Fermentor and method of performing fermentation process

Abstract

The present disclosure provides a fermenter comprising: a fluid accommodation space forming a closed loop flow path for a fermentation fluid; at least one substrate inlet for the entry of a substrate fluid; and at least one gas injection point for injecting a gas. The fermenter further comprises a fermentation outlet for removing the fermentation fluid from the space and at least one circulation member for circulating the fermentation fluid. At least a portion of the flow path forms a serpentine section comprising a section in a first set of sections and a section in a second set of sections connected in series wherein the first set of sections has a substantially vertical extension and the second set of sections has a substantially horizontal extension, the sections of the first set of sections are alternately arranged with the sections of the second set of sections. The segments in the first set of segments have a total length that exceeds 70% of the total flow length.

Description

Technical Field

[0001] This invention relates to a fermenter and a method for performing fermentation in the fermenter. Background Technology

[0002] Typically, in biotechnological processes, microorganisms are cultured in tanks, most commonly known as bioreactors, into which substrates necessary for their growth and the production of desired products are added.

[0003] This type of culture treatment typically occurs in an aqueous solution, often referred to as a fermentation broth / fermentation medium, which contains various substrates, such as carbon sources as well as nitrogen sources, phosphates, sulfates, and various other components, such as minerals, trace metals, and vitamins, depending on the fermenting microorganisms and the products to be produced in the culture treatment.

[0004] In many fermentation processes, microorganisms rely on oxygen for growth. Oxygen is typically added as a gas by pumping compressed air, pure oxygen, or oxygen-enriched air into the fermentation broth. All living cells require at least one carbon source to grow. In many conventional fermentation processes, carbon sources are added in fluid form, such as sugars (glucose, sucrose, maltose, starch, etc.), organic acids (e.g., acetic acid, citric acid, and succinic acid), or alcohols (e.g., methanol or ethanol). In some cases, depending on the requirements of the organisms being cultured, carbon sources are added to the fermentation broth as gases, such as methane, natural gas, biogas, ethane, propane, butane, or carbon dioxide, and some organisms also require non-carbon sources, such as hydrogen (H2) and ammonia.

[0005] A significant amount of energy is typically used in conventional fermenters to ensure that sufficient gaseous substrate is dissolved in the fermentation fluid. Summary of the Invention

[0006] One object of embodiments of the present invention is to provide an improved fermenter.

[0007] One object of embodiments of the present invention is to provide an improved method for performing fermentation processes.

[0008] According to a first aspect, the present invention provides a fermenter comprising:

[0009] A fluid containment space that forms a closed-loop flow path for the fermentation fluid;

[0010] At least one substrate inlet, the at least one substrate inlet being used for substrate liquid to enter the space;

[0011] At least one gas injection point, said at least one gas injection point being used to inject gas into the fermentation fluid;

[0012] A fermentation outlet, wherein the fermentation outlet is used to remove fermentation fluid from the space;

[0013] At least one circulation component, said at least one circulation component being used to circulate the fermentation fluid in the flow path;

[0014] The flow path has a total flow length, which is the distance along the flow path from the starting point to the ending point.

[0015] At least a portion of the flow path forms a meandering segment, the meandering segment comprising segments from a first group of segments and segments from a second group of segments connected in series, wherein the first group of segments has a predominantly vertical extension and the second group of segments has a predominantly horizontal extension, the first group of segments comprising at least four segments and the second group of segments comprising at least three segments, and wherein the segments in the first group of segments and the segments in the second group of segments are arranged alternately.

[0016] The main vertical extension is within a range of 50-130 degrees relative to the horizontal, and the main horizontal extension is within a range of + / -40 degrees relative to the horizontal.

[0017] And wherein the segment in the first group of segments has a total length exceeding 50% of the total flow length.

[0018] A fermenter is a reactor suitable for fermentation processes, which can be defined as the growth or maintenance of living cells under aerobic, anaerobic, or partially aerobic conditions to obtain a desired product. The desired product can be the cells themselves, substances produced by the cells, or substances transformed by the cells.

[0019] The fermenter includes a fluid-containing space that forms a flow path for the fermentation fluid. The fermentation fluid includes at least one fermenting microorganism and can be a fermentation liquid or a fermentation gas. Microbial fermentation can be performed using a pure culture of only one fermenting microbial species or a mixture / association of different microorganisms. The flow path can form a closed loop to allow the fermentation fluid to circulate within the closed loop, for example, in continuous circulation. During the circulation of the fermentation fluid, gaseous and / or liquid substances can be added to the fluid-containing space, and / or a portion of the fermentation fluid can be removed from the fluid-containing space.

[0020] As an example, the at least one fermenting microorganism may be yeast, filamentous fungi, or bacteria. The at least one fermenting microorganism may be selected from the group consisting of at least the following: *Saccharomyces cerevisiae*, *Saccharomyces boulardii*, *Candida tropicalis*, *Candida utilis*, *Kluyveromyces lactis*, *Pichia pastoris*, *Saccharomyces cerevisiae*, *Yeastra lipolytica*, *Schizosaccharomyces cerevisiae*, *Hansenula polymorpha*, *Cytomyces delavayi*, *Zygosaccharomyces rouxii*, *Aspergillus niger*, *Aspergillus oryzae*, *Aspergillus nidus*, *Aspergillus soysarum*, *Trichoderma reesei*, *Penicillium*, *Fusarium*, *Rhizopus*, *Neurospora crassa*, *Mucor*, *Penicillium chrysogenum*, *Thermophilus lateralis*, *Beauveria bassiana*, *Lactobacillus acidophilus*, *Lactobacillus brevis*, *Lactobacillus delavayi*, *Lactobacillus casei*, *Lactobacillus fermentum*, *Lactobacillus helveticus*, *Lactobacillus plantarum*, *Lactobacillus reuteri*, *Lactobacillus rhamnosus*, *Lactococcus oryzae*, *Streptococcus thermophilus*. Bacteria, including *Methylcoccus capsulatum*, *Methylcurvularia sporangiosum*, *Methylcystis* fungi, *Methylcytophyte*, *Methylcurvularia rubra*, *Methylmicrobes*, *Methylmonas* bacteria, *Methylcurvularia sporogenes*, *Thermophilic Methylthermia*, *Methylmolybacillus motiformis*, *Methyltrophic Methylophilus*, *Paracinococcus denitrificans*, *Pseudomonas putida*, *Autotrophic Flavobacterium*, *Bacillus* bacteria, *Escherichia coli*, *Desulfovibrio* bacteria, methanogenic archaea, *Pyrogenus* bacteria, *Acetobacter wulleri*, aquatic thermophiles, thermophilic hydrogen bacteria, acid-producing alkali-producing bacteria, *Anaerobic Bacillus dansneri*, *Acetobacter acetobacter*, *Glucosinolates* bacteria, *Clostridium* bacteria, *Propionibacterium* bacteria, *Mycoplasma* bacteria, *Pediococcus* bacteria, *Shewanella putrefactive*, *Campobacter* bacteria, *Clostridium butyricum*, *Bacillus terreus* bacteria, and *Enterococcus* bacteria. Other fermentation microorganisms can also be selected.

[0021] The products of fermentation treatment can be at least one of the following: biomass, single-cell protein, gas, enzyme, peptide, hormone, vitamin, organic acid, chemical precursor, alcohol and other metabolites or organic chemicals.

[0022] The fermenter includes at least one substrate inlet for substrate liquid to enter the space. The substrate liquid is added as a source for the growth of fermentative microorganisms. In one embodiment, the substrate liquid may include a substrate, such as sugars (glucose, sucrose, maltose, starch, etc.), organic acids (e.g., acetic acid, citric acid, and succinic acid), or alcohols (e.g., methanol or ethanol). Alternatively or alternatively, other substrates may be added. The substrate liquid may also include inorganic salts and trace metals. The substrate inlet may include a closing mechanism for opening and closing the substrate inlet. In one embodiment, the closing mechanism may be configured to be manually operated. The closing mechanism may alternatively or additionally be configured to be automatically operated. Adjustments (e.g., closing and / or opening the substrate inlet) may be made in response to computer-implemented and / or computer-generated setpoints.

[0023] The at least one substrate inlet can also be used to add, for example, pH-adjusting chemicals to improve fermentation conditions for microorganisms. To monitor the condition of the fermentation fluid supplied in the space, the fermenter may also include at least one pH sensor and / or at least one temperature sensor arranged in the fluid containment space.

[0024] The fermenter also includes at least one gas injection point for injecting gas into the fermentation fluid. The gas may be a substrate gas for microbial growth. In one embodiment, the gas may include 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. Depending on the microorganisms and / or products, other gases may be used alternatively, or additional gases may be used in addition to these.

[0025] At least one of the at least one gas injection point can be configured to inject pressurized gas. This can help increase the pressure in the tubular portion of the flow path.

[0026] The fermenter includes a fermentation outlet for removing fermentation fluid from the space. The removed fermentation fluid may include the generation of fermentation products. In one embodiment, the fermenter may include a recirculation inlet configured to recirculate a portion of the removed fermentation fluid back into the fluid containment space. In another embodiment, the fermentation outlet may be in fluid communication with a substrate inlet, thereby allowing a portion of the removed fermentation broth to enter the fluid containment space through the substrate inlet.

[0027] In one embodiment, the extracted fermentation broth, along with its contained biomass, dissolved gases, dissolved metabolites, and other extracellular chemicals and enzymes, can be pumped from the fermentation outlet into a storage tank. After the tank, the fermentation broth can be conveyed to a separator, such as a centrifuge, decanter, or filtration unit (e.g., a filter drum), to separate a biomass-rich concentrated phase and a dilute phase (supernatant), the dilute phase potentially containing most of the extracellular and dissolved components and minerals. Depending on the final product, the concentrated phase can be further processed to obtain the final protein product or further separated into nucleic acids, vitamins, fatty acids, or other proliferating cell components. The mineral- and nutrient-rich dilute supernatant can be recycled back to the fermenter, or the contained extracellular enzymes, peptides, vitamins, hormones, organic acids, and other dissolved molecules can be further purified and concentrated.

[0028] The fermenter includes at least one circulation component for circulating fermentation fluid in a flow path. As an example, the at least one circulation component may be an in-line pump arranged in the flow path. At least one of the at least one circulation components may include a pump, such as an impeller pump, a Roots pump, or a turbine pump, which may also be used as a pressure control means in the fermenter.

[0029] The fermenter may also include at least one mixing element for dispersing the gas injected into the fermentation fluid. As an example, the mixing element may be a static mixer. In an alternative embodiment, the mixer may be a dynamic mixer. It should be understood that the fermenter may include one or more static mixers and / or one or more dynamic mixers. Gas dispersion can promote microbial growth because it makes the gas accessible to microorganisms when dissolved in the fermentation fluid. Fermentation treatment can be enhanced by arranging multiple mixing elements along the flow path, as the transfer of gaseous substrate to the fermentation fluid is often a limiting step in the metabolism and growth rate of the organism. Therefore, the limiting step can be counteracted or partially counteracted by applying multiple mixing elements along the flow path. This can solve, or at least partially solve, the problem of dissolving sufficient gaseous nutrients in the fermentation fluid at a sufficiently high rate to meet the needs of the microorganisms.

[0030] Using very small bubbles for gas injection, or if the pressure inside the fermenter is higher than atmospheric pressure, or if the temperature in the fermentation fluid is lowered, can improve the rate of gaseous material transfer from the gas phase to the liquid phase. However, significantly lowering the temperature generally results in slower growth, leading to lower productivity in the fermentation process.

[0031] Bubbles in a liquid tend to merge together to form larger bubbles, which reduces the rate at which gaseous substances transfer from the gas phase to the liquid phase. This can be counteracted by applying multiple mixing elements along the flow path.

[0032] The flow path has a total flow length, which is the distance along the flow path from the start point to the end point. Since the flow path can form a closed loop, in some embodiments, the start and end points of the flow path can be located at the same position along the flow path.

[0033] At least a portion of the flow path forms a meandering segment, the meandering segment comprising segments from a first group of segments and a second group of segments connected in series, wherein the first group of segments has a predominantly vertical extension and the second group of segments has a predominantly horizontal extension. The predominantly vertical extension is within a range of 50-130 degrees relative to the horizontal, and the predominantly horizontal extension is within a range of + / -40 degrees relative to the horizontal.

[0034] It should be understood that a main vertical extension within the range of 50-130 degrees relative to the horizontal means that the tangent to the centerline of the corresponding segment is within the range of 50-130 degrees relative to the horizontal. Similarly, it should be understood that a main horizontal extension within the range of + / -40 degrees relative to the horizontal means that the tangent to the centerline of the corresponding segment is within the range of + / -40 degrees relative to the horizontal.

[0035] The first group of segments comprises at least four segments, and the second group of segments comprises at least three segments, wherein the segments in the first group of segments are arranged alternately with the segments in the second group of segments.

[0036] As an example, a fermenter may include four basic vertical sections and three basic horizontal sections arranged alternately. A first basic vertical section may be attached at one end to a last (fourth) basic vertical section via another basic horizontal section. In an alternative embodiment, the first basic vertical section may be attached at one end to the last (fourth) basic vertical section via a tank. In another example, the fermenter may include four sections and three basic horizontal sections arranged alternately at an angle of 50 degrees relative to the horizontal.

[0037] It should be understood that at least four segments having a main vertical extension in the range of 50-130 degrees relative to the horizontal can be arranged at different angles relative to the horizontal; that is, the angle of each of the at least four segments need not be the same. It should also be understood that at least three segments having a main horizontal extension in the range of + / -40 degrees relative to the horizontal can be arranged at different angles relative to the horizontal; that is, the angle of each of the at least three segments need not be the same.

[0038] The segments in the first group have a total length exceeding 70% of the total flow length, for example, exceeding 75% of the total flow length, exceeding 80% of the total flow length, or even exceeding 90% of the total flow length.

[0039] Since the risk of gas-liquid phase separation is likely to occur primarily in the second group of sections—that is, sections with horizontal extension or more closely horizontal—this risk can be significantly reduced when sections in the first group constitute more than 70% of the total flow length. The higher hydrostatic pressure and lower phase separation tendency in the first group of sections can result in gas-liquid mass transfer being 3-5 times more efficient in the first group than in the second group.

[0040] To further reduce the risk of gas-liquid phase separation, the length of each segment in the second group may be less than six times the diameter of the segment in the second group, for example, less than five times the diameter, for example, less than four times the diameter, for example, less than three times the diameter, for example, less than two times the diameter. In the context of this disclosure, the term "diameter" should be understood as the maximum dimension of the tubular portion transverse to the flow path.

[0041] Furthermore, by providing a fermenter comprising a first set of segments including at least four sections and a second set of segments including at least three sections, the total height of the fermenter can be reduced compared to a U-shaped fermenter having the same volume, the same diameter of the tubular segments, and comprising two basic vertical segments connected by a single horizontal segment, wherein the height is the vertical dimension of the fermenter from ground level to the highest point above ground level.

[0042] Furthermore, by maintaining the diameter of the tubular portion and the fluid flow velocity along the flow path, the volume of the flow path can be doubled, for example, without requiring one or more much larger circulation pumps, and without requiring much larger material thickness for the pipes / pipes that can be used to form the flow path.

[0043] The volume can be increased by adding one or more segments to the alternating first and second group sections. This increases the length of the flow path, thereby increasing the contact time between the gas and the fermentation fluid, which can increase the utilization rate of gaseous nutrients.

[0044] This flexible design of alternating sections can also facilitate the expansion of existing fermenters, as one or more sections can be added to the first and second groups of sections. When the length of the flow path is increased by adding more alternating sections, the average pressure in the flow path can be increased (see also Example 1), thereby increasing gas-liquid mass transfer and thus improving the efficiency of the fermenter.

[0045] In one embodiment, the fermenter may include four substantially vertical segments and three substantially horizontal segments arranged alternately. The substantially vertical segments may be attached to a first end of a substantially horizontal segment via a bend. However, the first portion of the bend may belong to a first group of segments, while the second portion of the bend may belong to a second group of segments, because the tangent through the centerline of the first portion may be within a range of 50 degrees relative to the horizontal, and the tangent through the centerline of the second portion may be within a range of + / - 40 degrees relative to the horizontal. A second bend may connect a second end of a substantially horizontal segment to a second substantially vertical segment. To minimize the length of the second group of segments, in this embodiment, the substantially horizontal segment and the two substantially vertical segments may be attached to each other via two bends, wherein the substantially horizontal segment; i.e., the segment in the second group, is formed by portions of two bends forming a transition from one segment in the first group to a second segment in the first group. In this embodiment, a segment with a substantially horizontal extension may be formed by a short transition from one segment with a substantially vertical extension to the next segment with a substantially vertical extension.

[0046] In one embodiment, the fluid containment space may consist of a tank and a tubular section in fluid connection, wherein a first outlet connects the tank to the tubular section to provide an outlet flow path for the fermentation fluid from the tank to the tubular section, and a first inlet connects the tubular section to the tank to provide an inlet flow path for the fermentation fluid from the tubular section to the tank. The flow path is formed by a portion of the meandering section, which may be formed by the tubular section or a portion of the tubular section. The tubular section may be formed by multiple pipes / ducts attached to each other to form a hollow path for the fermentation fluid. In the tubular section, a long contact time between the gas and the fermentation fluid can be achieved because the gas can be present in both the first set of sections and the second set of sections. This can increase the utilization rate of the gas in the fermentation fluid.

[0047] The tank can be arranged horizontally above the tubular section relative to the ground on which the fermenter can stand. In one embodiment, a first inlet and a first outlet can be arranged at the bottom portion of the tank, with the bottom portion facing the ground. This facilitates flow from the tank into the tubular section and flow from the tubular section into the tank. Excess waste gases (such as CO2 produced by metabolism) can be released from the liquid within the tank.

[0048] A fermenter that includes a fluid-containing space with a tank and tubular sections can have a horizontal extension that roughly corresponds to the horizontal extension of the tank, since all or at least a significant portion of the meandering sections can be located below the tank.

[0049] The first circulation component can be arranged in the flow path. The first circulation component can be arranged in the first section of the first set of sections, which is the section immediately following the first outlet. By arranging the first circulation component at the upper end of the first section, the first circulation component ensures flow from the tank to the tubular portion.

[0050] The first gas injection point can be additionally arranged at the upper end of the first section, which has a predominantly vertical extension, in the first set of sections. This allows for a nearly uncompressible injection of gas into the fermentation fluid, requiring little to no compressor, as the gas only needs to overcome, for example, a few meters of hydrostatic pressure.

[0051] As an example, the first circulation component can be arranged between the first outlet and the first gas injection point.

[0052] The fermenter may also include a first pressure control device for controlling the pressure in the flow path. The first pressure control device may be arranged in the last section of a first set of sections, immediately preceding the first inlet to the tank. By arranging the first pressure control device close to the first inlet to the tank, it helps maintain the pressure in the tubular section above atmospheric pressure and above the pressure in the tank. The first pressure control device also helps maintain the pressure in the tank below the pressure in the tubular section, allowing for effective degassing of the fermentation fluid in the tank.

[0053] The pressure in the tank can be higher than atmospheric pressure, for example, 0.5-5 bar, thereby increasing the pressure in the tubular part of the fermenter to further increase the transfer of gaseous nutrients from the gas phase to the liquid phase.

[0054] As an example, the first pressure control device may include at least one of a valve (such as a pressure control valve), a static mixer, a hydrocyclone, a pump (such as an impeller pump), a perforated plate, and a tubular portion in which the diameter or cross-section of the tubular portion where the first pressure control device is placed is narrowed.

[0055] The fermenter may further include: an ion sensor or analyzer for sensing at least one of phosphate, sulfate, ammonium, nitrate, and hydrogen ions (pH); an oxygen sensor for measuring dissolved oxygen concentration; and at least one temperature sensor for sensing temperature. The sensors may be disposed in the flow path of the circulating fermentation fluid. In a preferred embodiment, the sensors may be arranged in the region of the substrate inlet and / or gas injection point or in an attached bypass arrangement.

[0056] Sensors and analyzers can transmit signals to a data processing system, where the received signals can optionally be processed. The flow rates of injected gases, water, minerals, and pH control measures supplied via the substrate inlet and / or gas injection point can be calculated and optimized via pre-programmed quantities. Alternatively, temperature can be regulated by a temperature control device, such as a heating / cooling jacket, which, as an example, can be installed near the tank's first inlet. The jacket can have fittings for circulating the heating or cooling medium through it. In another alternative, temperature can be regulated by applying a bypass flow, thus providing the possibility of cooling the fermentation fluid.

[0057] The fermentation outlet for removing the fermentation fluid from the fluid containment space can preferably be located within the tank. However, in alternative embodiments, the fermentation outlet can be located within a tubular section, for example, in the lower portion of one of the sections.

[0058] The cross-sectional shape of the tubular portion, which is transverse to the flow path, can be generally circular or elliptical to increase the volume of the flow path relative to the flow direction. In alternative embodiments, the cross-sectional shape can be polygonal, rectangular, hexagonal, or other shapes.

[0059] The cross-sectional shape of the flow path can be substantially uniform along sections of the flow path, for example, substantially uniform along meandering sections. As an example, a flow path with a circular cross-sectional shape can have a constant diameter along most sections. However, at least one section of the flow path may also include one or more sections with a conical shape or one or more sections with shorter or longer regions that narrow or widen, as this can be used to regulate pressure along the flow path.

[0060] In one embodiment, the internal volume of the flow path can range from 1 liter to 1000 m³. 3 Within the range, preferably in the range of 0.2-500m 3 Within a range, for example, in the range of 25-250m 3 Within the range.

[0061] In one embodiment, at least a portion of the meandering segment may form a spiral shape.

[0062] The fermenter may also include additional openings. These additional openings may be located in the upper portion of the flow path for adding and / or removing gases, such as for degassing CO2 and / or excess CH4 and / or other gases (such as CO, H2, etc.). As an example, the additional openings may be positioned above the liquid surface in the tank, which can create a gas flow to flush the volume above the liquid surface (headspace) in the tank in parallel, counter-current, or cross-current with the liquid flow.

[0063] The fermenter may also include multiple flow-modifying elements arranged in the flow path. These flow-modifying elements can be configured to alter the flow of the fermentation fluid. Preferably, the flow-modifying elements are arranged within a tubular section. As an example, the flow-modifying elements may include static or dynamic mixers for effectively dispersing gas in the liquid. The static or dynamic mixers may be placed in the region of the gas injector, for example, immediately below the gas injector. The static or dynamic mixers may be arranged at regular intervals along a first section of the flow path. The flow rate of the fermentation fluid in the flow path can be adjusted to be sufficiently high to carry the injected gas through the static mixer and through the flow path. Thus, gas bubbles can be carried along with the fermentation fluid through the downstream first section to the lower end and further through the second section to the upstream first section, such that the gas bubbles are redispersed multiple times in the liquid by means of the regularly spaced static mixing elements. Alternatively or additionally, the flow-modifying elements may be one or more sections of the tubular section with a smaller diameter, since reducing the diameter of the tubular section of the flow path can also modify the flow through the tubular section.

[0064] According to a second aspect, the present invention provides a method for performing a fermentation process in a fermenter, the fermenter comprising:

[0065] A fluid containment space that forms a closed-loop flow path for the fermentation fluid;

[0066] At least one substrate inlet, the at least one substrate inlet being used for substrate liquid to enter the space;

[0067] At least one gas injection point, said at least one gas injection point being used to inject gas into the fermentation fluid;

[0068] A fermentation outlet, wherein the fermentation outlet is used to remove fermentation fluid from the space;

[0069] At least one circulation component, said at least one circulation component being used to circulate the fermentation fluid in the flow path;

[0070] The flow path has a total flow length, which is the distance along the flow path from the starting point to the ending point.

[0071] At least a portion of the flow path forms a meandering segment, the meandering segment comprising segments from a first group of segments and segments from a second group of segments connected in series, wherein the first group of segments has a predominantly vertical extension and the second group of segments has a predominantly horizontal extension, the first group of segments comprising at least four segments and the second group of segments comprising at least three segments, and wherein the segments in the first group of segments and the segments in the second group of segments are arranged alternately.

[0072] The main vertical extension is within a range of 50-130 degrees relative to the horizontal, and the main horizontal extension is within a range of + / -40 degrees relative to the horizontal.

[0073] Furthermore, the segments in the first group of segments have a total length exceeding 50% of the total flow length.

[0074] The fermentation fluid includes at least one fermenting microorganism, and the method includes the following steps:

[0075] The substrate liquid is added to the fermentation fluid in the space;

[0076] Inject the gas into the fermentation fluid;

[0077] The fermentation fluid is circulated along the flow path;

[0078] A portion of the fermentation fluid is removed through the fermentation outlet.

[0079] The method further includes a first step: increasing the pressure in the first region of the space relative to the pressure in the second region of the space to increase gas mass transfer.

[0080] The substrate liquid can be added via at least one substrate inlet. The substrate liquid may include a substrate, such as sugar, and may also include nutrients and pH-adjusting chemicals. Furthermore, the temperature of the fermentation fluid can be controlled during circulation in the flow path. Cultivation can occur simultaneously with the circulation of the fermentation fluid. The second region may be a tank forming part of the fluid-containing space. The first region may be part of a tubular section.

[0081] It should be understood that those skilled in the art will readily recognize that any feature described in connection with the first aspect of this disclosure can also be combined with the second aspect of this disclosure, and vice versa.

[0082] The fermenter according to the first aspect of this disclosure is well-suited for performing the method steps according to the second aspect of this disclosure. Therefore, the above description regarding the fermenter also applies to the method.

[0083] The method may further include a subsequent second step: reducing the pressure in the second region relative to the pressure in the first region to trigger the release of trapped gas from the fermentation fluid in the tank. The released gas can be removed from the tank via a valve.

[0084] The method may include an additional step of recycling a portion of the extracted fermentation fluid back into the space. The fermentation fluid can be extracted through a fermentation outlet. The extracted fermentation broth, along with its contained biomass, dissolved gases, dissolved metabolites, and other extracellular chemicals and enzymes, can be pumped from the fermentation outlet into a storage tank. After the tank, the fermentation broth can be conveyed to a separator to separate a biomass-rich concentrated phase and a dilute phase (supernatant), the dilute phase of which may contain most of the extracellular and dissolved components and minerals. In an alternative embodiment, the extracted fermentation broth and its contents can be directly pumped to the separator. In one embodiment, the mineral- and nutrient-rich dilute supernatant component can be recycled back into the fermenter.

[0085] The method may include the step of controlling the pressure in the flow path to a level above atmospheric pressure by increasing the pressure and by closing the fermentation outlet. The increased pressure in the flow path; that is, the increased pressure in the tubular section and / or in the tank, can increase the mass transfer of at least one injected gas from the gas phase to the liquid phase in the flow path. By closing the fermentation outlet, for example by a valve, the removal of gaseous components from the flow path can be prevented.

[0086] The gas injected through the at least one gas injection point may include 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.

[0087] The at least one microorganism may belong to the group of organisms capable of metabolizing carbon dioxide and / or hydrogen. Attached Figure Description

[0088] Embodiments of this disclosure will now be described in further detail with reference to the accompanying drawings, in which:

[0089] Figure 1 An embodiment of the fermenter is shown;

[0090] Figure 2 An alternative embodiment of the fermenter is shown;

[0091] Figure 3 A U-shaped fermenter is shown;

[0092] Figure 4 An embodiment of the fermenter is shown;

[0093] Figure 5 As shown Figure 3 Gas-liquid phase separation in the U-shaped fermenter shown;

[0094] Figure 6 As shown Figure 4 Gas-liquid phase separation in the fermenter shown;

[0095] Figure 7 As shown Figure 3 The U-shaped fermenter shown and as Figure 4 Comparison of non-uniformity in the fermenters shown;

[0096] Figure 8A and Figure 8B As shown Figure 3 The U-shaped fermenter shown and as Figure 4 A comparison of pressures in the fermenters shown;

[0097] Figure 9 As shown Figure 3 The processing data of the U-shaped fermenter shown; and

[0098] Figure 10 As shown Figure 4 The processing data of the fermenter shown. Detailed Implementation

[0099] It should be understood that the detailed description and specific examples, while illustrating embodiments of the present disclosure, are given by way of illustration only, as various variations and modifications within the scope of the present disclosure will become apparent to those skilled in the art from the detailed description.

[0100] Figure 1 An embodiment of fermenter 1 is shown, and Figure 2 An alternative embodiment of fermenter 1 is shown.

[0101] Fermenter 1 includes a fluid containment space 2 that forms a closed-loop flow path for the fermentation fluid. The flow direction in the flow path is indicated by arrows. In the two embodiments shown, the fluid containment space 2 consists of a tank 4 and a tubular portion 6, which are fluidly connected and form a flow path for the fermentation fluid to flow from the tank 4 to the tubular portion 6 and back to the tank 4.

[0102] The fermentation fluid contains at least one fermenting microorganism, which may be yeast, filamentous fungi, or bacteria. The at least one fermenting microorganism may be selected from the group consisting of at least the following: *Saccharomyces cerevisiae*, *Saccharomyces boulardii*, *Candida tropicalis*, *Candida utilis*, *Kluyveromyces lactis*, *Pichia pastoris*, *Saccharomyces cerevisiae*, *Yeastra lipolytica*, *Schizosaccharomyces cerevisiae*, *Hansenula polymorpha*, *Cytomyces delavayi*, *Zygosaccharomyces rouxii*, *Aspergillus niger*, *Aspergillus oryzae*, *Aspergillus nidus*, *Aspergillus soysarum*, *Trichoderma reesei*, *Penicillium*, *Fusarium*, *Rhizopus*, *Neurospora crassa*, *Mucor*, *Penicillium chrysogenum*, *Thermophilus lateralis*, *Beauveria bassiana*, *Lactobacillus acidophilus*, *Lactobacillus brevis*, *Lactobacillus delavayi*, *Lactobacillus casei*, *Lactobacillus fermentum*, *Lactobacillus helveticus*, *Lactobacillus plantarum*, *Lactobacillus reuteri*, *Lactobacillus rhamnosus*, *Lactococcus oryzae*, *Streptococcus thermophilus*. Bacteria, including *Methylcoccus capsulatum*, *Methylcurvularia sporangiosum*, *Methylcystis* fungi, *Methylcytophyte*, *Methylcurvularia rubra*, *Methylmicrobes*, *Methylmonas* bacteria, *Methylcurvularia sporogenes*, *Thermophilic Methylthermia*, *Methylmolybacillus motiformis*, *Methyltrophic Methylophilus*, *Paracinococcus denitrificans*, *Pseudomonas putida*, *Autotrophic Flavobacterium*, *Bacillus* bacteria, *Escherichia coli*, *Desulfovibrio* bacteria, methanogenic archaea, *Pyrogenus* bacteria, *Acetobacter wulleri*, aquatic thermophiles, thermophilic hydrogen bacteria, acid-producing alkali-producing bacteria, *Anaerobic Bacillus dansneri*, *Acetobacter acetobacter*, *Glucosinolates* bacteria, *Clostridium* bacteria, *Propionibacterium* bacteria, *Mycoplasma* bacteria, *Pediococcus* bacteria, *Shewanella putrefactive*, *Campobacter* bacteria, *Clostridium butyricum*, *Bacillus terreus* bacteria, and *Enterococcus* bacteria. Other fermentation microorganisms can also be selected.

[0103] The fermenter 1 also includes at least one substrate inlet 8 in the substrate liquid entry space 2 and at least one gas injection point 10 in the gas injection fermentation fluid.

[0104] The substrate liquid is added as a source of growth for the fermenting microorganisms. The substrate liquid may include substrates such as sugars (glucose, sucrose, maltose, starch, etc.), organic acids (e.g., acetic acid, citric acid, and succinic acid), or alcohols (e.g., methanol or ethanol). Alternatively or alternatively, other substrates may be added. The substrate liquid may also include inorganic salts and trace metals.

[0105] Substrate inlet 8 can also be used to add, for example, pH-adjusting chemicals to improve fermentation conditions for microorganisms.

[0106] The at least one gas injection point 10 is used to inject gas into the fermentation fluid. The gas can be a substrate gas for microbial growth. The gas may include 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.

[0107] The fermenter 1 also includes a fermentation outlet 12 for removing fermentation fluid from the space 2. In the illustrated embodiment, the fermentation outlet 12 is located at the bottom of the tank 2. The product of the fermentation treatment may be at least one of biomass, single-cell protein, gas, enzyme, peptide, hormone, vitamin, organic acid, chemical precursor, alcohol and other metabolites or organic chemicals, which may be removed from the fermenter 1 together with the removed fermentation fluid.

[0108] Fermenter 1 includes a circulation member 14 for circulating the fermentation fluid in the flow path. In the illustrated embodiment, the circulation member 14 is a loop pump arranged in the flow path. The circulation member 14 also serves as a pressure control means in fermenter 1.

[0109] The fermenter 1 also includes a plurality of mixing elements 16 for dispersing the gas injected into the fermentation fluid. In the illustrated embodiment, the plurality of mixing elements are in the form of a static mixer.

[0110] The first outlet 18 connects the tank 4 to the tubular section 6 to provide an outlet flow path for the fermentation fluid from the tank 4 to the tubular section 6, and the first inlet 20 connects the tubular section 6 to the tank 4 to provide an inlet flow path for the fermentation fluid from the tubular section 6 to the tank 4.

[0111] At least a portion of the flow path forms a meandering segment, which includes segments 22 from a first group of segments and segments 24 from a second group of segments connected in series. The first group of segments has a predominantly vertical extension, and the second group of segments has a predominantly horizontal extension. The predominantly vertical extension is within a range of 50-130 degrees relative to the horizontal, and the predominantly horizontal extension is within a range of + / -40 degrees relative to the horizontal. The first group of segments includes at least four segments 22, and the second group of segments includes at least three segments 24. The segments 22 in the first group and the segments 24 in the second group are arranged alternately.

[0112] exist Figure 1 In the illustrated embodiment, the fermenter 1 includes four basic vertical sections 22 belonging to a first group of sections and three basic horizontal sections 24 belonging to a second group of sections, with the sections 22 and 24 of the first and second groups arranged alternately. Figure 2 In the embodiment shown, the fermenter 1 includes six basic vertical sections 22 belonging to a first group of sections and five basic horizontal sections 24 belonging to a second group of sections, with the sections 22 and 24 in the first and second groups arranged alternately.

[0113] In both embodiments, the basic vertical segment 22 is attached to the first end of the basic horizontal segment 24 via a bend 26. The first portion of the bend 26 may belong to a first group of segments, while the second portion of the bend 26 may belong to a second group of segments, because the tangent to the centerline of the first portion can be within a range of 50-130 degrees relative to the horizontal, and the tangent to the centerline of the second portion can be within a range of + / -40 degrees relative to the horizontal. The second bend 26 connects the second end of the basic horizontal segment 24 to the second basic vertical segment 22.

[0114] The flow path has a total flow length, which is the distance along the flow path from the starting point to the ending point, where the starting point can be located at the outlet 18 and the ending point can be located at the inlet 20. Segment 22 in the first group of segments has a total length exceeding 70% of the total flow length.

[0115] To monitor the condition of the fermentation fluid provided in space 2, the fermenter 1 also includes at least one pH sensor 28 and at least one temperature sensor 30 arranged in the fluid containing space 2.

[0116] A temperature regulating device 32 in the form of a heating / cooling jacket is installed at the basic vertical section 24 near the inlet 20 to the tank 4.

[0117] A pressure control device 34 is disposed in the final, substantially vertical section 24 near the inlet 20. In the illustrated embodiment, the pressure control device 34 is in the form of narrowing the diameter of a portion of the section 24. In addition to the circulation member 14 and the pressure control device 34, the pressure in the flow path can be increased by injecting pressurized air or other gases via the gas injection point 10.

[0118] By flushing the volume above the liquid surface in tank 4 with flushing gas via flushing valve 36 arranged in the upper part of tank 4, gas separation in tank 4 can be improved.

[0119] The degassing valve 38 in tank 4 can be used to release gases that may have been released from the fermentation liquid.

[0120] Example

[0121] The following examples illustrate three different uses and applications of this disclosure.

[0122] Example 1

[0123] Example 1 describes the production of single-cell protein (SCP) via fermentation of *Methylococcus capsulatum*, optionally in combination with acid-producing alkali-producing bacteria, *Bacillus dansii*, and *Bacillus stoichioides*, using methane, natural gas, or biogas as both carbon and energy sources. Ammonia is used as the nitrogen source. In addition to these gaseous substrates, the cultivation of *Methylococcus capsulatum* requires water, phosphates, sulfates, and various minerals, including calcium, potassium, iron, and copper (added to the mineral mixture as sulfates, phosphates, nitrates, or chlorides). Sodium hydroxide and sulfuric acid are used for pH adjustment.

[0124] The overall bioreaction process for producing biomass is shown in the following stoichiometric equation:

[0125] ax CH4 + bx O2 + cx NH3 → CH 1.68 O 0.36 N 0.22 (biomass) + dx CO2 + ex H2O

[0126] The two main inputs (methane and oxygen) are present in the gas phase, while microorganisms and biological reactions occur in the liquid phase of the fermenter.

[0127] The transfer of methane and oxygen from the gas phase to the liquid phase is a major limiting factor in biomass productivity.

[0128] The above processing is as follows Figure 3 The fermentation process is shown in the U-shaped fermenter 100 according to the present disclosure and is carried out in the fermenter 1 according to the present disclosure, wherein, as shown in the U-shaped fermenter 100 according to the present disclosure, the fermentation process is carried out in the U-shaped ... the fermentation Figure 4 Four additional vertical sections 22 and three additional horizontal sections 24 are introduced into the tubular flow section 6 of the fermenter 1 shown. Figure 4 The fermenter 1 shown and forming part of this example therefore includes a flow path, a portion of which forms a meandering section comprising segments 22 of a first group of segments and segments 24 of a second group of segments connected in series, wherein the first group of segments has a predominantly vertical extension and the second group of segments has a predominantly horizontal extension. The first group of segments comprises six segments 22 and the second group of segments comprises five segments 24, the segments in the first group of segments and the segments in the second group of segments are arranged alternately, and the segments in the first group of segments have a total length exceeding 70% of the total flow length. Figure 4 The fermenter 1 shown is similar to Figure 2 Fermenter 1 is shown. Since there are three U-shaped sections in this fermenter 1, it is designated as a 3U fermenter or 3U bioreactor throughout this example. Two fermenters 1, 110 ( Figure 3 and 4The heights of the sections are the same, and the diameters of the tubular flow sections are also the same. In this example, additional vertical and horizontal sections increase the volume from 100 liters in the U-shaped fermenter to 170 liters in the 3U fermenter.

[0129] Already targeted Figure 3 The U-shaped fermenter 100 shown and Figure 4 CFD simulations were performed on the 3U fermenter 1 shown. In the U-shaped fermenter 100, the CFD simulations indicate that the large horizontal section 124 leads to non-uniformity in gas dispersion in the liquid phase, resulting in gas-liquid phase separation caused by the buoyancy of methane and oxygen bubbles 150, such as... Figure 5 As shown, the basic vertical section 122 and horizontal section 124 are schematically illustrated along with the normalized volume fraction. CFD simulations also show that almost complete phase separation occurs at position P in the horizontal section of the U-shaped annulus, at a distance P corresponding to two pipe diameters from the vertical section along the flow direction. This results in lower gas transfer efficiency in the horizontal region.

[0130] In the 3U fermenter 1, the shorter horizontal section in the second set of sections leads to Figure 6 The CFD simulations shown illustrate lower inhomogeneity, with the essentially vertical segment 22 and horizontal segment 24 schematically shown together with normalized volume fractions. The simulations indicate that, despite the 3U fermenter being 70% larger than the U-shaped reactor, the 3U fermenter 1 exhibits 40% lower inhomogeneity along the length of the tubular section 6 compared to the U-shaped reactor. Figure 7 As shown. The lower non-uniformity in the 3U fermenter increases gas-liquid transport capacity, thus improving the mass transfer efficiency of the 3U design compared to the U-shaped design. Figure 7 A comparison (%) of the non-uniformity between the U-shaped fermenter 100 and a fermenter 1 (3U) according to the present disclosure having six basic vertical sections 22 (first group) and five basic horizontal sections 24 (second group) is shown. The non-uniformity of the U-shaped fermenter is approximately 11%, while the non-uniformity of the 3U fermenter is approximately 7%. Therefore, despite a 70% increase in volume, the 3U design has a 40% lower non-uniformity.

[0131] Figure 5 and Figure 6 Showing the target Figure 3 The U-shaped fermenter 100 shown and Figure 4 The results of a CFD simulation performed on the 3U fermenter 1 are shown. The CFD simulation shows the normalized gas volume fraction from 0 to 5, as indicated by the indicator bars inserted below / above the schematically shown fermenters 1 and 100, respectively. Figure 5 It is also shown that the buoyancy of the gas bubbles 150 causes gas-liquid phase separation in the longer horizontal section 124 of the U-shaped fermenter 100.

[0132] exist Figure 5 The diagram shows a CFD simulation of a U-shaped fermenter 100. In the lower portion of the horizontal section 124 of the U-shaped ring, volume F1 (below the dashed line) has a normalized volume fraction less than 1. In the main volume portion F2 of the fundamental vertical section 122 of the U-shaped ring, the normalized volume fraction is approximately 1. The upper portion of the fundamental horizontal section 124 comprises two volumes: an upper volume F4 extending along the upper wall section and an intermediate volume F3 extending along the upper volume, wherein the normalized volume fraction in the upper volume F4 is greater than 4, and the normalized volume fraction in the intermediate volume F3 is between 3 and 4. The gas volume fraction is normalized using an average value; that is, perfect homogeneity would give 1.0 across the entire volume.

[0133] exist Figure 6 In the CFD simulation, a low trend of gas-liquid phase separation was observed in the fermenter 1 according to the present disclosure, in which the fermenter has six vertical sections 22 and five horizontal sections 24. Figure 6 The diagram shows a CFD simulation for a 3U fermenter 1. In the lower portion of the horizontal section 24, volume F1 (below the dashed line) has a normalized volume fraction less than 1. In the main volume portion F2 of the essentially vertical section 22, the normalized volume fraction is approximately 1. The upper portions of the upper essentially horizontal section 24 each comprise two volumes: an upper volume F4 located at the upper wall section and a lower volume F3 along the upper volume. The normalized volume fraction in the upper volume F4 is approximately 4, and the normalized volume fraction in the middle volume F3 is between 2.75 and 4. In the lower portion of the upper essentially horizontal section 24, the normalized volume fraction is approximately 1. The gas volume fraction is normalized using the average value; that is, perfect homogeneity would give 1.0 across the entire volume.

[0134] The horizontal white line at the tubular portion of the 3U fermenter 1 indicates the location of components (such as static and / or dynamic mixers) that can be arranged along the flow path. It should be understood that such components can also be positioned at other locations along the flow path.

[0135] Because the additional horizontal and vertical sections 24 and 22 in the 3U design are placed in the lower part of the tubular part 6 of the fermenter 1, the average pressure in the 3U fermenter 1 will be 17% higher than that in the U-shaped fermenter 100. Figure 8A and Figure 8B A comparison of 50m was shown. 3 U-shaped fermenter 100 and 85m 3 CFD simulations of a 3U-shaped fermenter were performed. Both fermenters have the same height. The higher average pressure results in a higher gas-liquid mass transfer driving force in the 3U design compared to the U-shaped fermenter. Figure 8AThe absolute pressure in each fermenter is shown by the values ​​indicated by the indicator bars inserted above the schematically shown fermenters 1 and 100. In the U-shaped fermenter 100, the pressure at the upper portion P1' of the first basic vertical leg 122 is in the range of 5.5 bar, while the pressure at the upper portion P2' of the second basic vertical leg 122 is in the range of 3 bar. The pressure decreases from the upper portion P1' of the first leg to the upper portion P2' of the second leg. The average pressure of the U-shaped fermenter 100 is approximately 4.5 bar (see [reference]). Figure 8B In the 3U fermenter 1, the pressure at the upper portion P1 of the first basic vertical leg 22 is in the range of 6 bar, while the pressure at the upper portion P2 of the last basic vertical leg 22 is in the range of 4 bar. The pressure decreases from the upper portion P1 of the first leg to the upper portion P2 of the last leg. The average pressure of the 3U fermenter 1 is approximately 5.4 bar (see [link to relevant documentation]). Figure 8B ).

[0136] exist Figure 8A In the diagram, the horizontal and vertical white lines at the tubular portions of the U-shaped fermenter 100 and the 3U fermenter 1 indicate the positions of elements (such as static and / or dynamic mixers) that can be arranged along the flow path. It should be understood that such elements can also be positioned at other locations along the flow path.

[0137] exist Figure 8B In this example, the average pressure (in bar) of the U-shaped fermenter 100 is compared with the average pressure of the U3 fermenter 1, showing that the average pressure in the 3U fermenter is 17% higher.

[0138] With the same liquid flow rate of 1 m / s for the fermentation fluid, the residence time in the 3U fermenter 1 is significantly higher compared to the U-shaped design 100. The higher residence time increases the gas-liquid contact time and results in better mass transfer. Furthermore, the shorter basic horizontal section 24 (second set of sections) in the 3U bioreactor provides better phase flow compared to the two-phase flow observed in the basic horizontal section 124 of the U-shaped fermenter in this example.

[0139] Since the diameters and flow rates of the tubular sections 6 and 106 are the same for both the U-shaped fermenter 100 and the 3U fermenter 1 in this example, the two fermenters can use the same pump type and size. In this example, this reduces the power consumption of the circulation pump per fermenter volume in the 3U fermenter 1 compared to the U-shaped fermenter 100.

[0140] After cleaning and sterilizing the two fermenters 1 and 100 in this example, fill the fermenters with water and necessary nutrients (see [link]). Figure 3 and Figure 4), and add the inoculum culture of *Methylococcus capsulatum*, optionally together with the co-fermenting organisms. Liquid circulation is initiated by applying a pressure of approximately 2 bar at gas injection points 10, 110 near the first outlets 18, 118 of tanks 4, 104 at the upper portion of the tubular sections 6, 106 of fermenters 1, 100, and by activating the first loop pumps 14, 114. Before the circulating fermentation fluid enters tanks 4, 104, the pressure is reduced to atmospheric pressure by pressure control devices 34, 134. Then, just below the first circulation member 14, 114 (in the form of a pump), feed gas (methane, natural gas, or biogas and ammonia, atmospheric air, and oxygen) is introduced into the fermentation liquid in the tubular section of the fermenter until a stable state with a biomass content of 1-4% (w / w) solids is achieved. Then, the fermentation liquid is circulated through the fermentation outlet (12, see...) Figure 2 Remove the fermentation liquid, and simultaneously reflux it at a rate of 0.05 to 0.4 h. -1 The dilution rate is used to supply water and mineral solutions. Then, based on the online measurement of ammonium, phosphate, nitrate, nitrite, oxygen, methane, etc. by sensors in the system, the supply of gases, nutrients, and pH adjustment measures are performed.

[0141] In this example, carbon dioxide is produced by the fermenting organisms. To avoid the accumulation of carbon dioxide at an inhibitory concentration in the fermentation broth, the carbon dioxide is separated from the fermentation broth in tanks 4 and 104 and released via vent valves 38 and 138. This degassing of the fermentation broth ensures that the first circulation components 14 and 114 operate on the fermentation fluid with low gas content, thereby improving the efficiency of the circulation components.

[0142] During steady-state fermentation, oxygen saturation was maintained below 10% (measured at the end of the tubular flow section), pH was controlled at approximately pH 6.8, and temperature was maintained at approximately 43°C.

[0143] The fermenter dimensions, key processing conditions, and amounts of added and removed substances are shown in Table 1 below.

[0144] Table 1: Examples of single-cell protein (SCP) production compared to a 100L U-shaped fermenter (3U) with a volume of 170L according to this disclosure.

[0145] The obtained stem cell weight concentration and dilution rate were the same in both fermentation treatments described above (see [link]). Figure 9 and Figure 10 Therefore, the volumetric productivity of biomass (kg / m³) 3 The yield (kg / h) is the same in both fermenters 1 and 100, and therefore the total productivity (kg / h) in fermenter 1 is 70% higher than that in fermenter 100. (See Table 1 and...) Figure 9 and Figure 10 It can be seen that, compared with the U-shaped fermenter 100, the methane flow rate (Nm³) per fermenter volume in the 3U fermenter 1 is higher. 3 / m 3 The lower energy consumption per minute ( / min) means that the 3U fermenter converts methane into biomass more efficiently. It is also noted that the energy per fermenter volume of the circulating pump is lower in the 3U fermenter, resulting in higher overall productivity with less energy consumption.

[0146] Figure 9 The processing data from a U-shaped fermenter 100 with a volume of 100L is shown, while Figure 10 The processing data from a 3U fermenter 1 with a volume of 170L are shown.

[0147] The data processed includes:

[0148] • A: DCW: Stem cell weight (g / l)

[0149] • B: Productivity: Amount of output per unit time

[0150] • C: NH3 flow rate: Ammonia entering (NL / min)

[0151] • D: Dilution rate (h) -1 )

[0152] • E: O2 flow rate: Oxygen intake (NL / min)

[0153] • F: Methane flow rate: Methane gas intake (NL / min)

[0154] The processing data for DCW (A), productivity (B), dilution rate (D), and O2 flow rate (E) are normalized according to the minimum and maximum operating windows of the fermenter used in this example.

[0155] Fermentation fluids taken from fermenters 1 and 100 are processed using standard downstream processing equipment, including an inactivation step in which more than 95% of bacterial cells are inactivated to obtain solid single-cell protein powder with a solid content (TS) of about 94%, which may optionally be granulated before further use.

[0156] Table 2 shows the average component values ​​of single-cell protein (SCP) products.

[0157] Table 2: Average component values ​​of single-cell protein (SCP) products.

[0158] Example 2

[0159] Example 2 describes the production of extracellular amylase by fermenting a genetically modified strain of Bacillus licheniformis, where glucose is used as both the carbon and energy source, compressed atmospheric air as the oxygen source, and ammonia is used both as a nitrogen source and as a means of controlling pH. In this example, the genetically modified strain can convert up to 25% of the metabolized glucose into extracellular amylase.

[0160] This type of processing is typically carried out industrially in conventional fermenters, such as stirred tank bioreactors in batch, fed-batch, or continuous modes. Most typically, in fed-batch mode, all the required nutrients and minerals are present in the fermentation broth at the start of fermentation, and the carbon source (here, glucose) is metered into the fermenter in fluid form. Compressed air and nitrogen are supplied in gaseous form during fermentation to support the growth of biomass and the production of amylase, according to the following general stoichiometric equation:

[0161]

[0162] CH2O 0.5 N 0.2 It represents cellular material, extracellular amylase, and other organic matter produced by microorganisms during their growth and replication.

[0163] Due to the low gas-liquid mass transfer efficiency in stirred tank reactors, the supply of oxygen to the microorganisms in the fermentation broth becomes a limiting parameter for microbial growth and amylase production. To increase the availability of oxygen for the fermenting microorganisms in the form of higher concentrations of dissolved oxygen in the fermentation broth, more oxygen can be added via a higher flow rate of compressed air through an injector located at the bottom of the stirred tank reactor. However, since 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 aeration in the fermenter, typically in industrial applications up to 100m³. 3Two-thirds of the total energy consumption of the reactor (see: Ali Jasinha, Jerome Ramirez, and Ian O'Hara, "Advancing Precision Fermentation: Reducing Energy Consumption in Industrial-Scale Bioreactors through Mechanistic Modeling," *Computer & Chemical Engineering*, Vol. 188, 2024). Increasing the flow rate of compressed air further increases the risk of agitator flooding. In stirred tank reactors, the agitator transfers energy to the fermentation liquid via an impeller to ensure proper mixing of the liquid and to increase gas-liquid mass transfer by breaking up bubbles into smaller bubbles. As the volume fraction of bubbles increases, the energy transfer efficiency from the agitator to the liquid decreases significantly (see "Voltage Power Consumption of a Pilot-Scale 550-L Fermenter with Upward-Push Hydrofoil B2 Impeller under Extremely High Viscosities"—ScienceDirect), and eventually, a state where almost no energy is transferred from the impeller to the liquid can be achieved, resulting in flooding. A third means of increasing gas-liquid mass transfer and thus oxygen availability is to increase the pressure in the stirred tank reactor. However, this will also increase the energy required for aeration and the energy required to overcome the pressure in the fermenter by adding other substances (such as sugars, acids, and alkalis).

[0164] To prevent the accumulation of glucose in the fermentation broth, which can lead to the inhibition of amylase production through so-called glucose inhibition, the glucose dosage must be stoichiometrically balanced relative to oxygen availability. This is typically done by adjusting the glucose dosage rate using signal feedback from a dissolved oxygen probe, where the dissolved oxygen setpoint is usually around 10%.

[0165] In this example, the above treatment is carried out as continuous fermentation in tubular fermenter 1 (see [reference]). Figure 1 , Figure 2 and Figure 4 The flow path 6 forms a meandering segment, the meandering segment including segments 22 of a first group of segments and segments 24 of a second group of segments connected in series, wherein the first group of segments has a predominantly vertical extension and the second group of segments has a predominantly horizontal extension, the first group of segments including at least four segments 22 and the second group of segments including at least three segments 24, and wherein the segments in the first group of segments and the segments in the second group of segments are arranged alternately, the segments in the first group of segments having a total length exceeding 70% of the total flow length according to the present disclosure.

[0166] The fluid containment space is filled with a liquid substrate containing a nitrogen source, nutrients, vitamins, and minerals required by the microorganisms, including 1% w / w glucose, and the fermentation fluid is inoculated with a pure preculture of Bacillus licheniformis strain. The preculture can be obtained from shake flasks, vials, or a laboratory fermenter. The pH and temperature are adjusted to optimal conditions for the microbial strain. Circulation pump 14 is started, and compressed air is continuously added to the reactor; in this example, ammonia is used to maintain pH rather than being supplied at a fixed volumetric flow rate. As the microorganisms begin to grow (which can be assessed by several parameters such as CO2 production, ammonia consumption, and changes in dissolved oxygen concentration), an 80% w / w glucose liquid solution and a liquid solution of the remaining substrate components (including nutrients, minerals, and vitamins) are continuously metered into the fermenter, and the fermentation fluid is removed from the reactor at the same rate as the glucose and nutrients are metered into the reactor. As in Example 1, carbon dioxide consumed metabolically from the carbon source (here, glucose) is degassed in tank 4, which is connected to the tubular section 6 of the fermenter, and released via valve 38 in tank 4.

[0167] As in conventional stirred tank reactors, the dissolved oxygen concentration in the reactor is maintained above 0% by automatically controlling the glucose dosage based on dissolved oxygen sensor readings, with a setpoint of 10% for dissolved oxygen.

[0168] Compared to a stirred tank reactor, in this example, air is introduced after the first circulation component 14, meaning a high volume fraction of air can be added without the risk of overflowing the circulation pump 14. Furthermore, in this example, air is introduced at the beginning of the first basic vertical tubular section 22, where the hydrostatic pressure is lowest, thereby reducing the energy required to compress the air. At a height of 8m and a diameter of 3.7m, the 85m... 3 In the stirred tank reactor, 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 contrast, the reactor with an 85m³ capacity according to this disclosure... 3 The average pressure in fermenter 1 of the specified volume is 5.4 bar, such as Figure 8B As shown, this is an important driving force for gas-liquid mass transfer, because the solubility of a gas in equilibrium with a liquid is proportional to the pressure (as stated by Henry's Law).

[0169] Compared to stirred tank reactor processing, the transfer of oxygen from the gas phase to the liquid phase is more efficient in the fermenter 1 according to this disclosure, which means that more oxygen is transferred to the fermentation fluid per unit time.

[0170] Given that the above treatments are stoichiometric in terms of nutrient supply and consumption, the yields of biomass and extracellular amylase per unit of substrate are the same in both cases. However, due to the higher gas-liquid mass transfer, the biomass and amylase productivity per unit time in the tubular fermenter 1 according to this disclosure is higher than that in a stirred tank reactor, and this is achieved with lower energy consumption.

[0171] Example 3

[0172] Example 3 describes the fermentation of hydrogenophilic bacteria to produce single-cell proteins from carbon dioxide and hydrogen.

[0173] The overall bioreaction process for producing biomass is shown in the following stoichiometric equation:

[0174] 2.1 H2 + CO2 + O,2NH3 = CH 1.8 O 0.5 N 0.2 (Biomass) + 1.5 H2O

[0175] At 30°C and atmospheric pressure, hydrogen is more than 20 times less soluble in water than oxygen, while carbon dioxide is 40 times more soluble. In this process, the extremely low solubility of hydrogen is a limiting parameter for efficient biomass production.

[0176] Fermentation is carried out in fermenter 1 (see Figure 1 , 2 The process is carried out in (4), wherein at least a portion of the flow path 6 forms a meandering segment comprising segments 22 of a first group of segments and segments 24 of a second group of segments connected in series, wherein the first group of segments has a predominantly vertical extension and the second group of segments has a predominantly horizontal extension, the first group of segments comprising at least four segments and the second group of segments comprising at least three segments, wherein the segments in the first group of segments and the segments in the second group of segments are arranged alternately, and segment 22 of the first group of segments has a total length exceeding 70% of the total flow length according to this disclosure. The fermentation process is initiated with the same basic steps as described in Example 1, but with the addition of carbon dioxide and hydrogen instead of methane and oxygen. As shown in the general stoichiometric equation above, no gas is produced other than the biomass. Since degassing is not required in tank 4, the exhaust valve 38 in the tank is closed, and the fermentation fluid circulates in the tubular section 6 and the flow path in tank 4 without losing the hydrogen and carbon dioxide added to the fluid containment space 2 of fermenter 1, thereby ensuring very high hydrogen utilization and high yield of single-cell protein per volume of added hydrogen.

Claims

1. A fermenter comprising: A fluid containment space that forms a closed-loop flow path for the fermentation fluid; At least one substrate inlet, the at least one substrate inlet being used for substrate liquid to enter the space; At least one gas injection point, said at least one gas injection point being used to inject gas into the fermentation fluid; A fermentation outlet, wherein the fermentation outlet is used to remove fermentation fluid from the space; At least one circulation component, said at least one circulation component being used to circulate the fermentation fluid in the flow path; The flow path has a total flow length, which is the distance along the flow path from the starting point to the ending point. At least a portion of the flow path forms a meandering segment, the meandering segment comprising segments from a first group of segments and segments from a second group of segments connected in series, wherein the first group of segments has a predominantly vertical extension and the second group of segments has a predominantly horizontal extension, the first group of segments comprising at least four segments and the second group of segments comprising at least three segments, and wherein the segments in the first group of segments and the segments in the second group of segments are arranged alternately. The main vertical extension is within a range of 50-130 degrees relative to the horizontal, and the main horizontal extension is within a range of + / -40 degrees relative to the horizontal. And wherein the segment in the first group of segments has a total length exceeding 70% of the total flow length.

2. The fermenter according to claim 1, wherein the length of each segment in the second group of segments is less than six times the diameter of the segments in the second group of segments.

3. The fermenter according to claim 1 or 2, wherein the fluid containment space is composed of a tank and a tubular portion in fluid connection, and wherein... The first outlet connects the tank to the tubular section to provide a flow path for the fermentation fluid from the tank to the outlet of the tubular section, and The first inlet connects the tubular portion to the tank to provide a flow path for the fermentation fluid from the tubular portion to the inlet of the tank.

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

5. The fermenter according to any one of the preceding claims further includes at least one mixing element arranged in the flow path for dispersing gas injected into the fermentation fluid.

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

7. The fermenter according to claims 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 following the first outlet.

8. The fermenter of claim 7, wherein the first circulation member is arranged between the first outlet and one of the at least one gas injection point.

9. The fermenter according to any one of claims 3-8, further comprising a first pressure control device for controlling pressure in the flow path, the first pressure control device being arranged in the last section of the first group of sections, the last section being the section immediately preceding the first inlet.

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

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

12. The fermenter according to claim 11, wherein at least one segment of the flow path forms a cross-section that is wider or narrower transversely to the flow path.

13. The fermenter according to any one of the preceding claims, wherein the internal volume of the flow path is between 1 liter and 1000 m³. 3 Within the range, preferably in the range of 0.2-500m 3 Within the range.

14. The fermenter according to any one of the preceding claims, wherein at least a portion of the meandering section is formed in a spiral shape.

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

16. The fermenter according to any one of the preceding claims further includes an additional opening disposed in the upper portion of the flow path for adding and / or removing gas.

17. The fermenter according to any one of the preceding claims further includes a plurality of flow modification elements arranged in a flow path, the flow modification elements being configured to alter the flow of the fermentation fluid.

18. A method of performing a fermentation process in a fermenter, the fermenter comprising: A fluid containment space that forms a closed-loop flow path for the fermentation fluid; At least one substrate inlet, the at least one substrate inlet being used for substrate liquid to enter the space; At least one gas injection point, said at least one gas injection point being used to inject gas into the fermentation fluid; A fermentation outlet, wherein the fermentation outlet is used to remove fermentation fluid from the space; At least one circulation component, said at least one circulation component being used to circulate the fermentation fluid in the flow path; The flow path has a total flow length, which is the distance along the flow path from the starting point to the ending point. At least a portion of the flow path forms a meandering segment, the meandering segment comprising segments from a first group of segments and segments from a second group of segments connected in series, wherein the first group of segments has a predominantly vertical extension and the second group of segments has a predominantly horizontal extension, the first group of segments comprising at least four segments and the second group of segments comprising at least three segments, and wherein the segments in the first group of segments and the segments in the second group of segments are arranged alternately. The main vertical extension is within a range of 50-130 degrees relative to the horizontal, and the main horizontal extension is within a range of + / -40 degrees relative to the horizontal. Furthermore, the segments in the first group of segments have a total length exceeding 70% of the total flow length. Fermentation fluid includes at least one fermenting microorganism. The method includes the following steps: The substrate liquid is added to the fermentation fluid in the space; Inject the gas into the fermentation fluid; The fermentation fluid is circulated along the flow path; A portion of the fermentation fluid is removed through the fermentation outlet. The method further includes a first step: increasing the pressure in the first region of the space relative to the pressure in the second region of the space to increase gas mass transfer.

19. The method of claim 18, further comprising a subsequent second step: reducing the pressure in the second region relative to the pressure in the first region to induce gas release.

20. The method of claim 18 or 19, further comprising the step of recycling a portion of the extracted fermentation fluid back into the space.

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

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

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

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

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

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