[0112] The two major inputs (methane and oxygen) are present in the gas phase, while the microorganisms and the biological reactions are taking place in the liquid phase of the fermentor.

[0113] The transfer of methane and oxygen from the gas phase to the liquid phase is the main limitation of the productivity of biomass.

[0114] The above process is carried out in a U-shaped fermentor 100, as illustrated in Fig. 3 and not being in accordance with the present disclosure and in a fermentor 1 in accordance with the present disclosure where four additional vertical sections 22 and three additional horizontal sections 24 are introduced in the tubular flow part 6 of the fermentor 1 as shown in Fig. 4. The fermenter 1 illustrated in Fig. 4 and forming part of this example, thus comprises a flow path, where a part of the flow path forms a meandering section comprising serially connected sections 22 of a first group of sections and sections 24 of a second group of sections, wherein the first group of sections has a predominantly vertical extend and the second group of sections has a predominantly horizontal extend. The first group of sections comprises six sections 22, and the second group of sections comprises five sections 24, the sections of the first group of sections being arranged alternately sections of the second group of sections, and the sections of the first group of sections have a total length exceeding 70% of the total flow length. The fermentor 1 illustrated in Fig. 4 is similar to the fermentor 1 illustrated in Fig. 2. As three U-shaped sections are thus present in this fermentor 1 it will be designated the 3U fermentor or 3U bioreactor throughout this example. The height of the two fermentors 1, 110 (Fig. 3 and 4) is the same as well as the diameter of the tubular flow parts is the same. In this example, the additional vertical and horizontal sections increase the volume from 100 litres in the U-shaped fermentor to 170 litres in the 3U fermentor.

[0115] CFD simulations have been carried out for the U-shaped fermentor 100 illustrated in Fig. 3 and the 3U fermentor 1 illustrated in Fig. 4. In the U-shaped fermentor 100, CFD simulations show that the large horizontal section 124 creates non-uniformity of the gas dispersion in the liquid phase leading to gas-liquid phase separation caused by the buoyancy of the methane and oxygen gas bubbles 150 as depicted in Fig. 5, where the substantially vertical sections 122 and the horizontal section 124 are schematically illustrated together with a normalised volume fraction. CFD simulations also show that at a position P in the horizontal part of the U-shaped loop at a distance in the flow direction from the vertical section corresponding to two diameters of the pipe, an almost entire phase separation has occurred. This results in a less efficient transfer of gasses in the horizontal zone.

[0116] In the 3U fermentor 1, the shorter horizontal sections of the second group of sections lead to lower non-uniformity shown by CFD simulations in Fig. 6, where the substantially vertical sections 22 and the horizontal section 24 are schematically illustrated together with a normalized volume fraction. The simulations show that the 3U fermentor 1 has a 40% lower non-uniformity over the length of the tubular part 6 compared to the U-shaped reactor as shown in Fig. 7 despite the 3U fermentor being 70% larger than the U-shape reactor. The lower non-uniformity in the 3U fermentor increases the gas-liquid transport capability and hence the mass transfer efficiency of the 3U design over the U-shape design. Fig. 7 illustrates a comparison of non-uniformity (in %) in a U-shaped 100 vs a fermentor 1 (3U) according to the present disclosure having six substantially vertical sections 22 (first group) and five substantially horizontal sections 24 (second group). The non-uniformity of the U-shaped fermentor is app. 11%, whereas the non-uniformity of 3U fermentor is app. 7%. Thus, the 3U design has 40% lower non-uniformity despite a 70% higher volume. Figs. 5 and 6 illustrate results of CFD simulations carried out for the U-shaped fermentor 100 illustrated in Fig. 3 and the 3U fermentor 1 illustrated in Fig. 4. The CFD simulations illustrate a normalised gas volume fraction from 0 to 5 as indicated in the indicator bar inserted below / above the schematically illustrated fermentors 1, 100, respectively. Fig. 5 further illustrates buoyancy of the gas bubbles 150 resulting in gas-liquid phase separation in the longer horizontal section 124 of the U-shapes fermentor 100.

[0117] In Fig. 5 illustrating CFD simulations for the U-shaped fermentor 100, the normalised volume fraction is below 1 in the volume Fl in the lower part of the horizontal part 124 of the U-loop (below the dotted line). In the major volume part F2 of the substantially vertical sections 122 of the U-loop, the normalised volume fraction is approximately 1. The upper part of the substantially horizontal part 124 comprises two volumes, an upper volume F4 extending along the upper wall section and an intermediate volume F3 extending along the upper volume, where the normalised volume fraction in the upper volume F4 is above 4, and where the normalised volume fraction in the intermediate volume F3 is between 3 and 4. The gas volume fraction is normalised with mean value, i.e., the perfect uniformity would give 1.0 in the entire volume.

[0118] In Fig. 6, the CFD simulation shows a low tendency for gas-liquid phase separation in a fermentor 1 according to the present disclosure which in this example has six vertical sections 22 and five horizontal sections 24. In Fig. 6 illustrating CFD simulations for the 3U fermentor 1, the normalised volume fraction is below 1 in the volume Fl in the lower part of the horizontal sections 24 (below the dotted lines). In the major volume part F2 of the substantially vertical sections 22, the normalised volume fraction is approximately 1. The upper part of the upper substantially horizontal parts 24 each comprises two volumes, an upper volume F4 at the upper wall section and a lower volume F3 along the upper volume, where the normalised volume fraction in the upper volume F4 is approximately 4, and where the normalised volume fraction in the intermediate volume F3 is between 2.75 and 4 In the lower part of the upper substantially horizontal parts 24, the normalised volume fraction is approximately 1. The gas volume fraction is normalised with mean value, i.e., the perfect uniformity would give 1.0 in the entire volume.

[0119] The horizontal white lines at the tubular parts of the 3U fermentor 1 indicate positions for elements which may be arranged along the flow path, such as static and / or dynamic mixers, etc. It should be understood that such elements may also be positioned at other positions along the flow path.

[0120] As the additional horizontal and vertical sections 24, 22 in the 3U design is placed in the lower part of the tubular part 6 of the fermentor 1, the average pressure will be 17% higher in the 3U fermenter 1 compared to the U-shaped fermenter 100. Figs. 8A and 8B illustrate CFD simulations comparing a 50 m3U-shaped fermenter 100 with an 85 m33U-shaped fermenter 1. The two fermenters are of the same height. The higher average pressure results in a higher driving force for gas-liquid mass transfer in the 3U design over the U-shaped fermenter. In Fig. 8A, the absolute pressure in each of the fermenters are illustrated with values indicated in the indicator bar inserted above the schematically illustrated fermenters 1, 100, respectively. In the U-shaped fermenter 100, the pressure at the upper part Pl' of the first substantially vertical leg 122 is in the range of 5.5 bar, whereas the pressure at the upper part P2' of the second substantially vertical leg 122 is in the range of 3 bar. The pressure decreases from the upper part Pl' of the first leg to the upper part P2' of the second leg. The average pressure of the U-shaped fermentor 100 is approximately 4.5 bar (see Fig. 8B). In the 3U fermentor 1, the pressure at the upper part Pl of the first substantially vertical leg 22 is in the range of 6 bar, whereas the pressure at the upper part P2 of the last substantially vertical leg 22 is in the range of 4 bar. The pressure decreases from the upper part Pl of the first leg to the upper part P2 of the last leg. The average pressure of the 3U fermentor 1 is approximately 5.4 bar (see Fig. 8B).

[0121] In Fig. 8A, the horizontal and vertical white lines at the tubular parts of the U-shaped and the 3U fermentor 100, 1 indicate positions for elements which may be arranged along the flow path, such as static and / or dynamic mixers, etc. It should be understood that such elements may also be positioned at other positions along the flow path.

[0122] In Fig. 8B, the average pressure (in bar) of the U-shaped fermentor 100 of this example is compared to the average pressure of the U3 fermentor 1 of this example, illustrating an average pressure being 17% higher in the 3U fermentor.

[0123] With an identical liquid flow velocity of 1 m / s for the fermentation fluid, the residence time in the 3U fermentor 1 is much higher compared to the U-shaped design 100. Higher residence time increases gas-to-liquid contact time and mass transfer becomes higher. In addition, the shorter substantially horizontal sections 24 (second group of sections) in the 3U bioreactor provides better phase flow compared to a two-phase flow observed in substantially horizontal part 124 of the U-shaped fermentor of the present example.

[0124] As the diameter of the tubular sections 6, 106 and the flow velocity in this example is identical for the U-shaped fermentor 100 and the 3U fermentor 1, the same pump types and pump sizes can be used for the two fermentors. In the present example, this reduces the electricity consumption by the circulation pump per fermentor volume in the 3U fermentor 1 compared to the U-shaped fermentor 100. After cleaning and sterilizing the two fermenters 1, 100 of the present example, the fermenters (see Figs. 3 and 4) are filled with water and the necessary nutrient salts and an inoculation culture of M capsulatus, optionally together with the above-mentioned cofermenting organisms, is added. A pressure of approximately 2 bars is applied via a gas injection point 10, 110 at the upper part of the tubular part 6, 106 of the fermentors 1, 100 close to the first outlet 18, 118 from the tank 4, 104 and liquid circulation is started by starting the first in-line pump 14, 114. The pressure is reduced to atmospheric pressure by the pressure controlling device 34, 134 before the circulating fermentation fluid enters the tank 4, 104. The feed gases (methane, natural gas, or biogas and ammonia, atmospheric air and oxygen) are then introduced into the fermentation liquid in the tubular part of the fermentor just below the first circulation member 14, 114 in the form of a pump, until a steady state with a biomass content of 1-4% (w / w) solids is obtained. Then withdrawal of fermentation liquid through the fermentation outlet (12, see Fig. 2) is initiated simultaneously with supplying water and the mineral solution at a dilution rate of 0.05 to 0.4 h-1. Then the supply of gases, nutrient salts and pH adjustment means is performed based on in-line measurements of ammonium, phosphate, nitrate, nitrite, oxygen, methane etc. by the sensors in the system.

[0125] In this example, carbon dioxide is produced by the fermenting organisms. To avoid building up inhibitory concentrations of carbon dioxide in the fermentation liquid, the carbon dioxide is separated from the fermentation liquid in the tank 4, 104 and released via an exhaust valve 38, 138. This degassing of the fermentation liquid ensures that the first circulating member 14, 114 acts on a fermentation fluid with a low content of gasses thereby increasing the efficiency of the circulating member.

[0126] During steady-state fermentation, oxygen saturation is maintained at a level below 10% measured at the end of the tubular flow section, the pH value is controlled to around pH 6.8, and the temperature is maintained around 43°C.

[0127] The fermenter dimensions, key process conditions and amounts of added and removed substances are shown in Table 1 below.

[0128] Table 1 : Example of Single Cell Protein (SCP) production in a 100L U-shaped fermentor compared to a fermentor (3U) according to the present disclosure with a volume of 170L

[0129] The obtained dry cell weight concentration and the dilution rate is the same in both above- mentioned fermentation processes (see Figs. 9 and 10). Thereby the volumetric productivity of biomass (kg / m3 / h) is the same in both fermentors 1, 100, and the total productivity (kg / h) in the 3U fermentor 1 is thus 70% higher than in the U-shaped fermentor 100. It is seen from Table 1 and Figs. 9 and 10, that the methane flow per fermentor volume (Nm3 / m3 / min) is lower in the 3U fermentor 1 compared to the U-shaped fermentor 100 meaning that the 3U fermentor is more efficient for converting methane to biomass. It is also noticed that the energy for the circulation pump is lower per fermentor volume in the 3U fermentor and thereby that a higher total productivity is obtained with less energy consumption.

[0130] Fig. 9 illustrates process data from a U-shaped fermentor 100 with a volume of 100L, whereas Fig. 10 illustrates process data from a 3U fermentor 1 with a volume of 170L.

[0131] The process data includes:

[0132] • A: DCW: Dry Cell Weight (g / l)

[0133] • B: Productivity: amount of product per unit time

[0134] • C: NH3 flow: inlet of ammonia gas (NL / min)

[0135] • D: Dilution rate (h-1)

[0136] • E: O2 flow: inlet of oxygen gas (NL / min)

[0137] • F: Methane flow: inlet of methane gas (NL / min) The process data for DCW (A), Productivity (B), Dilution rate (D), and O2 flow (E) are normalized according to minimum and maximum operating windows of the fermentors used for this example.

[0138] The fermentation fluid withdrawn from the fermentor 1, 100 is processed using standard downstream processing equipment including an inactivation step where >95% of the bacterial cells are inactivated to obtain a solid single cell protein powder having a content of solids (TS) of approximately 94% that optionally can be pelletized prior to further use.

[0139] The average composition values of the Single Cell Protein (SCP) product are shown in table 2.

[0140] Table 2: Average composition values of the Single Cell Protein (SCP) product.

[0141] Example 2

[0142] Example 2 describes production of an extracellular amylase by fermentation of a genetically modified strain of Bacillus licheniformis using glucose as carbon and energy source and compressed atmospheric air as an oxygen source, and ammonia gas is used as both nitrogen source and as a means for controlling pH. For this example, the genetically modified strain can convert up to 25% of the metabolized glucose into the extracellular amylase.

[0143] This type of process is most often carried out industrially in traditional fermentors, such as stirred tank bioreactors in batch, fed-batch or continuous mode, most typically in fed-batch mode where all needed nutrient salts and minerals are present in the fermentation liquid at the start of the fermentation, and where the carbon source, here glucose, is dosed into the fermentor in a fluid form, and compressed air and nitrogen are provided in gaseous form during the fermentation process to support the growth of the biomass and the production of the amylase according to the below generic stoichiometric equation: aC6Hi206 + PNH3 + 8O2 — * 8CH2O0.5N0.2 + cpCOz + yH20 + r|H Where CH2O0.5N0.2 represents cellular material, the extracellular amylase and other organic matter produced by the microorganism as it grows and replicates.

[0144] As the gas to liquid mass transfer is inefficient in a stirred tank reactor, the supply of oxygen to the microorganisms in the fermentation liquid becomes the limiting parameter for the microbial growth and the amylase production. To increase the availability of oxygen for the fermenting microorganisms in the form of a higher concentration of dissolved oxygen in the fermentation liquid, more oxygen via a higher flow of compressed air can be added via a sparger located at the bottom of the stirred tank reactor. As the compressed air is added at the bottom of the reactor where the hydrostatic pressure is highest, a significant amount of energy is consumed for the aeration of the fermentor, in the range of two-thirds of the total energy consumption for an industrial 100 m3reactor (cf. Ali Jahanian, Jerome Ramirez, Ian O'Hara, Advancing precision fermentation: Minimizing power demand of industrial scale bioreactors through mechanistic modelling, Computers & Chemical Engineering, Volume 188, 2024). Increasing the flowrate of compressed air further leads to a risk of flooding the agitator. In a stirred tank reactor, the agitator transfers energy via impellers to the fermentation liquid to ensure proper mixing of the liquid and to increase the gas-liquid mass transfer by means of breaking the air bubbles into smaller bubbles. When the volume fraction of air-bubbles increases, the efficiency of energy transfer from the agitator to the liquid decreases significantly (cf. Gassed and ungassed power draw in a pilot scale 550 litre fermentor retrofitted with up-pumping hydrofoil B2 impellers in media of different viscosity and with very high power draw - ScienceDirect) and eventually a situation of flooding can be obtained where almost no energy from the impellers are transferred to the liquid. A third means of increasing the gas-liquid mass transfer and hence the oxygen availability is to increase the pressure in the stirred tank reactor. This, however, will also increase the energy needed for aeration and energy for other inlets like sugars, acids and bases to overcome the pressure in the fermenter.

[0145] To avoid an accumulation of glucose in the fermentation liquid which, via the so-called glucose repression, can lead to inhibition of the amylase production, the dosing of glucose must be stoichiometrically balanced versus the availability of oxygen. This is typically carried out by using the signal from a dissolved oxygen probe to feed-back regulate the rate of the glucose dosing with a set-point of dissolved oxygen typically around 10%

[0146] In this example, the above process is carried out as a continuous fermentation in a tubular fermentor 1 (see Figs. 1, 2, and 4) where at least a part of the flow path 6 forms a meandering section comprising serially connected sections 22 of a first group of sections and sections 24 of a second group of sections, where the first group of sections has a predominantly vertical extend and the second group of sections has a predominantly horizontal extend, the first group of sections comprising at least four sections 22, and the second group of sections comprising at least three sections 24, and where sections of the first group of sections are arranged alternately sections of the second group of sections, the sections of the first group of sections having a total length exceeding 70% of the total flow length according to the present disclosure.

[0147] The fluid containment space is filled up with a liquid substrate containing a nitrogen source, the needed nutrient salts, vitamins and minerals required by the microorganism, including 1% w / w of glucose, and the fermentation fluid is inoculated with a pure preculture of the Bacillus licheniformis strain. The preculture can be obtained from a shake flask, a vial, or a laboratory fermentor, pH and temperature is adjusted to the optimal conditions for the microbial strain, the circulation pump 14 is started, and compressed air is continuously added to the reactor, whereas ammonia gas in this example is used to maintain the pH and not supplied at a fixed volumetric rate. When the microorganisms have started to grow, which can be evaluated by several parameters such as CO2 production, ammonia consumption, changes in dissolved oxygen concentration etc., a 80% w / w liquid solution of glucose as well as a liquid solution of remaining substrate components including nutrient salt, minerals and vitamins, is continuously being dosed into the fermentor, and fermentation fluid is withdrawn from the reactor in the same rate as glucose and nutrients are dosed into the reactor. As in Example 1, the carbon dioxide resulting from the metabolic consumption of carbon source (here glucose) is degassed in the tank 4 connecting the tubular sections 6 of the fermentor and released via a valve 38 in the tank 4.

[0148] As in the traditional stirred tank reactor, a concentration of dissolved oxygen in the reactor is maintained higher than 0% by controlling the dosing of glucose automatically based on the reading of a dissolved oxygen sensor with a setpoint for dissolved oxygen of 10%.

[0149] In contrast to the stirred tank reactor, the air is in this example introduced after the first circulating member 14 meaning that a high volumetric fraction of air can be added without the risk of flooding the circulation pump 14. The air is in the present example furthermore introduced in the beginning of the first substantially vertical tubular section 22 where the hydrostatic pressure is lowest, thereby reducing the energy needed for compression of the air. In a stirred tank reactor of 85 m3with a height of 8 m and a diameter of 3.7 m, the hydrostatic pressure at the bottom of the tank is 0.8 bar. If a headspace pressure of 0.5 bar is applied, the average pressure in the stirred tank reactor is 0.65 bar. In comparison, the average pressure in a fermentor 1 according to the present disclosure having a volume of 85 m3, has an average pressure of 5.4 bar as shown in Fig. 8B which is an important driving force for gas-liquid mass transfer because the solubility of a gas in equilibrium with a liquid is directly proportional to the pressure (as stated in Henry's Law). Relative to the stirred tank reactor process, the transfer of oxygen from the gas phase to the liquid phase is more efficient in the fermentor 1 according to the disclosure, meaning that more oxygen is transferred to the fermentation fluid per time unit.

[0150] Given that the above process is stoichiometrically balanced regarding the supply and consumption of nutrients, the yield of biomass and extracellular amylase per unit substrate is the same in both situations. However, the productivity of biomass and amylase per time unit is higher in the tubular fermentor 1 in accordance with the present disclosure compared to the stirred tank reactor due to the higher gas-liquid mass transfer, and this is obtained with a lower energy consumption.

[0151] Example 3

[0152] Example 3 describes fermentation of Hydrogenobacter thermophilus to produce single cell protein from carbon dioxide and hydrogen.

[0153] The overall biological reaction to produce biomass looks like the below stoichiometry:

[0154] 2.1 H2+ CO2+ 0,2 NH3= CH1.8O0.5N0 2 (biomass) + 1.5 H2O

[0155] At 30 degrees C and atmospheric pressure, the solubility of hydrogen in water is more than 20 times lower while the solubility of carbon dioxide is 40 times higher than that of oxygen. In this process, the very low solubility of hydrogen is the limiting parameter for an efficient biomass productivity.

[0156] The fermentation process is carried out in a fermentor 1 (see Fig. 1, 2, and 4) where at least a part of the flow path 6 forms a meandering section comprising serially connected sections 22 of a first group of sections and sections 24 of a second group of sections, where the first group of sections has a predominantly vertical extend and the second group of sections has a predominantly horizontal extend, the first group of sections comprising at least four sections, and the second group of sections comprising at least three sections, and where sections of the first group of sections are arranged alternately sections of the second group of sections, the sections 22 of the first group of sections having a total length exceeding 70% of the total flow length according to the disclosure. The fermentation process is started up with the same basic steps as described in Example 1, however with addition of carbon dioxide and hydrogen rather than methane and oxygen. As shown in the generic stoichiometric equation above, no gasses are produced besides the biomass. With no need for degassing in the tank 4, the vent valve 38 in the tank is closed, and the fermentation fluid is circulated in the flow path in the tubular part 6 and the tank 4 without loss of hydrogen gas and carbon dioxide which are added to the fluid containment space 2 of the fermentor 1, ensuring a very high utilisation of the hydrogen and a high yield of single cell protein per added volume of hydrogen.

Claims

CLAIMS1. A fermentor comprising :- a fluid containment space forming a closed loop flow path for a fermentation fluid;- at least one substrate inlet for inlet of a substrate liquid to the space;- at least one gas injection point for injection of gas into the fermentation fluid;- a fermentation outlet for withdrawing fermentation fluid from the space;- at least one circulation member for circulation of the fermentation fluid in the flow path; wherein the flow path has a total flow length being a distance along the flow path from a starting point to an end point, wherein at least a part of the flow path forms a meandering section comprising serially connected sections of a first group of sections and sections of a second group of sections, wherein the first group of sections has a predominantly vertical extend and the second group of sections has a predominantly horizontal extend, the first group of sections comprising at least four sections, and the second group of sections comprising at least three sections, and wherein sections of the first group of sections are arranged alternately sections of the second group of sections, wherein the predominantly vertical extend is in the range of 50-130 degrees from horizontal, and wherein the predominantly horizontal extend is in the range of + / -40 degrees from horizontal, and wherein the sections of the first group of sections have a total length exceeding 70% of the total flow length.

2. The fermentor according to claim 1, wherein a length of each of the sections in the second group is less than six times a diameter of the sections in the second group.

3. The fermentor according to claim 1 or 2, wherein the fluid containment space is constituted by a tank and a tubular part being in fluid connection, and wherein- a first outlet connects the tank to the tubular part to provide an outlet flow path for the fermentation fluid from the tank to the tubular part, and- a first inlet connecting the tubular part to the tank to provide an inlet flow path for the fermentation fluid from the tubular part to the tank.

4. The fermentor according to claim 3, wherein the first inlet and the first outlet are arranged at a bottom part of the tank, the bottom part facing a ground on which the fermentor is arranged.

5. The fermentor according to any of the preceding claims, further comprising at least one mixing element arranged in the flow path for dispersion of gas injected in the fermentation fluid.

6. The fermentor according to any of the preceding claims, wherein a first circulation member of the at least one circulation member is arranged in the flow path.

7. The fermentor according to claim 3 and 6, wherein the first circulation member is arranged in a first section of the first group of sections, the first section being the section immediately after the first outlet.

8. The fermentor according to claim 7, wherein the first circulation member is arranged between the first outlet and one of the at least one gas injection points.

9. The fermentor according to any of claims 3-8, further comprises a first pressure controlling device for controlling a pressure in the flow path, the first pressure controlling device being arranged in a last section of the first group of sections, the last section being the section immediately before the first inlet.

10. The fermentor according to any of claims 3-9, wherein the fermentation outlet is arranged in the tank.

11. The fermentor according to any of the preceding claims, wherein a cross-sectional shape of the flow path is substantially uniform along sections of the flow path.

12. The fermentor according to claim 11, wherein at least one section of the flow path forms a widening or narrowing cross-section transverse to the flow path.

13. The fermenter according to any of the preceding claims, wherein an inner volume of the flow path is in the range of 1 litre to 1000 m3, preferably in the range of 0.2-500 m3.

14. The fermenter according to any of the preceding claims, wherein at least a part of the meandering section forms a helical shape.

15. The fermenter according to any of the preceding claims, wherein at least one of the at least one gas injection points is configured for injection of a pressurized gas.

16. The fermenter according to any of the preceding claims, further comprising an additional opening, the additional opening being provided in an upper part of the flow path for adding and / or removal of gas.

17. The fermenter according to any of the preceding claims, further comprising a plurality of flow modification elements arranged in the flow path, the flow modification element being configured for changing a flow of the fermentation fluid.

18. A method of performing a fermentation process in a fermenter comprising :- a fluid containment space forming a closed loop flow path for a fermentation fluid;- at least one substrate inlet for inlet of a substrate liquid to the space;- at least one gas injection point for injection of gas into the fermentation fluid;- a fermentation outlet for withdrawing fermentation fluid from the space;- at least one circulation member for circulation of the fermentation fluid in the flow path; wherein the flow path has a total flow length being a distance along the flow path from a starting point to an end point, wherein at least a part of the flow path forms a meandering section comprising serially connected sections of a first group of sections and sections of a second group of sections, wherein the first group of sections has a predominantly vertical extend and the second group of sections has a predominantly horizontal extend, the first group of sections comprising at least four sections, and the second group of section comprising at least three sections, andwherein sections of the first group of sections are arranged alternately sections of the second group of sections, wherein the predominantly vertical extend is in the range of 50-130 degrees from horizontal, and wherein the predominantly horizontal extend is in the range of + / -40 degrees from horizontal, and wherein the sections of the first group of sections have a total length exceeding 70% of the total flow length, the fermentation fluid comprising at least one fermenting microorganism, the method comprising the steps of:- adding a substate liquid to the fermentation fluid in the space;- injecting gas into the fermentation fluid;- circulating the fermentation fluid along the flow path;- withdrawing a portion of the fermentation fluid via the fermentation outlet, wherein the method further comprises a first step of increasing pressure in the space in a first zone relative to a pressure in a second zone in the space to increase mass transfer of the gas.

19. The method according to claim 18, further comprising a second subsequent step of decreasing pressure in the second zone relative to the pressure in the first zone to initiate liberation of gas.

20. The method according to claim 18 or 19, further comprising a step of recycling a part of withdrawn fermentation fluid to the space.

21. The method according to any claims 18-20, further comprising a step of controlling pressure in the flow path at a level above atmospheric pressure by increasing pressure and closing the fermentation outlet.

22. The method according to any of claims 18-21, wherein the gas comprising at least one of atmospheric air, pure oxygen, oxygen enriched atmospheric air, methane, natural gas, biogas, ethane, propane, butane, carbon dioxide, carbon monoxide, hydrogen (H2), and ammonia gas.

23. The method according to any of claims 18-22, wherein the least one fermenting microorganism is a yeast, a filamentous fungi, or a bacteria.

24. The method according to any of claims 18-23, wherein the at least one microorganism belongs to a group of organisms capable of metabolising carbon dioxide and / or hydrogen.

25. The method according to any of claims 18-24, wherein a product of the fermentation process is a product from a group consisting of biomass, single cell protein, gasses, enzymes, peptides, hormones, vitamins, organic acids, chemical precursors, alcohols, and other metabolites or organic chemicals.

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