Multi-stage fermentation process

Abstract

A multi-stage fermentation process for protein and / or lipid production is described. The process comprises a first fermentation and a second fermentation. The first fermentation comprises gas fermentation by a first microorganism to produce a first product stream comprising an organic acid and / or a conjugate base thereof. The process further comprises transferring the first product stream to the second fermentation. The second fermentation comprises fermentation of the conjugate base of said organic acid of the first product stream by a second microorganism to provide a second product stream comprising protein and / or lipid. The process further comprises controlling the pH of the second fermentation by addition of a nitrogen-containing acid and / or controlling the pH of the second fermentation at a pH of greater than 8.5.

Description

[0001] Multi-Stage Fermentation Process

[0002] This application claims priority from GB2501757.5 filed 6 February 2025, the contents and elements of which are herein incorporated by reference for all purposes.

[0003] Field of the Invention

[0004] The present invention relates to a multi-stage fermentation process for biomass production

[0005] Background

[0006] Many industrial processes produce carbon dioxide (CO2), and solutions for effective carbon capture from such processes are being sought. Acetogenic bacteria are a group of bacteria capable of utilising CO2 (or carbon monoxide (CO)) as the sole carbon source, or a combination of CO2 and CO as carbon sources. The use of acetogenic bacteria has therefore been proposed for capturing CO2 from industrial processes. At the same time, these bacteria can also be used to produce useful chemicals, such as acetic acid. Additionally, to meet the nutritional demand of the world’s growing population, while minimizing the environmental burden of traditional agricultural processes, sources of alternative proteins and fats are being sought. Proteins and fats derived from fermentation of microorganisms are one possible source of such alternative proteins. However, a substrate must be provided to grow these microorganisms on -typically this is sugar, or a sugar-based byproduct.

[0007] Growth of such bacteria and microorganisms is typically conducted by fermentation processes using aqueous fermentation broths. During such fermentation processes, acid and / or base is often added to the fermentation broth in order to maintain the pH of the fermentation broth within a range suitable for growth of said bacteria. Additionally, biomass growth is typically reliant on provision of a nitrogen source that can be assimilated by the bacteria, so ammonia is often added to the fermentation broth.

[0008] Once the concentration of a desired fermentation product in the fermentation broth has reached a target value, fermentation broth can be drawn from the fermentation vessel and the fermentation product extracted therefrom through one or more separation processes.

[0009] In conducting fermentation processes and the downstream separation of fermentation products, waste streams containing high concentrations of salts and nitrogenous compounds are often produced The present invention has been devised in light of the above considerations.

[0010] Summary of the Invention

[0011] In a first aspect, there is provided a multi-stage fermentation process for protein and / or lipid production, the process comprising a first fermentation and a second fermentation, wherein: the first fermentation comprises gas fermentation by a first microorganism to produce a first product stream comprising anorganic acid and / or a conjugate base thereof; the process further comprises transferring the first product stream to the second fermentation; the second fermentation comprises fermentation of the conjugate base of said organic acid of the first product stream by a second microorganism to provide a second product stream comprising a protein and / or lipids; and the process further comprises controlling the pH of the second fermentation by addition of a nitrogen-containing acid.

[0012] In the second fermentation, the conjugate base of the organic acid is consumed as the carbon source. Accordingly, absent pH control, the pH of the second fermentation would increase as more of the conjugate base of the organic acid is consumed during fermentation and the free organic acid present dissociates to maintain the acid equilibrium. Therefore, another acid (e.g. an inorganic acid) is typically added to the second fermentation in order to maintain the pH at the target pH throughout fermentation. Typically, a nitrogen source also needs to be added to the second fermentation to support metabolism and growth of the second microorganism. A common nitrogen source for fermentations is ammonia. However, if addition of ammonia (or another basic nitrogen source) is not closely matched to the consumption rate by the second fermentation, its addition and accumulation can further contribute to increasing the pH of the second fermentation. Consequently, an increased amount of acid would need to be added to maintain the pH at the target pH. This addition of large amounts of (inorganic) acid to the second fermentation for pH control would also result in the outflow of large quantities of salts (e.g. inorganic salts) from the second fermentation in the second product stream. This is particularly the case when the fermentation process is run continuously. Thus, by controlling the pH using a nitrogencontaining acid, that nitrogen-containing acid can advantageously also act as the nitrogen source for the second fermentation. In this way, it is possible to reduce the quantity of salts (e.g. inorganic salts) produced by the multi-stage fermentation in comparison to the typical arrangement discussed above. Controlling the pH of the second fermentation may comprise controlling the pH of the second fermentation at a pH of greater than 8.5. For example, the pH of the second fermentation may have a target pH of about 9. Running the second fermentation at a high pH reduces the amount of acid that is required to maintain the pH of the second fermentation at this target pH. This in turn reduces the material consumption of the second fermentation and the quantity of waste salts in the second product stream that the protein and / or lipid product may then need to be separated from.

[0013] Controlling the pH of the second fermentation using the nitrogen-containing acid may comprise controlling the flowrate of the nitrogen-containing acid to the second fermentation based on one or more of: the pH of the second fermentation, the target pH of the second fermentation, the nitrogen accumulation rate in the second fermentation, and the flowrate of the nitrogen-containing acid to the second fermentation.

[0014] Controlling the pH of the second fermentation using the nitrogen-containing acid may comprise: determining the pH of the second fermentation, determining a difference between the pH of the second fermentation and the target pH of the second fermentation, and determining a change in flowrate of the nitrogen-containing acid into the second fermentation based on said difference. The process may further comprise implementing said change in flowrate of the nitrogen-containing acid.The nitrogen-containing acid and the first product stream may be the sole nitrogen sources for the second fermentation. The first product stream may contain a nitrogen source that was added to the first fermentation in excess as a nitrogen source for the first fermentation (i.e. unassimilated nitrogen source from the first fermentation may be passed to the second fermentation in the first product stream).

[0015] Alternatively, the nitrogen-containing acid may be the sole nitrogen source for the second fermentation (i.e. there may be no excess nitrogen source in the first fermentation, such that the first product stream does not act as a nitrogen source for the second fermentation). Such operation of the multi-stage fermentation process reduces the quantity of salts produced by the multi-stage fermentation in comparison to if additional, basic, nitrogen sources are added to the second fermentation.

[0016] Alternatively, the process may further comprise providing a supplementary nitrogen source to the second fermentation in addition to the nitrogen-containing acid and, where applicable, the first product stream. The supplementary nitrogen source may not be a nitrogen-containing acid. The supplementary nitrogen source may be a nitrogen-containing base, e.g. ammonium hydroxide. Adding the supplementary nitrogen source can favour biomass production in the second fermentation, for example, by providing a nitrogen source that the second microorganism is able to assimilate at a greater rate than the nitrogen-containing acid. The amount of nitrogen (i.e. moles of nitrogen atoms) added to the second fermentation by the supplementary nitrogen source may be greater than the amount of nitrogen added to the second fermentation by the nitrogen-containing acid. Additionally, providing the supplementary nitrogen source to the second fermentation that is not a nitrogen-containing acid may allow further nitrogen to be added to the second fermentation without decreasing the pH of the second fermentation. The process may comprise controlling the flowrate of the supplementary nitrogen source to the second fermentation based on one or more of: the nitrogen accumulation rate in the second fermentation, the flowrate of the nitrogencontaining acid into the second fermentation, the pH of the second fermentation and a target pH of the second fermentation, and the pH of the supplementary nitrogen source.

[0017] Controlling the pH of the second fermentation may further comprise addition of a nitrogen-free acid to the second fermentation. This can allow the pH of the second fermentation to be adjusted independently of the concentration of nitrogen sources in the second fermentation, and thus facilitates close matching of the nitrogen source supply and consumption in the second fermentation (to minimise nitrogen concentration in the fermentation broth in the second product stream) without restricting or interfering with pH control. The nitrogen-free acid may be a nitrogen-free acid other than the organic acid produced in the first fermentation. The nitrogen-free acid may be an inorganic acid, for example, sulphuric acid or hydrochloric acid. Reducing nitrogen concentration in the fermentation broth in the second product stream is desirable in order to facilitate disposal of the remnants of the second product stream after the extraction of the protein and / or lipids therefrom.

[0018] Controlling the pH of the second fermentation using the nitrogen-free acid may comprise controlling the flowrate of the nitrogen-free acid to the second fermentation based on one or more of: the pH of the second fermentation, the target pH of the second fermentation, the nitrogen accumulation rate in the second fermentation, and the flowrate of the nitrogen-containing acid to the second fermentation. Such a process allows the pH of the second fermentation to be controlled independently of the nitrogenaccumulation rate in the second fermentation. More specifically, controlling the pH of the second fermentation by addition of nitrogen-free acid may comprise: determining the pH of the second fermentation, determining a difference between the pH of the second fermentation and the target pH of the second fermentation, determining the nitrogen accumulation rate in the second fermentation, and determining a change in flowrate of the nitrogen-containing acid and / or a change in flowrate of the nitrogen-free acid based on said difference and the nitrogen accumulation rate. Where the process further comprises providing a supplementary nitrogen source to the second fermentation, controlling the pH of the second fermentation may comprise determining said difference between the pH of the second fermentation and the target pH of the second fermentation, determining the nitrogen accumulation rate in the second fermentation, and determining a change in one or more of: the flowrate of the nitrogencontaining acid; the flowrate of the nitrogen-free acid; and the flowrate of the supplementary nitrogen source based on said difference and the nitrogen accumulation rate. In this way, the flowrates of the nitrogen-containing acid, the nitrogen-free acid and the supplementary nitrogen source can be set to move the pH of the second fermentation towards the target pH of the second fermentation and move the nitrogen accumulation rate towards zero.

[0019] The nitrogen-containing acid may be nitric acid. Nitrate ions produced by dissociation of nitric acid can be readily assimilated by microorganisms suitable for use as the second microorganism (e.g. a Geobacillus) and nitric acid is a strong (i.e. low pKa) nitrogen-containing acid.

[0020] The concentration of salts (e.g. inorganic salts, such as ammonium-based salts) in the second product stream may be less than or equal to 20 mM, or less than or equal to 15 mM, for example, between 13 mM and 15 mM. Such concentrations of salts in the second product stream mean that once the biomass has been separated from the second product stream to provide the aqueous phase stream, the aqueous phase stream can be easily disposed of. The salts in the second product stream may comprise a cation present in the supplementary nitrogen source added to the second fermentation vessel or an inorganic base added to the first fermentation vessel. The salts in the second product stream may comprise an anion present in the nitrogen-containing acid added to the second fermentation vessel or an anion present in the nitrogen-free acid added to the second fermentation vessel. The concentration of salts (e.g. inorganic salts, such as ammonium-based salts) in the second product stream may be greater than or equal to 5 mM, greater than or equal to 10 mM, or greater than or equal to 13 mM. Preferably, the concentration of salts (e.g. inorganic salts, such as ammonium-based salts) in the second product stream is less than or equal to 20 mM.

[0021] The process may further comprise controlling the pH of the first fermentation. The pH of the first fermentation may be controlled by the addition of an exogenous base (e.g. an inorganic base, such as NaOH or NH3) to the first fermentation. The production of organic acid in the first fermentation may, absent pH control, result in a reduction of the pH of the first fermentation, and accordingly, the pH of the first fermentation can be controlled to a target pH by addition of base thereto. The process may comprise maintaining the first fermentation at a pH of less than 7. The process may comprise maintaining the first fermentation at a pH of less than 6, less than 5.2, less than 5 or less than 4. The method may comprise maintaining the first fermentation at a pH of greater than 4, greater than 4.5, greater than 5, greater than5.2 or greater than 6. The method may comprise maintaining the first fermentation at a pH of less than 7 and greater than 4, for example, maintaining the first fermentation at a pH of less than 6 and greater than 4.5, or maintaining the first fermentation at a pH of less than 5.2 and greater than 4.5, e.g. less than 5 and greater than 4.5.

[0022] The protein produced by the second fermentation in the multi-stage fermentation process may be in the form of extracellular protein produced by the second microorganism and / or an intracellular protein product of the second microorganism, or the microorganism may be single-cell protein, such that the protein produced is biomass from the second fermentation. The lipid produced by the second fermentation in the multi-stage fermentation process is typically in the form of intracellular lipids produced by the second microorganism, however, the lipids produced may also, or alternatively, comprise an extracellular lipid. The second microorganism may be a different species of microorganism to the first microorganism. By way of example, the first microorganism may be capable of metabolising carbon monoxide (CO) and / or carbon dioxide (CO2) into one or more organic acids and / or conjugate bases thereof, optionally in the presence of hydrogen gas (H2). In contrast, the second microorganism may be capable of metabolising the conjugate base of the organic acid produced by the first fermentation.

[0023] The process may comprise providing one or more gases to the first fermentation as a substrate, e.g. the first microorganism may grow on said one or more gases as the main carbon source. The one or more gases may comprise CO2, CO or a mixture thereof. The one or more gases may further comprise hydrogen (H2). The one or more gases may be syngas (i.e. a mixture comprising CO and H2, and optionally further comprising CO2, methane (CH4) and / or one or more trace gases). The one or more gasses may be sparged into the first fermentation. In this way, the first fermentation can produce the target organic acid(s) from byproduct gases from industrial processes and / or from gasification of biomass. The first microorganism may be selected from the group consisting of: thermophilic acetogens; thermophilic propionibacteria; thermophilic butyric acid bacteria; thermophilic lactic acid bacteria; thermophilic succinic acid bacteria; and thermophilic hydrogen oxidising bacteria. Such microorganisms are capable of producing organic acids and withstanding such acids in high concentrations in the first fermentation broth. The first microorganism may be selected from the group consisting of: Moorella, Thermoanaerobacter, Thermoanaerobacterium, Acetogenum, Carboxydothermus, and Acetobaterium. Preferably, the microorganism is Moorella thermoacetica. In another preferred embodiment, the microorganism is Thermoanaerobacter kivui.

[0024] The organic acid may be one or more organic acids selected from the group consisting of acetic acid, butyric acid; propionic acid; lactic acid; succinic acid; pyruvic acid; tartaric acid; iso-butyric acid; hydroxybutyric acid; and hydroxyvaleric acid. Preferably, said one or more organic acids may be selected from the group consisting of acetic acid, butyric acid; propionic acid; lactic acid; and succinic acid. Most preferably, said one or more organic acids consist of acetic acid.

[0025] The first microorganism may be thermophilic. The first fermentation may comprise maintaining the temperature of the fermentation broth above 45°C, e.g. about 60°C. Such temperatures may increase the production rate of the organic acid and / or the conjugate base thereof in the first fermentation.The second microorganism may be thermophilic, and the second fermentation may optionally comprise maintaining the temperature of the fermentation broth above 45°C, e.g. about 60°C. Such temperatures may increase the production rate of protein and / or lipids in the second fermentation. Alternatively, the second microorganism may be mesophilic, and the second fermentation may comprise maintaining the temperature of the fermentation broth at or below 45°C, e.g. about 37°C.

[0026] The second microorganism may be able to assimilate nitrate ions as a nitrogen source for fermentation. The second microorganism is able to grow at a pH of the second fermentation of greater than 8.5. The second microorganism may be a fungus (e.g. a yeast) or a bacterium. The second microorganism may be a Geobacillus or Bacillus, e.g. Bacillus subtilis.

[0027] The first fermentation may be conducted in a first fermentation vessel. The second fermentation may be conducted in a second fermentation vessel. The first fermentation vessel may be distinct from the second fermentation vessel. A fermentation vessel may be any tank that performs a biocatalytic conversion reaction (e.g. an enzymatic reaction or microorganism-based reaction). A fermentation vessel may have a set of monitored and (further optionally) controlled parameters; the set of parameters may include one or more of temperature, pH, liquid level, optical density, and pressure.

[0028] The multi-stage fermentation may be operated as a continuous process. The present invention is particularly advantageous in the context of a continuous fermentation process due to the quantity of salt that would otherwise be produced if a nitrogen-containing acid was not used for pH control.

[0029] The process may comprise providing a continuous liquid nutrient feed to the first fermentation vessel. The liquid nutrient feed may comprise an aqueous solution of salts (e.g. magnesium salts, calcium salts, and / or chloride salts), a nitrogen source (e.g. yeast extract or urea), trace elements, and vitamins in order to support maintenance and growth of the first microorganism.

[0030] The first fermentation may be anaerobic. The second fermentation may be aerobic or microaerobic. Accordingly, the process may further comprise supplying oxygen to the second fermentation vessel (e.g. sparging oxygen into the second fermentation vessel). The process may comprise continually supplying oxygen to the second fermentation vessel. Microaerobic may be defined as having a dissolved oxygen content in the second fermentation broth of greater than or equal to 0.1 % and / or less than or equal to 15 %. Aerobic may be defined as having a dissolved oxygen content in the second fermentation broth of greater than 15 %. Alternatively, the second fermentation may be anaerobic, and the process may comprise supplying an electron acceptor other than oxygen (e.g. nitrate, iron (III), manganese (IV), sulphate, hydrogen, carbon monoxide or carbon dioxide) to the second fermentation vessel.

[0031] The process may further comprise a biomass removal step on the first product stream to remove biomass therefrom. In this way, the second fermentation is not contaminated with the first microorganism. The biomass resulting from the biomass removal step may be recycled to the first fermentation in order to maintain the biomass density of the first fermentation. The biomass removal step may be conducted by centrifugation, sedimentation, and / or membrane separation. The biomass removal step may provide a biomass-depleted first product stream.The process may further comprise a concentration step on the first product stream (or the biomass-depleted first product stream). The concentration step may remove water from the first product stream, for example, the concentration step may be configured such that the only liquid compound removed from the first product stream between the first fermentation and the second fermentation is water. The concentration step may increase the total concentration of acetic acid and the conjugate base thereof in the first product stream by more than 50% (i.e. more than 1.5 times the concentration prior to the concentration step), more than 75%, or more than 100%. The concentration step may increase the total concentration of acetic acid and the conjugate base thereof in the first product stream by less than 150%. The concentration step may be conducted by one or more of the techniques selected from the group consisting of: membrane filtration, nanofiltration, and evapoconcentration. Preferably, the concentration step is conducted by membrane filtration (e.g. reverse osmosis). The concentration step may provide a concentrated first product stream. It can be understood that one or more compounds dissolved in the first product stream may be removed in solution with the water removed from the first product stream (e.g. dissolved salts or acetate in solution in the water removed by the concentration step). However, the concentration of the one or more compounds dissolved in the water removed by the concentration step may be lower than the concentration of said one or more compounds dissolved in the first product stream. The biomass removal step and / or concentration step may be the only intermediate step(s) between the first fermentation and the second fermentation.

[0032] Alternatively, the liquid composition of the first product stream may be unchanged between the first fermentation and the second fermentation. For example, the biomass removal step may be present, and the concentration step may not be present.

[0033] The second fermentation may produce carbon dioxide. The process may further comprise recycling the carbon dioxide (e.g. a portion, or all, of the carbon dioxide) produced by the second fermentation to the first fermentation, for example, gas comprising carbon dioxide may be drawn from the second fermentation vessel and recycled to the first fermentation vessel. In this way, a by-product of the second fermentation can be recycled to the first fermentation where it provides the substrate for the fermentation. This can reduce the carbon dioxide emissions from the multi-stage fermentation process. The second fermentation may be aerobic and accordingly gas drawn from the second fermentation vessel may comprise carbon dioxide and oxygen. The process may further comprise a carbon dioxide separation unit configured to separate carbon dioxide from other gaseous species (e.g. oxygen) contained in gas drawn from the second fermentation vessel. A carbon dioxide stream from the carbon dioxide separation unit may be recycled to the second fermentation. The carbon dioxide stream may comprise substantially no oxygen (e.g. less than about 100 ppm). The carbon dioxide separation unit may provide a waste gas stream comprising the other gaseous species (e.g. oxygen) separated from the carbon dioxide stream. The waste gas stream may be recycled to the second fermentation vessel. The carbon dioxide separation unit may comprise an adsorption unit, a membrane separation unit, or a cryogenic distillation unit. In this way, the carbon dioxide produced by the second fermentation can be recycled to the first fermentation unit without introducing oxygen into the first fermentation vessel, such that the first fermentation remains anaerobic and / or the first microorganism is not inhibited.The process may further comprise a separation step comprising separation of the second product stream into a biomass stream and an aqueous stream. The separation step may be conducted by one or more of the techniques selected from the group consisting of: membrane filtration, nanofiltration, centrifugation, settling, sedimentation, or acoustic separation. The aqueous stream (e.g. a portion, or all, of the aqueous stream) may be recycled back to the first fermentation and / or the second fermentation. The aqueous stream may comprise one or more nutrients, and accordingly recycling the aqueous stream back to the fermentations can reduce the consumption of virgin nutrients in the process.

[0034] The total productivity of the organic acid and conjugate base thereof in the first fermentation may be greater than or equal to 8 mM / hour, greater than or equal to 12 mM / hour, greaterthan or equal to 17 mM / hour, greater than or equal to 25 mM / hour, greater than or equal to 33 mM / hour, greater than or equal to 40 mM / hour, or greater than or equal to 50 mM / hour. The total productivity may be calculated based on the organic acid produced by the microbial fermentation over a time period of 1 hour, 6 hours, 12 hours, 1 day, 2 days or 1 week. The total productivity may be defined as the productivity of both the free acid and dissociated / conjugate base forms of the organic acid.

[0035] The total concentration of the organic acid and the conjugate base thereof in the first product stream, prior to a concentration step, where present, may be greaterthan 0.25 M, for example, greaterthan or equal to 0.6 M, or greater than or equal to 1.0 M. The total concentration of the organic acid and the conjugate base thereof in the first product stream, prior to a concentration step, where present, may be less than 1.15 M. The total concentration of the organic acid and the conjugate base thereof in the first product stream, prior to a concentration step, where present, may be greaterthan or equal to 0.25 M and less than or equal to 1.15 M, e.g. greater than or equal to 0.6 M and less than or equal to 1.15 M.

[0036] One or more alcohols may be produced in small amounts as a by-product of the first fermentation. Said one or more alcohols may be selected from the group consisting of ethanol; butanol; propanol; propylene glycol; butane-1,4-diol; butane-1,2,3,4-tetrol; isobutanol; butane-1,3-diol; butane-1,2-diol; and pentane-1 ,4-diol. Preferably, said one or more alcohols may be selected from the group consisting of ethanol, butanol, propanol, propylene glycol and butane-1,4-diol. Most preferably, said one or more alcohols consist of ethanol.

[0037] Where the first fermentation produces one or more alcohols as a by-product, the ratio of the concentration of one or more organic acids (i.e. the total organic acid concentration) in the first product stream to the concentration of the one or more alcohols in the second product stream may be greaterthan or equal to 5:1, greater than or equal to 10:1, greater than or equal to 20:1. Preferably, said ratio is greater than or equal to 20: 1 , more preferably greater than or equal to 50: 1. Said ratio may be less than or equal to 1000:1. In one embodiment, the first fermentation may produce substantially no alcohol.

[0038] Where the first fermentation produces one or more alcohols as a by-product, the concentration of the one or more alcohols in the first product stream, prior to a concentration step, where present, may be less than or equal to 0.25 M, less than or equal to 0.15 M, less than or equal to 0.05 M, or less than or equal to 0.02 M, preferably less than or equal to 0.001 M. The concentration of the one or more alcohols in the first product stream, prior to a concentration step, where present, may be greaterthan or equal to 0.0001M. The first product stream may contain substantially no alcohol (e.g. where the first fermentation produces substantially no alcohol).

[0039] The dilution rate of the first fermentation vessel may be about 0.1 hr1, for example, greater than or equal to 0.05 hr1and less than or equal to 0.12 hr1. The dilution rate of the second fermentation vessel may be about 0.15 hr1, for example, greater than or equal to 0.12 hr1and less than or equal to 0.25 hr1, e.g. less than or equal to 0.22 hr1. As the protein and / or lipids product from the second fermentation is present in the fermentation broth in the second fermentation (either intracellular or extracellular), the production rate of the protein and / or lipids by the process is a function of the dilution rate, with such dilution rates supporting desired production rates.

[0040] The first fermentation may be an industrial scale process. By way of example the first fermentation may be at at least 1 m3scale, at least 5 m3scale, at least 50 m3scale, at least 150 m3scale, or at least 250 m3scale. For example, the fermentation broth in the first fermentation may have a volume of at least 1 m3, at least 5 m3, at least 50 m3, at least 150 m3, or at least 250 m3The fermentation broth in the first fermentation may have a volume of less than 500 m3 scale, less than 250 m3less than 150 m3, less than 50 m3or less than 5 m3The fermentation broth in the first fermentation broth may have a volume between 1 m3and 250 m3, for example between 5 m3and 150 m3, e.g. between 50 m3and 100 m3The second fermentation may be an industrial scale process. By way of example the second fermentation may be at at least 1 m3scale, at least 5 m3scale, at least 50 m3scale, at least 150 m3scale, or at least 250 m3scale. For example, the fermentation broth in the second fermentation may have a volume of at least 1 m3, at least 5 m3, at least 50 m3, at least 150 m3, or at least 250 m3The fermentation broth in the second fermentation may have a volume of less than 500 m3 scale, less than 250 m3less than 150 m3, less than 50 m3or less than 5 m3The fermentation broth in the second fermentation broth may have a volume between 1 m3and 250 m3, for example between 5 m3and 150 m3, e.g. between 50 m3and 100 m3.

[0041] In a second aspect, there is provided a multi-stage fermentation process for protein and / or lipid production, the process comprising a first fermentation and a second fermentation, wherein: the first fermentation comprises gas fermentation by a first microorganism to produce a first product stream comprising an organic acid and / or a conjugate base thereof; the process further comprises transferring the first product stream to the second fermentation; the second fermentation comprises fermentation of the conjugate base of said organic acid of the first product stream by a second microorganism to provide a second product stream comprising protein and / or lipid; and the process further comprises controlling the pH of the second fermentation at a pH of greater than 8.5. Running the second fermentation at a high pH reduces the amount of acid that is required to be added to maintain the pH of the second fermentation at this set point. This in turn reduces the material consumption of the second fermentation and the quantity of waste in the second fermentation that the protein and / or lipid product must be separated from. The invention includes the combination of the aspects and optional features described except where such a combination is clearly impermissible or expressly avoided.Summary of the Figures

[0042] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

[0043] Figure 1 is a process flow diagram for a multi-stage fermentation process according to the first aspect; and

[0044] Figure 2 is a schematic of the flows into and out of the second fermentation.

[0045] Detailed Description of the Invention

[0046] Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0047] Figure 1 is a process flow diagram for a multi-stage fermentation process for producing protein and / or lipids. The process comprises a first fermentation taking place in a first fermentation vessel F1 and a second fermentation taking place in a second fermentation vessel F2. The process illustrated in Figure 1 is configured to be run continuously, but can also be run in a batchwise or fed-batchwise manner, if desired.

[0048] The first fermentation facilitates the conversion of carbon dioxide (CO2) and hydrogen gas (H2) into acetic acid using a first microorganism such as Moorella thermoacetica or Thermoanaerobacter kivui, which is capable of metabolising CO2 into acetate in the presence of H2. A gas feed 6 is used to continuously sparge a mixture comprising CO2 and H2 into the first fermentation vessel F1, for example at a 0.2 VVM (volume of gas per unit volume of medium per minute). A continuous liquid nutrient feed is also provided to the fermentation vessel via liquid feed 7. The liquid nutrient feed comprises an aqueous solution of salts (e.g. magnesium salts, calcium salts, and / or chloride salts), a nitrogen source (e.g. yeast extract or urea), trace elements, and vitamins in order to support maintenance and growth of the first microorganism.

[0049] Metabolism of CO2 by M. thermoacetica or T. kivui in the presence of H2 as an energy source results in the production of acetate (i.e. the conjugate base form of acetic acid). As a result of the pKa of acetic acid and the pH in the first fermentation vessel F1, both acetate and free acetic acid (i.e. the protonated form of acetic acid) will be present in the fermentation broth. It can be appreciated that the total concentration of acetic acid (i.e. the sum of the concentration of acetate and free acetic acid) in a solution is independent of the pH of that solution.

[0050] The process comprises temperature and pH control in order to maintain these at appropriate values for the microorganism (e.g. a pH of about 6.5 and a temperature of about 60°C). The temperature control is not illustrated in Fig. 1. The pH control is executed by controlling the flow of base stream 11; The production of acetate by M. thermoacetica or T. kivui requires a base (e.g. an inorganic base, such asNaOH or NH3) to be added to the first fermentation vessel F1 via the base stream 11 in order to maintain the pH of the fermentation broth, which would otherwise fall.

[0051] A first product stream 1 is continuously drawn from the first fermentation vessel F1. The first product stream 1 comprises cellular biomass and an aqueous solution comprising acetate and free acetic acid. Accordingly, the first product stream 1 is passed to a biomass removal unit S1 (e.g. a filtration or centrifugation unit) in which the cellular biomass is removed from the aqueous solution. The retained cellular biomass from the biomass removal unit S1 is recycled to the first fermentation vessel F1 in the first fermentation biomass recycle stream 10. The liquid from the biomass removal unit S1 is then passed to a concentration unit S2 as a filtered first product stream T. The concentration unit S2 is configured to concentrate the filtered first product stream T by removing water therefrom (e.g. via evapoconcentration or membrane separation). By way of example, the filtered first product stream T may have a total acetic acid concentration of 0.6 M, and the concentrated first product stream 1” may have a total acetic acid concentration of double that in the filtered first product stream 1 ’ (i.e. 1.2 M).

[0052] The concentrated first product stream 1” is then passed to the second fermentation unit F2.

[0053] In the second fermentation unit F2, a second fermentation facilitates the consumption of the acetate fed into the second fermentation unit F2 for the production of protein (e.g. extracellular or intracellular protein) and / or lipids. The second fermentation can be conducted by a second microorganism such as Bacillus subtilis under aerobic mesophilic conditions, with a temperature of about 37°C and oxygen continuously sparged into the second fermentation vessel F2 via oxygen feed 9.

[0054] To support growth and protein / lipid production in the second fermentation vessel F2, pH is controlled at a predetermined set point of around 8.5 for Bacillus subtilis. Due to consumption of acetate as the fermentation substrate, absent pH control, the pH in the second fermentation vessel F2 would be expected to rise. Feeding of further acetic acid by the concentrated first product stream 1” contributes towards maintaining the desired pH. However, in practice, a further acid source is used for reliable pH control (e.g. to be able to account for upstream process disturbances and for start-up of the process by providing a further control variable). Additionally, to favour protein and / or lipid formation, a nitrogen source should be added into the second fermentation vessel.

[0055] In conventional fermentation processes, ammonia is used as a nitrogen source for fermentation, as ammonia is readily assimilated by a range of microorganisms. However, ammonia is also basic.

[0056] Consequently, in the context of the present process, if addition of ammonia (or another basic nitrogen source) is not closely matched to the consumption rate by the second fermentation, its addition can further contribute to increasing the pH of the second fermentation. In turn, an increased amount of acid would need to be added to maintain the pH at the target pH, and this would contribute to greater salt outflows from the second fermentation, which is undesirable. Thus, the present process comprises addition of nitric acid (as an example of a nitrogen-containing acid) to the second fermentation vessel F2 via nitrogen-containing acid stream 3, which then acts both as the nitrogen source for the second fermentation and also the acid source for pH control. In this way, it is possible to reduce the quantity ofsalts (e.g. inorganic salts) that is produced by the multi-stage fermentation compared to a process in which a basic nitrogen is the sole nitrogen source added to the second fermentation vessel F2.

[0057] As illustrated in Fig. 1, in addition to the nitrogen-containing acid stream 3, the process is also configured to allow a supplementary nitrogen source (in the form of ammonium hydroxide in Fig. 1 ) to be added to the second fermentation vessel F2 via the supplementary nitrogen source stream 4. Due to the different rates at which different nitrogenous compounds are assimilated by microorganisms, having a supplementary nitrogen source can allow total nitrogen assimilation by Bacillus subtilis to be increased to support protein and / or lipid production, whilst still reducing salt production in the second fermentation compared to if the only nitrogen source was a base.

[0058] Additionally, the process flow diagram in Fig.1 illustrates the presence of a nitrogen-free acid stream 5 feeding into the second fermentation vessel F2, through which sulphuric acid can be supplied to the second fermentation. Providing the nitrogen-free acid stream 5 in addition to the nitrogen-containing acid stream 3 allows the pH of the second fermentation to be adjusted independently of the concentration of nitrogen sources in the second fermentation, and thus facilitates close matching of both I) the nitrogen supply and consumption and II) the pH to the target pH in the second fermentation with reduced crosstalk.

[0059] The control of the second fermentation is discussed further in relation to Fig. 2 below.

[0060] A second product stream 2 is continuously drawn from the second fermentation vessel F2. The second product stream 2 may comprise a mixture of biomass and intracellular and extracellular products (e.g. intracellular and extracellular protein) and an aqueous solution. Accordingly, the second product stream 2 is passed to a separation unit S3 (e.g. a filtration or centrifugation unit) in which the cellular biomass (containing intracellular proteins and / or lipids) and the extracellular proteins and / or lipids are separated from the aqueous solution. The aqueous solution passes into an aqueous stream 2” that Fig. 1 illustrates as being recycled back to the first fermentation vessel F1. It can be understood that the aqueous stream 2” (e.g. a portion, or all, of the aqueous stream recovered from the separation unt S3) may be recycled to the first fermentation vessel F1 or the second fermentation vessel F2. The aqueous stream 2” comprises one or more nutrients, and accordingly recycling the aqueous stream 2” back to the first and / or second fermentation can reduce the consumption of virgin nutrients in the process.

[0061] Gas is also drawn from the headspace of the second fermentation vessel F2. The second fermentation may produce CO2 and is operated aerobically; accordingly, the gas drawn from the second fermentation vessel F2 comprises a mixture of CO2 and oxygen. In order to reduce CO2 emissions from the multi-stage fermentation process, the CO2 drawn from the second fermentation vessel F2 is recycled to the first fermentation vessel F1. However, because the first fermentation vessel is operated anaerobically, the process further comprises a carbon dioxide separation unit S4 (e.g. an adsorption unit) that receives the gas drawn from the headspace of the second fermentation vessel F2, separates the oxygen from the CO2, and provides a CO2 stream 8 that is recycled to the first fermentation vessel F1.

[0062] Fig. 2 a schematic of the flows into and out of the second fermentation vessel F2. The concentrated first product stream 1” provides an inflow of acetate (MAC, in) and nitrogen (MNJH) into the second fermentationvessel F2. The oxygen feed 9 provides an inflow of oxygen (Mo2 n) into the second fermentation vessel F2, which operates under aerobic conditions. The nitrogen-containing acid stream 3 provides an inflow of acid for pH control and nitrogen (MN-acid n) into the second fermentation vessel F2. The supplementary nitrogen source stream 4 provides an inflow of additional nitrogen source (i.e. in addition to the nitrogen from the concentrated first product stream 1” and the nitrogen from the nitrogen-containing acid stream 3) (MNx n). The nitrogen-free acid stream 5 provides an inflow of nitrogen-free acid (Mnxjn) into the second fermentation vessel F2. The second product stream 2 that is drawn from the second fermentation vessel F2 provides an outflow of acetic acid (Mac, out), nitrogen (MN.OUO, and biomass (Mx,out) out of the second fermentation vessel F2. Additionally, gas may be drawn from the second fermentation vessel, leading to an outflow of oxygen (Mo2,out) and CO2 (Mco2,out)from the second fermentation vessel F2.

[0063] The second fermentation in the second fermentation vessel F2 has an acetate consumption rate (rAc), a nitrogen consumption rate ( ) and a biomass growth rate (p).

[0064] In operating the second fermentation, the target is to maximise the production of biomass (i.e. maximise Mx,out). Biomass production is a function of the biomass growth rate (p) as set by the dilution rate of the second fermentation vessel F2 (which can be set independently of the dilution rate of the first fermentation vessel F1). Additionally, it is desired that all of the acetic acid (both acetate and free acetic acid) fed to the second fermentation should be consumed by the second fermentation (i.e. MAc,out= 0) in order to prevent loss of the carbon source.

[0065] The concentrated first product stream 1” that is fed to the second fermentation vessel F2 is at pH 6 and contains a high concentration of acetate. The fed acetate (MAC, in) is consumed by the second microorganism at rate TAC, which is a factor of the biomass growth rate (p) (amongst other factors), As acetate is consumed, additional acid needs to be added to the second fermentation vessel F2 in order to maintain the fermentation pH. The pH is measured online and dynamically maintained at a set point pH through the dose-controlled addition of acid from the nitrogen-containing acid stream 3 (MN-acid,in) and / or the nitrogen-free acid stream 5 (Mnxjn). A high set point pH can be selected in order to reduce the amount of acid (MN-acid n + Mnx n) that needs to be added to main fermentation pH at the set point pH (i.e. Mn-acidjn + Mnxjn is a function of the rate of acid consumption (TAG) and the set point pH).

[0066] When adding acid for pH control, the conjugate base of the added acid typically remains in solution and is fed off in a waste salt. As discussed above, to reduce salt production in the second fermentation vessel F2, at least one of the acids added to the second fermentation vessel F2 for pH control in the present process is nitrogen-containing acid HNO3. The conjugate base nitrate ion (NO3 ) of nitric acid can be assimilated by the microorganism in the second fermentation, and typically the inflow of nitrogen, MNJH, from the first fermentation vessel F1 is insufficient, and biomass production can be increased by assimilation of the nitrogen provided by the nitrate ions. The overall nitrogen consumption rate of the biomass, TN, is a function of the biomass growth rate, p. When using a nitrogen-containing acid for pH control and nitrogen supply, three scenarios can arise:

[0067] I. When MN-acidjn (mol / h) + MNJH (mol / h) = rN (mol / h), no additional nitrogen source is needed (i.e.

[0068] MNXJH = 0), and pH control is conducted with just the nitrogen-containing acid (Mnxjn = 0).II. When MN-acid,in (mol / h) + MN H (mol / h) > rN (mol / h), nitrogen will accumulate in the second fermentation vessel F2 and be fed out in the second product stream 2 (i.e. MN.OUI > 0).

[0069] III. When MN-acid,in (mol / h) + MN H (mol / h) < (mol / h), there is insufficient nitrogen to sustain the desired biomass growth rate ( ), and the realised biomass growth rate will fall overtime.

[0070] Operation under scenario I is preferred, as it reduces salt production in the second fermentation vessel F2 and reduced the amount of nitrogen in waste streams from the process. However, through the use of the supplementary nitrogen source stream 4 and the nitrogen-free acid stream 5 and dynamic control loops, stable performance of the process under scenarios II and III is also possible.

[0071] Under scenario II, in order to reduce MN.OUI back towards zero, the flowrate of nitrogen-containing acid (MN-acidjn) should be reduced, and in order to simultaneously maintain the pH at the set point pH, an additional nitrogen-free acid (e.g. H2SO4) should be supplied to the second fermentation vessel F2 via nitrogen-free acid stream 5 (i.e. Mnx n > 0).

[0072] Under scenario III, in order to maintain the desired biomass growth rate , a supplementary nitrogen source that can be assimilated by the microorganism (e.g. NH4OH) should be supplied to the second fermentation vessel F2 via the supplementary nitrogen source stream 4 (i.e. MNX H > 0). Where the supplementary nitrogen source is basic and its addition leads to a fall in pH, corresponding pH control may be provided by addition of nitrogen free acid to the second fermentation vessel F2 via nitrogen-free acid stream 5 (i.e. Mnx n > 0).

[0073] The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

[0074] While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.

[0075] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations.

[0076] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0077] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example + / - 10%.

[0078] Examples

[0079] Example 1

[0080] To demonstrate the growth of a protein producer on defined minimal medium with acetate as sole carbon source and nitrate as sole nitrogen source, the growth of Bacillus licheniformis DSM13 under such conditions was investigated.

[0081] The medium was prepared according to the following recipe:

[0082] - 4.5 g / L MgCI2*6H2O,

[0083] - 0.05 g / L CaCI2*2H2O,

[0084] - 1 g / L KH2PO4,

[0085] - 1 g / L NaCI,

[0086] - 0.24 g / L MgSO4,

[0087] - 2 g / L NaNO3,

[0088] supplemented with a trace element mix and a vitamin solution.

[0089] Acetate was added to the medium as the carbon source, and the pH was adjusted to pH 9.0. After sterilisation, 25 ml of medium was dispensed into a 250 mL shake flask and inoculated with a 1:50 dilution of an overnight preculture comprising Bacillus licheniformis DSM13. Cultures were left in a shaking incubator to grow at 37°C. Growth was measured by determined cell dry weight, and acetate consumption measured with standard HPLC practice.

[0090] The above protocol resulted in the formation of 1.68 gCdw / L biomass, with full acetate consumption. Microscopy confirmed live cells under the tested conditions.

[0091] Example 2

[0092] Spent gas fermentation broth provided from the first fermentation was used to assess the growth of different fungi on such a broth.

[0093] A representative gas fermentation was run in accordance with that described in WO2024074714A1, which is hereby incorporated by reference.The spent broth from the first fermentation was filtered (0.2 pm pore size) to remove residual biomass. The filtered spent broth was dispensed into 250 ml shake flasks, each flask receiving 25 ml. Upon inoculation 1:50 with an overnight preculture comprising each specific strain, the flasks were left to grow at appropriate temperatures (see table 1). Growth was measured by determined cell dry weight, and acetate consumption measured with standard HPLC practice. The results for different fungi are presented in Table 1.

[0094] Table 1

[0095]

[0096] Table 1 demonstrates that three of the fungi investigated were able to accumulate biomass on spent broth from gas fermentation with minimal intervention between process steps (only filtering).

[0097] Example 3

[0098] Protein production by a microorganism on a medium with acetate as the sole carbon source, nitrate as the sole nitrogen source, and at pH 9 has been investigated.

[0099] B. licheniformis was used as the organism, and was transformed with a green fluorescent protein (GFP)-expression plasmid according to standard procedures (as set out in Bacillus Licheniformis Transformation Protocol, Protocol No. 4308915.505 - 08 / 2003).

[0100] The same medium recipe as in Example 1 was used to grow the wild type (WT) strain and the strain with the GFP-expression plasmid. The strains were grown in a BioT ek Synergy H1 ™ plate reader and the medium was either supplemented with nitrate ions, or not supplemented with a nitrogen source as a control.

[0101] Growth of the strains was measured by light absorbance at 630 nm and GFP absorption measured with excitation at 485 nm and absorbance at 515 nm. Table 2 reports the maximum optical density (OD) and GFP absorbance as averages of the values from biological quadruplicates. Note that the GFP strains reached the maximum absorbance of the equipment, at 100000 units.

[0102]

[0103] Substantially higher Max OD was observed for both the WT strain and GFP strain when supplying nitrate as the sole nitrogen source in comparison to the control of no nitrogen source.

Claims

Claims:

1. A multi-stage fermentation process for protein and / or lipid production, the process comprising a first fermentation and a second fermentation, wherein:the first fermentation comprises gas fermentation by a first microorganism to produce a first product stream comprising an organic acid and / or a conjugate base thereof;the process further comprises transferring the first product stream to the second fermentation; the second fermentation comprises fermentation of the conjugate base of said organic acid of the first product stream by a second microorganism to provide a second product stream comprising protein and / or lipids; andthe process further comprises controlling the pH of the second fermentation by addition of a nitrogen-containing acid.

2. The multi-stage fermentation process according to claim 1, wherein the pH of the second fermentation is controlled at a pH of greater than 8.5 by addition of the nitrogen-containing acid.

3. A multi-stage fermentation process for protein and / or lipid production, the process comprising a first fermentation and a second fermentation, wherein:the first fermentation comprises gas fermentation by a first microorganism to produce a first product stream comprising an organic acid and / or a conjugate base thereof;the process further comprises transferring the first product stream to the second fermentation; the second fermentation comprises fermentation of the conjugate base of said organic acid of the first product stream by a second microorganism to provide a second product stream comprising protein and / or lipid; andthe process further comprises controlling the pH of the second fermentation at a pH of greater than 8.5.

4. The multi-stage fermentation process according to claim 3, wherein the process comprises controlling the pH of the second fermentation by addition of nitrogen-containing acid.

5. The multi-stage fermentation process according to any one of claims 1 , 2 or 4, wherein:the nitrogen-containing acid and the first product stream are the sole nitrogen sources in the second fermentation; orthe nitrogen-containing acid is the sole nitrogen source in the second fermentation.

6. The multi-stage fermentation process according to any one of claims 1 , 2, or 4 wherein the process further comprises providing a supplementary nitrogen source to the second fermentation in addition to the nitrogen-containing acid.

7. The multi-stage fermentation process according to any one of the preceding claims, wherein controlling the pH of the second fermentation further comprises addition of a nitrogen-free acid to the second fermentation.

8. The multi-stage fermentation process according to any one of claims 1, 2, 4, 5, 6, or 7 as dependent on any one of claims 1 , 2, 4, 5, or 6, wherein the nitrogen-containing acid is nitric acid.

9. The multi-stage fermentation process according to any one of the preceding claims, wherein the process further comprises a biomass removal step on the first product stream to remove biomass therefrom.

10. The multi-stage fermentation process according to any one of the preceding claims, wherein the process further comprises a concentration step on the first product stream.

11. The multi-stage fermentation process according to claim 10, wherein the only liquid compound removed from the first product stream between the first fermentation and the second fermentation is water.

12. The multi-stage fermentation process according to any one of claims 1 to 7, wherein the liquid composition of the first product stream is unchanged between the first fermentation and its transfer to the second fermentation.

13. The multi-stage fermentation process according to any one of the preceding claims, wherein:the second fermentation produces carbon dioxide; andthe process further comprises recycling the carbon dioxide produced by the second fermentation to the first fermentation.

14. The multi-stage fermentation process according to any one of the preceding claims, wherein the process further comprises a separation step comprising separation of the second product stream into a biomass stream and an aqueous stream.

15. The multi-stage fermentation process according to claim 14, wherein the process further comprises recycling the aqueous stream to the first fermentation and / or the second fermentation.

16. The multi-stage fermentation process according to any preceding claim, wherein the total productivity of the organic acid and conjugate base thereof in the first fermentation is greater than or equal to 8 mM / hour.

17. The multi-stage fermentation process according to any preceding claim, wherein the total concentration of the organic acid and the conjugate base thereof in the first product stream, prior to a concentration step, where present, is greater than 0.25 M.

18. The multi-stage fermentation process according to any preceding claim, wherein the concentration of salts in the second product stream is less than or equal to 20 mM.

19. The multi-stage fermentation process according to any preceding claim, wherein:the second fermentation is aerobic and / or microaerobic; andthe process further comprises supplying oxygen to the second fermentation.

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