Fermentation system and method

Abstract

A fermentation system, the system comprising: a vessel; a recirculation loop, connected to the vessel, comprising: three or more inlet conduits, provided inside the vessel, configured to recirculate fluid into the vessel and to induce helical fluid flows within the vessel; an aeration system, comprising an in-line diffuser provided in the recirculation loop; a feed system, and a process control system, wherein, in use, the fermentation system is configured for the production of biological material.

Description

[0001] Fermentation system and method

[0002] FIELD

[0003] The present disclosure relates to a fermentation system and an associated method of producing biological material. The disclosure includes methods of re-engineering / retrofitting brewing fermenters for use in the production of biological material, and the resulting fermentation systems.

[0004] BACKGROUND

[0005] Conventional production and supply systems are not sustainable to address global population growth and environmental concerns. Furthermore, there is a growing demand for plant-based or animal-free products to replace conventional sources of protein from animals. In light of this, scaled food biotechnology is an area of interest for the large-scale production of exogenous bio-products including but not limited to proteins.

[0006] Fermentation is commonly used within the food processing industry and has the potential to allow the production of a wide range of biological materials. These biological materials, for example proteins, may form the basis for foodstuffs to replace conventional livestock based proteins, such as eggs and dairy.

[0007] Currently biotechnology precision fermentation production processes for the production of biological materials like proteins makes use of complex, fully aerobic aseptic fed-batch fermenters to create high yields of biological material. These processes are relatively small scale (e.g. in terms of total precision fermentation capacity), have high capital and operating costs. Additionally, they are operationally highly complex with operations optimized depending on the target biological material.

[0008] One challenge is the scale up of such complex precision fermentation processes to a commercial scale that allows the production of useful food products at a competitive cost.

[0009] SUMMARY

[0010] According to a first aspect of the invention there is provided a fermentation system, the system comprising: a vessel; a recirculation loop, connected to the vessel, comprising: three or more inlet conduits, provided inside the vessel, configured to recirculate fluid into the vessel and to induce helical fluid flows within the vessel; an aeration system, comprising an in-line diffuser provided in the recirculation loop; a feed system, and a process control system, wherein, in use, the fermentation system is configured for the production of biological material.

[0011] The vessel may be a low pressure vessel (e.g. a brewing fermenter). A low pressure vessel is defined as a vessel pressure rated up to 1.5 bar(g)

[0012] 15216541-1The provision of a recirculation loop with three or more inlet conduits and an aeration system, comprising an in-line diffuser, results in an increased oxygen transfer rate to the contents of the fermentation system, allowing the use of low pressure vessels for partial aerobic fermentation (for example, precision fermentation). Low pressure vessels are widely-available and economic.

[0013] The inlet conduits agitate fluid within the vessel by recirculating the fluid, without introduction of further structures, simplifying partial-aerobic fed batch fermentation system production and / or retrofit of existing brewing fermenters.

[0014] The inlet conduits are configured to induce helical fluid flows within the pressure vessel, which are effective in promoting mixing within the pressure vessel.

[0015] The use of three or more inlet conduits increases the local fluid velocities throughout the vessel, enhancing mixing and jet penetration and thus oxygen transfer efficiency.

[0016] The distal portions of the inlet conduits may be angled at 25 degrees or more below the horizontal.

[0017] Distal potions of the inlet conduits may be angled at 75 degrees or less below the horizontal.

[0018] The distal portions may be angled at 40 degrees or more below the horizontal. The distal portions may be angled at 60 degrees or less below the horizontal.

[0019] The angle between a distal portion and the rest of the inlet conduit may be around 45 degrees.

[0020] The angles relative to the horizontal refer to their orientation in the nominal upright position of the system.

[0021] The angle of the distal portions improves penetration of the recirculation flow below the outlet level. The outlet level is the level along the height of the vessel at which outlets of the conduits are situated

[0022] Distal potions of the inlet conduits may be tangentially oriented relative to sides of the vessel.

[0023] Fluid outlets of the inlet conduits may be positioned at a level corresponding to 5% or more of the vessel height.

[0024] Advantageously, the level of the outlets corresponding to 5% or more of the total tank height may avoid direct suction of the recirculation flows back into the recirculation loop through the vessel bottom. This may ensure that the recirculation flow can circulate throughout the vessel.

[0025] 15216541-1Fluid outlets of the inlet conduits may be positioned at a level corresponding to 30% or less of the vessel height.

[0026] Advantageously, the level of the outlets corresponding to 30% or less of the total tank height may minimise the formation of stagnant oxygen-deficient zones near the vessel bottom. In addition, the gas holdup of the system is increased.

[0027] The distal portion of each inlet conduit may be angled relative to the rest of the inlet conduit.

[0028] Advantageously, the provision of angled distal portions allows inlet vertical conduits to induce flows with a tangential component.

[0029] The fluid outlets of each inlet conduit may be disposed up to half the vessel radius from the vessel wall.

[0030] The fluid outlets of each inlet conduit may be radially situated around one third of the vessel radius from the vessel walls.

[0031] The inlet conduits may be mounted through a top plate for the vessel.

[0032] The inlet conduits may be mounted through a side wall of the vessel.

[0033] The aeration system may be configured to supply oxygen-enriched air.

[0034] Oxygen-enriched air denotes any gas containing a greater oxygen concentration than atmospheric air. Alternatively, Oxygen-enriched air may denote gases with an oxygen concentration greater than 25%. Alternatively, Oxygen-enriched air may denote gases with an oxygen concentration greater than 30%. Alternatively, Oxygen-enriched air may denote gases with an oxygen concentration greater than 40%. It will be understood that the term ‘air’ used in the following description is used as a generic designation for any gas mixture comprising oxygen, and not necessarily gas with an atmospheric composition.

[0035] Advantageously, by increasing oxygen concentration of the gas, the gas-liquid interfacial area can be reduced for a given oxygen transfer rate, allowing a reduction in the flowrate of introduced gas and agitation power at a reduced internal pressure. Increasing oxygen concentration of the gas reduces the volume of gas needed to achieve a given oxygen transfer rate (OTR). Lowering the pressure allows the use of lower power compressors and use or repurposing of low pressure vessels such as brewing fermenters for use in partially-aerobic processes. In addition to reduced pressure, the increased oxygen concentration reduces the agitation power requirement for a given OTR.

[0036] The vessel may be pressure rated up to 1 bar. The pressure vessel may be pressure rated up to 1.5 bar (e.g. brewing fermenters). In some cases, the vessel may be pressure rated up to 3 bar.

[0037] 15216541-1The vessel may be pressure rated at 0.8 bar or less. The vessel may be pressure rated at 0.6 bar or more.

[0038] The vessel may have a volume of 50 m3 or more. The vessel may have a volume of 1000 m3 or less. The vessel may have a volume of 120 m3 or more. In an example, the vessel may have a volume of 800 m3.

[0039] The volume of the vessel may be determined by the desired output. Utilising existing large-scale fermenters such as those typically present within the brewing industry cuts initial capital expenditure.

[0040] The vessel may have a height to diameter ratio of 2:1 or more. The vessel may have a height to diameter ratio of 2.2:1 or more. The vessel may have a height to diameter ratio of 3:1 or less. The vessel may have a height to diameter ratio of 3.2:1 or less.

[0041] The vessel height to diameter ratio may be selected to achieve a maximum volume per footprint area for a given maximum tolerable hydrostatic pressure for fermentation. The vessel may be a brewing fermenter.

[0042] For example, the brewing fermenter may be a fermenter typically used for the production of beer. The brewing fermenter may be pressure rated up to 1 bar. The brewing fermenter may be pressure rated up to 1.5 bar. The brewing fermenter may be pressure rated up to 0.8 bar. During beer production, fermenters may operate at (gas) pressures between around 0.1 bar and 0.4 bar. A higher pressure rated brewing fermenter may also be utilised (for example, with a pressure rating up to 1.5bar).

[0043] The vessel may comprise a cylindrical brewing fermenter. The vessel may comprise a cylindro-conical or dished-head brewing fermenter. The term dished-head denotes vessel shapes where the cylindrical tank is capped at a bottom end by a convex surface (e.g. elliptical cap, spherical cap) which provides a concave inner surface.

[0044] Alternatively, the vessel may be a biofuel fermentation vessel.

[0045] Within the traditional brewing industry, anaerobic batch (or fed-batch) fermentation is used on a large scale with the purpose of minimising biological material production and optimising yields of alcohol. The brewing fermentation process may be considered to be lower in technical complexity with lower capital and operating expenditure compared to known biotechnology fermentation processes.

[0046] The claimed fermentation system (which may be used for partial-aerobic fed batch processes) bridges the gap between known highly complex (aseptic-)aerobic fermentation systems used in biotechnology applications and anaerobic fermentation used within the brewing industry.

[0047] 15216541-1A brewing fermenter adapted to allow for the large-scale production of biological material provides a cost-effective way in which to scale up production of biological material while minimising capital expenditure, operational complexity and operational expenditure. However, the challenges in adapting, for example retrofitting a brewing fermenter for such purposes can be significant given the operational differences between a complex aerobic fermentation and the anaerobic batch process used within the brewing industry. The aeration system may be configured to supply oxygen-enriched air at a volumetric rate of 1 VVM (volume of gas per volume of vessel per minute) or less.

[0048] The aeration system may be configured to supply a gas at a volumetric rate of 0.5 VVM or less.

[0049] The aeration system may be configured to supply gas at a volumetric rate around 0.25 VVM or more.

[0050] The volume of air required may be selected based upon an optimal oxygen transfer rate.

[0051] It will be understood that the term ‘air’ used in the following description is used as a generic designation for any gas mixture comprising oxygen.

[0052] The aeration system may be configured to supply pure oxygen.

[0053] The aeration system may comprise a filter. The filter may be configured to sterilise the air prior to the air entering the fermentation system. The filter may comprise a filter, filtration membrane, or the like having a pore size of 0.2 pm or more. The filter may comprise a filter, filtration membrane, or the like having a pore size of 0.45 pm or less. The aeration system may comprise at least one cooler arranged to cool air from an air or gas supply. The air cooler may be located downstream of the air supply. The air cooler may be located upstream of the air filter. The air cooler may be located downstream of the air filter.

[0054] The system may comprise a pressure relief valve. The pressure relief valve may be connected through a top plate of the vessel, if one is present.

[0055] In a typical brewing fermenter, a pressure relief valve is provided to allow the fermenter to cope with the volume of carbon dioxide generated by yeast during the brewing fermentation process.

[0056] The pressure relief valve may have a higher rating than those valves typically present on existing brewing fermenters. Typical brewing fermenter pressure relief valves are configured to open intermittently, releasing any pressure build-up.

[0057] The vessel of the present disclosure may comprise a pressure relief valve pressure rated up to the pressure rating of the vessel. The pressure release valve may be pressure

[0058] 15216541-1rated at most 20% below the pressure rating of the vessel. The pressure relief valve may be pressure rated 10% or more below the pressure rating of vessel. For example, a vessel pressure rated at 0.8 bar may be provided with a pressure relief valve pressure rated between 0.6 and 0.7 bar at most.

[0059] The vessel of the present disclosure may comprise a pressure relief valve configured to continuously exhaust gas. The pressure release valve may be actively controlled to regulate gas pressure within the vessel. In some embodiments, the flow capacity of the pressure relief valve may be up to four times that of a typical brewing fermenter pressure relief valve. In some embodiments, the flow capacity of the pressure relief valve may be twice that of a typical brewing fermenter pressure relief valve, or more. In some embodiments, the flow capacity of the pressure relief valve may be twice that of a typical brewing fermenter pressure relief valve, or more. In some embodiments, the flow capacity of the pressure relief valve may be ten times that of a typical brewing fermenter pressure relief valve, or less. In general, the pressure relief valve flow capacity should correspond to the air flow rate - for example, for 0.25 to 1 VVM flow rates, the pressure relief valve flow capacity should be 0.3-1.2VVM (incorporating a 20% safety margin).

[0060] The provision of a pressure release valve configured to continuously exhaust gas may allow the brewing fermenter to be used with the aeration system for the production of biological material, due to the higher volumetric gas flowrate.

[0061] The fermentation system of the present disclosure have a lower oxygen transfer rate than that required for a fully aerobic fed batch system. For example, a full aerobic fed batch system may typically have a peak oxygen transfer rate (OTR) of more than 150-250 mmol / L / h. The partial-aerobic fed batch fermentation system may have an OTR less than this.

[0062] The oxygen requirement may be related to the strain of microorganism used within the system. For example, some microorganism strains are more effective at producing biological materials at lower oxygen concentrations than others. The fermentation systems of the present disclosure may utilise an appropriate micro-organism (e.g. yeast) strain with a lower oxygen requirement.

[0063] The upper OTR threshold for a ‘reduced-oxygen’ system may be 120 mmol / L / h

[0064] The upper OTR threshold for a ‘low-oxygen’ system may be 90 mmol / L / h

[0065] The fermentation system may have an oxygen transfer rate of 120 mmol / L / h or less. The fermentation system may have an oxygen transfer rate of 50 mmol / L / h or more. The partial-aerobic systems of the present disclosure may comprise an oxygen transfer rate of 60 mmol / L / h.

[0066] 15216541-1By reducing the oxygen transfer rate and demand, a balance between operational complexity, cost and yield of biological material may be obtained. Whilst the yield rate of biological material of the fermentation system (when used with a partial aerobic fed batch process) may be lower compared to known fully aerobic batch systems, the capacity of low pressure vessel (e.g. a brewing fermenter) may be larger and as such, the partial-aerobic fed batch system of the present disclosure may allow for the production of larger volumes of biological material at reduced costs.

[0067] The partial-aerobic fed batch fermentation system of the present disclosure may be configured to yield biological material at a rate more than 50 % but less than 75% of a fully aerobic fed batch fermentation system. The total yield of biological material (per batch) may be equal to the yield of a fully aerobic fed batch fermentation system. The yield of biological material may be more than a traditional anaerobic batch fermentation system for brewing but less than that obtained from fully aerobic fed batch fermentation systems. Anaerobic batch systems typically yield about 5% biological material relative to fully aerobic fed batch systems.

[0068] The fermentation system of the present disclosure may be configured for the partial-aerobic fed batch production of any biological material. For example, the fermentation system may be configured for the production of biological material for use in or as food. The fermentation system may be configured for the production of at least one of the following biomass, yeast, filamentous fungi, bacteria, algae, recombinant proteins, carbohydrates, mood-enhancing polyphenolic compounds, small organic molecules with functional organoleptic properties, biological materials (e.g. bio-polymers) with structural or physical properties. The recombinant proteins may be bio-identical animal-free or plant-based proteins designs to replace or mimic dairy-based proteins, egg-based proteins, meat-based proteins, structural biomaterial proteins and the like.

[0069] The vessel may comprise an internal surface having an average surface roughness of 0.8 pm or less.

[0070] The internal surface may be configured for contact with any feedstocks and the fermentation broth. The internal surface may comprise a smooth, polished surface. The internal surface may comprise a surface roughness of 0.6 pm or less.

[0071] The vessel may be a brewing fermenter which may comprise welded seams. The welded seams internal to the brewing fermenter may comprise a surface roughness of 0.8 pm or less. In general, the maximum surface roughness of the internal surface and / orwelded seams may be 1.0 pm or less. T op and bottom parts of the internal surface may comprise a surface roughness of 0.6 pm or less.

[0072] The vessel whether a brewing fermenter or otherwise may be constructed at least partially from metal. For example, the vessel may be formed from steel. For example, the vessel may be formed from cold rolled stainless steel manufactured according to

[0073] 15216541-1European Standard 10028-72B. For example, the vessel may be formed from 304L or 316L stainless steel.

[0074] The vessel may comprise an internal surface coating configured to at least one of: improve thermal resistivity; improve chemical resistivity; to reduce the surface roughness; to reduce surface adhesion.

[0075] The internal surface coating may comprise polytetrafluoroethylene (PTFE).

[0076] The internal surface finish may comprise an average surface roughness (RA) of 30 pin (0.76 pm) or less. The internal surface finish may be optimised (e.g. by polishing) to reduce fouling and improve sterility of the vessel for use to produce biological material.

[0077] The system may further comprise a cooling system configured to maintain a fermentation broth at between about 15 °C to about 40 °C.

[0078] The cooling system may comprise a cooling jacket. The cooling jacket may be sized according to the vessel and the cooling requirements of the system.

[0079] The cooling jacket may comprise a single or double cooling jacket comprising a coolant. The coolant may comprise for example, a glycol-based coolant.

[0080] Reducing the fermentation process temperature may reduce the required oxygen transfer rate. This may be due to a reduced metabolic rate of microorganisms with a reduction in temperature. In addition, reducing the temperature increases the solubility of oxygen within the fermentation broth, further reducing the minimum volumetric flowrate of gas.

[0081] The system may comprise a heating system. The heating system may comprise a heating jacket. The heating jacket may be sized according to the vessel and the heating requirements of the system.

[0082] In some embodiments, heat input from a heating system may assist increasing the metabolic rate of the microorganisms and achieving the optimal temperature for biological material production.

[0083] The recirculation loop may be configured to condition contents of the vessel.

[0084] Conditioning contents may include adjusting thermal properties, composition and fluid dynamic properties. The recirculation loop may be configured to agitate a vessel interior.

[0085] The capacity of the one of more heat exchangers may depend upon the operational requirements of the system.

[0086] The recirculation loop may comprise inline heat exchange equipment.

[0087] 15216541-1The recirculation loop may allow for improved aeration of a fermentation broth contained in the vessel and improved mixing of the fermentation broth, thereby improving the yield of the fermentation system. The recirculation loop may allow for the introduction of nutrients into the fermentation broth. Additionally, the recirculation loop can serve as a single point of air introduction and reduces the requirement for additional aeration devices to be introduced into the system, thereby reducing possible sources of contamination.

[0088] The system may further comprise dedicated agitation systems. For example, the vessel may be fitted with an agitator arrangement. The vessel may be provided with an impeller arrangement. The vessel may be retrofitted with the agitation means.

[0089] The in-line air diffuser may also be configured for mixing of the fermentation broth. The injection of oxygen-enriched air into a flow of liquid (i.e. fermentation broth) in the recirculation loop disrupts the linear flow of liquid, causing mixing and thermal / compositional homogenisation of liquid within the recirculation loop. The direction of injection may be generally perpendicular to the flow of liquid.

[0090] The in-line air diffuser may be configured for the injection of oxygen or oxygen-enriched air. The in-line air diffuser may be configured to deliver oxygen whilst modulating the hydrostatic pressure of the fermentation broth. Accordingly, pressure shocks may be minimised, reducing the impact on the physiological condition of the biological material. The inline air diffuser may be a venturi injection device. The venturi air injection device may also be referred to as a turbo air venturi.

[0091] The aeration system may further comprise a rotary jet head, configured to agitate contents of the vessel.

[0092] The aeration system may further comprise a nanobubble generator, configured to introduce nanobubbles into the fermentation broth.

[0093] The aeration system may comprise a combination of at least one of: an air diffuser within the vessel, an in-line air diffuser, and a sparger positioned within the vessel.

[0094] The system may further comprise a clean in place (CIP) system configured to control the risk of contamination within the fermentation system.

[0095] The sterilisation system may be configured to disperse chemical sterilisation agents. The chemical sterilisation agents may be dispersed via a rotary jet head.

[0096] The sterilisation system may comprise a clean in place (CIP) system. Advantageously, the CIP system allows the fermentation system to be sterilised and cleaned between batches without major disassembly of the fermentation system.

[0097] 15216541-1The CIP system may comprise at least one cleaning solution reservoir and a pump fluidly connected with the fermentation system to allow for a cleaning solution to be circulated throughout the fermentation system, as required to maintain sterility.

[0098] Sterilisation agents may include at least one of ortho-phosphoric acid, sodium hydroxide, chlorine dioxide, ozone, hydrogen peroxide, ozone, hypochlorites, iodophors, peroxyacetic acid, quaternary ammonium compositions.

[0099] The CIP system may further comprise at least one heater to heat the at least one cleaning solution prior to circulation through the fermentation system.

[0100] The CIP system may further comprise a water reservoir, wherein the water reservoir is fluidly connected to the fermentation system. The water reservoir may allow for the fermentation system to be rinsed with water.

[0101] The CIP system may be fluidly connected to the recirculation loop and / or the rotary jet head.

[0102] In some embodiments, the sterilisation system may comprise a steam in place (SIP) system. The SIP system may comprise a steam injection means configured to supply steam to the fermentation system. Accordingly, steam may be circulated throughout the fermentation system as required to maintain sterility.

[0103] The process control system may comprise at least one of: a thermal sensor, a pH meter, a flow meter, a biological material concentration measurement device, an alcoholmeter, a hydrostatic pressure meter, an exhaust gas monitoring system, a foam sensor, an in-process compositional analysis system, and a foam control system.

[0104] The partial-aerobic fed batch fermentation system comprises a feed system. The feed system comprises at least one nutrient reservoir in fluid communication with the vessel. The at least one nutrient reservoir may be provided to allow nutrients to be fed to the vessel for the production of biological material.

[0105] The at least one nutrient reservoir may be configured to feed directly into the vessel. Alternatively, or in addition, the at least one nutrient reservoir may be in fluid communication with the recirculation loop.

[0106] The feed system may comprise an injection means configured to supply material from the at least one nutrient reservoir to the vessel and / or the recirculation loop. In an example, nutrients in gaseous form may be fed into the recirculation loop via an in-line air diffuser, where one is present in the system.

[0107] The at least one nutrient reservoir may comprise at least one of a buffer reservoir; a nitrogen feedstock reservoir; a carbon feedstock reservoir; an induction agent reservoir, micronutrients.

[0108] 15216541-1In particular, the nitrogen feedstock may comprise gaseous ammonia.

[0109] The at least one nutrient reservoir may comprise a mixing means. The mixing means may be provided to ensure homogeneity of the contents of the reservoir prior to supply to the vessel and / or the recirculation loop.

[0110] According to a second aspect of the invention, there is provided a method of retrofitting a brewing fermenter, the method comprising: modifying an brewing fermenter to comprise: a recirculation loop, connected to the vessel, comprising: three or more inlet conduits, provided inside the vessel, configured to recirculate fluid into the vessel and to induce helical fluid flows within the vessel; an aeration system, comprising an in-line diffuser provided in the recirculation loop; a feed system, and a process control system, wherein, in use, the fermentation system is configured for the production of biological material.

[0111] The brewing fermenter may be of a type used for anaerobic brewing.

[0112] The inlet conduits may be positioned such that distal portions of the inlet conduits are angled at 25 degrees or more below the horizontal.

[0113] Fluid outlets of the inlet conduits may be positioned at a level corresponding to 30% or less of the vessel height.

[0114] Modifying the brewing fermenter may comprise mounting the inlet conduits through a top plate for the vessel.

[0115] Modifying the brewing fermenter may comprise mounting inlet conduits through a side wall of the vessel.

[0116] The aeration system may be configured to supply oxygen-enriched air.

[0117] Modifying the brewing fermenter may comprise applying an internal surface coating to the brewing fermenter, wherein the internal surface coating is configured to at least one of: improve thermal resistivity; improve chemical resistivity; to reduce the surface roughness, and to reduce surface adhesion.

[0118] Modifying the brewing fermenter may comprise modifying the internal surface of the fermenter. Modifying the internal surface may comprise polishing the internal surface. For example, to provide an internal surface roughness of about 0.8 pm, or less than 0.8pm.

[0119] The brewing fermenter may be modified to comprise an aeration system comprises providing an aeration system configured to deliver a volume of oxygen-enriched air at up to around 1 VVM (volume air per volume of fermenter per minute).

[0120] 15216541-1The method of modifying the brewing fermenter to comprise an aeration system may comprise providing an aeration system configured to supply gas at a volumetric rate up to around 0.5 VVM.

[0121] The method of modifying the brewing fermenter to comprise an aeration system may comprise providing an aeration system configured to supply gas at a volumetric rate around 0.25 VVM or greater.

[0122] Modifying the brewing fermenter may comprise replacing any existing pressure relief valves with a pressure relief valve configured to continuously exhaust gas.

[0123] Modifying the brewing fermenter may comprise providing an adapted top plate, where a top plate is present. For example, a modified top plate arrangement configured to accommodate additional pipe work for the partial aerobic fed-batch process. The three or more inlet conduits may be mounted through the adapted top plate

[0124] Modifying the brewing fermenter may comprise adapting the fermentation vessel to receive additional pipe work and connections to render it suitable for a partial-aerobic fed-batch process.

[0125] The method may further comprise modifying existing fermentation recirculation devices for the delivery of oxygen to the brewing fermenter.

[0126] The existing fermentation recirculation device may be a rotary jet head.

[0127] Modifying the brewing fermenter to comprise a feed system may comprise providing at least one nutrient reservoir in fluid communication with the brewing fermenter. The feed system may be in direct fluid communication with a recirculation loop, where one is present. The at least one nutrient reservoir may be provided to allow nutrients to be fed to the brewing fermenter for the production of biological material.

[0128] The method of retrofitting the brewing fermenter may comprise providing a sterilisation system. The sterilisation system may be provided to minimise the presence of contaminants within the system, thereby ensuring product quality. The sterilisation system may comprise a clean in place (CIP) system. The sterilisation system may be configured to circulate a cleaning solution throughout the system. For example, the rotary jet head may be used to distribute cleaning solution over the brewing fermenter.

[0129] Modifying the brewing fermenter to comprise a process control system may comprise providing at least one of: a thermal sensor, a pH meter, a flow meter, a biological material concentration measurement device, an alcoholmeter, a hydrostatic pressure meter, an exhaust gas monitoring system, a foam sensor, an in-process compositional analysis system, a foam control system to be operatively associated with the brewing fermenter and / or the feed system and / or the aeration system. At least one of the feed system or aeration system may be controlled based on the output from the one of more sensors or meters provided as part of the process control system. The process control system may

[0130] 15216541-1comprise a feedback loop or system associated with the output from the one or more sensors or meters, wherein at least one of the feed system or aeration system is controlled by the feedback loop or system.

[0131] The method of retrofitting the brewing fermenter may further comprise providing a cooling system. This may be provided to maintain optimum fermentation temperatures within the brewing fermenter for the production of biological material. For example, the cooling system may be configured to maintain the fermentation broth at between about 15 °C and about 40 °C.

[0132] The method of retrofitting the brewing fermenter may further comprise providing an agitation system. The agitation system may comprise a recirculation loop operatively connected to the brewing fermenter. The agitation system may comprise or further comprise an agitation means provided within the brewing fermenter. For example, the brewing fermenter may be fitted with an agitator arrangement. The brewing fermenter may be provided with an impeller arrangement.

[0133] According to a third aspect of the invention there is provided a method of producing biological material, the method comprising: providing a fermentation system, the system comprising: a vessel; a recirculation loop, connected to the vessel, comprising: three or more inlet conduits, provided inside the vessel, configured to recirculate fluid into the vessel and to induce helical fluid flows within the vessel; an aeration system, comprising an in-line diffuser provided in the recirculation loop; a feed system; and a process control system; controlling the aeration system and feed system to produce biological material using a partial-aerobic fed batch process.

[0134] The method may further comprise configuring the aeration system to supply oxygen-enriched air.

[0135] The method may further comprise supplying oxygen-enriched air with the aeration system to the vessel at up to around 1 VVM (volume air per volume of the vessel per minute).

[0136] The method may comprise supplying a volume of gas with the aeration system at up to around 0.5 VVM.

[0137] The method may comprise supplying a volume of gas with the aeration system at around 0.25 VVM or greater.

[0138] The method may comprise supplying pure oxygen.

[0139] The method may further comprise supplying oxygen to the vessel using the aeration system to obtain an oxygen transfer rate of 120 mmol / L / h or less.

[0140] The method may comprise supplying oxygen to the vessel using the aeration system to obtain an oxygen transfer rate of 50 mmol / L / h or more.

[0141] 15216541-1The oxygen requirement may be related to the strain of microorganism used within the system. For example, some microorganism strains are more effective at producing biological materials at lower concentrations than others. The fermentation systems of the present disclosure may utilise an appropriate micro-organism strain with a lower oxygen requirement.

[0142] The method may further comprise controlling the temperature of the partial-aerobic fed batch process to between about 15 °C to 40 °C.

[0143] The method may further comprise controlling the pH of the fermentation broth in the range of about 2.5 to 9.5.

[0144] The partial-aerobic fed batch process may comprise a process cycle time of between about 48 to about 300 hours.

[0145] The method may comprise producing a cell density of about 100 g / l or less.

[0146] The method may comprise producing a cell density of about 70 g / l or less.

[0147] The method may comprise producing a cell density of about 50 g / l or more

[0148] The method may comprise feeding one or more growth nutrients from the feed system to the vessel (for example, a carbon source, a nitrogen source, micronutrients, cofactors, metabolic induction agents) at specified rates according to process conditions. For example, based on feedback from the process control system. The process control system may be configured to provide feedback and / or output measurements on process conditions such as but not limited to biological material growth, quality, the presence of biomarkers, flow rates, pH, temperature.

[0149] Features of different aspects of the invention may be combined together.

[0150] BRIEF DESCRIPTION OF DRAWINGS

[0151] These and other aspects of the present disclosure will now be described, by way of example only, with reference to the accompanying drawings in which:

[0152] Figure 1 shows a schematic drawing of a fermentation system according to the present disclosure;

[0153] Figure 2 shows a partially transparent perspective view of a vessel of the present disclosure;

[0154] Figure 3 shows a part of the fermentation system in isolation;

[0155] Figure 4 shows a schematic top view of the vessel and inlet conduits of the present disclosure; and

[0156] Figure 5 shows a schematic top view of a vessel and its attached inlet conduits according to another embodiment of the present disclosure.

[0157] 15216541-1DETAILED DESCRIPTION OF DRAWINGS

[0158] A fermentation system 10 according to the present disclosure is shown schematically in Figure 1. The fermentation system comprises a low pressure vessel 12 which is a modified brewing fermenter. The fermentation system further comprises an aeration system 20, a recirculation loop 50 and a feed system and is used for the production of biological material. The fermentation system may be operated in a partial aerobic fed batch process.

[0159] In use, the vessel 12 and recirculation loop 50 are at least partially filled with a fermentation broth, comprising a culture for producing biological material. The recirculation loop 50 is connected to the vessel 12, forming a fluid circuit. The recirculation loop 50 is configured to circulate the fermentation broth through the fluid circuit and to condition the fermentation broth. The aeration system 20 is configured to supply air or oxygen-enriched air (e.g. gas with an oxygen content of at least 25% by volume) to the fermentation system 10.

[0160] The vessel 12 is a cylindroconical brewing fermenter of a type that is typically used within the brewing industry for the production of alcoholic beverages. Thus, the fermentation system 10 may be produced by modifying an anaerobic batch brewing fermenter. The vessel may be pressure rated up to 1.5 bar (i.e. it is a low pressure vessel). The vessel may be pressure rated up to 1 bar. The vessel may be pressure rated up to 0.8 bar. For example, the vessel may be a fermenter typically used for the production of beer via anaerobic fermentation.

[0161] Alternatively, any suitable low-pressure vessel may be used, and not necessarily a brewing fermenter. For example, the low pressure vessel may be a dedicated-purpose vessel of any geometry (e.g. cylindrical, cylindroconical, dished-head or cuboidal). In a further example, the fermentation system may comprise a low pressure vessel produced by modifying a biofuel fermentation vessel. A low pressure vessel is defined here as a vessel pressure rated up to 1.5 bar.

[0162] The low pressure vessel 12, whether a brewing fermenter or otherwise, may be formed from steel, preferably 304L or 316L stainless steel. An internal surface of the low pressure vessel 12 may be coated in PTFE. The internal surface may comprise an average surface roughness of 0.8pm or less, including any welded seams of the low pressure vessel 12.

[0163] The fermentation system 10 (otherwise referred to simply as the system) comprises a pressure release valve 18. The pressure release valve 18 is connected through a top plate 60 of the low pressure vessel 12. The top plate 60 closes the non-conical top end of the low pressure vessel 12. The pressure release valve 18 regulates the pressure in the low pressure vessel 12 and acts as a gas exhaust for the system 10 by allowing gas supplied from the aeration system 20 and gas produced during fermentation to escape. Pressure release valve 18 may be an actively-controlled valve, being controlled to achieve a set gas pressure in the low pressure vessel 12. This may be achieved by

[0164] 15216541-1providing a pressure sensor to sense gas pressure in the low pressure vessel 12. The actively-controlled pressure release valve 18 may be controlled by an electronic controller that receives input from the pressure sensor.

[0165] The pressure relief valve may be rated up to 10 times higher than a pressure release valve typically provided for anaerobic batch fermentation processes. In an example, the pressure release valve may be rated at between 0.6 and 1.5 bar (e.g. 1 bar). In general, the pressure release valve may be rated around 10% lower than the vessel pressure rating or more than 10% lower. The pressure release valve may be rated at most around 20% lower than the vessel pressure rating

[0166] The aeration system 20 comprises a gas source 21, a compressor 22, a cooler 24 and a filter 26 connected in line. The gas source 21 supplies gas with an oxygen content similar to, or greater than, atmospheric air (i.e. around 21% v / v Oxygen or greater). For example, gas source 21 supplies gas with an oxygen content of at least 25% by volume. The gas supplied by gas source 21 with an oxygen content greater than atmospheric air may be referred to as oxygen-enriched air. It will be understood that the term ‘air’ used in the following description is used as a generic designation for any gas mixture comprising oxygen, and not necessarily gas with an atmospheric composition.

[0167] Embodiments in which the gas source 21 supplies gas with an oxygen content similar to atmospheric air may have a reduced oxygen transfer rate relative to embodiments in which the gas source 21 supplies gas with a higher oxygen content (i.e. oxygen-enriched air or pure oxygen). Due to a lower oxygen transfer rate, the microorganism culture being fermented may produce biological material (e.g. proteins) at a slower rate compared with when the gas is oxygen-enriched or pure oxygen.

[0168] In some embodiments pure oxygen may be supplied by the aeration system 20. Use of pure oxygen avoids inefficiency arising from compressing and cooling inert components of a gas mixture (e.g. nitrogen in air). Additionally, the use of pure oxygen significantly reduces the operating pressure relative to conventional aerobic fed batch systems. The operating pressure in embodiments utilising pure oxygen may be 1 bar or less. For example, the operating pressure may be 0.8 bar. In an example, aerobic fed batch systems may operate at 1.4 to 1.8 bar.

[0169] The aeration system 20 may be configured to supply gas at a volumetric rate of 1 VVM (volume of gas per volume of vessel per minute) or less. Alternatively, the aeration system may be configured to supply a gas at a volumetric rate of 0.5 VVM or less. The aeration system may be configured to supply gas at a volumetric rate of around 0.25 VVM or more. In an example, the aeration system may be configured to supply gas at a volumetric rate of around 0.3 VVM.

[0170] The volume of gas and gas composition required may be selected based upon an optimal oxygen transfer rate. The optimal oxygen transfer rate may be dependent on a particular strain of microorganism culture that is being fermented in order to generate biological

[0171] 15216541-1material. In an example, some strains may require less oxygen relative to others. Such strains may be more suited to the use of atmospheric air in the aeration system.

[0172] Partial-aerobic fed batch fermentation systems of the present disclosure may achieve an oxygen transfer rate of 120 mmol / L / h or less. Partial-aerobic fed batch fermentation systems of the present disclosure may achieve an oxygen transfer rate of 50 mmol / L / h or more. The air compressor 22 may have a power input of 1.2kW per 1000L vessel volume or more. The air compressor 22 may have a power input of 1.5kW per 1000L vessel volume or more. The air compressor 22 may have a power input of 2.5kW per 1000L vessel volume or less. The air filter 26 may comprise a filtration membrane having a pore size of 0.2 pm or more. The air filter 26 may comprise a filtration membrane having a pore size of 0.45 pm or less. The air cooler 24 is provided to cool air from the compressor 22.

[0173] The recirculation loop 50 is connected to the vessel 12, forming a fluid circuit. The recirculation loop 50 comprises three inlet conduits 62a, 62b, 62c, provided inside the vessel 12, which expel fluid into the vessel, recirculating fluid into the vessel and inducing helical fluid flows within the vessel. The recirculation loop 50 further comprises a heat exchanger 16 and a pump 17. The recirculation loop 50 draws fluid (i.e. fermentation broth) from an outlet 14 of the vessel, situated at the lower conical end of the vessel 12 toward the inlet conduits 62a-c, via the heat exchanger 16 and the pump 17. Flow in the recirculation loop 50 is induced, at least partially, by the pump 17. The recirculation loop 50 is configured to recirculate the contents of the vessel 12 (e.g. a fermentation broth), agitating the interior of the vessel. In addition, the recirculation loop 50 is configured to thermally condition (e.g. reduce the temperature of) contents of the vessel by means of the heat exchanger 16. The recirculation loop also comprises a harvesting outlet 72 for extracting biological material from the fermentation system 10 at the end of a batch or processing cycle.

[0174] The three inlet conduits 62a-c are mounted through the top plate 60 along with the pressure release valve 18. The inlet conduits 62a-c will be described in more detail below. In alternative embodiments four, five, six or more inlet conduits may also be provided. Additionally, or alternatively, the inlet conduits may be combined with more than one recirculation loop.

[0175] In this example, the aeration system 20 further comprises an inline air diffuser 27, and an air diffuser comprising a rotary jet head (or toftejorg) 28 located within the vessel 12 proximate to a floor of the vessel. Both air diffusers are connected to the outlet of the filter 26.

[0176] Ammonia may be supplied into the fermentation system through components of aeration system 20 (e.g. the filter 26). Gaseous ammonia is supplied by ammonia source 42 and introduced into the aeration system upstream of the filter 26. The ammonia may act as a nitrogen source for the fermentation process.

[0177] 15216541-1In an alternative, the gaseous ammonia may be introduced into the aeration system downstream of the filter. In general, gaseous ammonia may be introduced into the aeration system at any suitable point upstream of the inline air diffuser.

[0178] The (oxygen-enriched) air is introduced by inline air diffuser 27 into the recirculation loop 50 together with gaseous ammonia from ammonia source 42. The inline air diffuser 27 is upstream of the pump 16 and heat exchanger 16. The rotary jet head also introduces air and ammonia into the vessel 12 directly.

[0179] The ammonia source 42 may comprise a supply of ammonia water or liquid ammonia and apparatus configured to produce pressurised gaseous ammonia from the ammonia water or liquid ammonia. The ammonia water may be 25-30% ammonia by weight. The apparatus configured to produce pressurised gaseous ammonia may comprise a heat exchanger.

[0180] The inline air diffuser 27 is a venturi air injection device. Each of the air diffusers 27, 28 is configured to deliver oxygen and ammonia to the fermentation broth. They also act to agitate the broth, providing for improved aeration and mixing. The direction of gas injection may be generally perpendicular to the flow of liquid.

[0181] Air is delivered to the fermentation broth via the re-circulation loop 50, and the supply of air is controlled via automated control valves (not shown) based on feedback from a process control system. The recirculation loop 50 may allow for improved aeration of the fermentation broth and improved mixing of the fermentation broth, thereby improving the yield of the fermentation system.

[0182] Because the fermentation broth is aerated, the pump 17 is preferably a positive displacement pump. Positive displacement pumps create a lower shear force on the microorganisms, which aids their growth. In addition, positive displacement pumps operate more effectively than other pump types (e.g. centrifugal pumps) where the working fluid is highly viscous (e.g. fermentation broth with high cell density) and / or the working fluid contains a significant amount of gas (e.g. aerated / partially aerated fermentation broth). Other types of pumps may be used, but may not be as effective. The provision of the inline air diffuser 27 in the recirculation loop 50 creates a two-phase liquid-gas flow downstream of the inline air diffuser. The fermentation broth within the vessel 12 undergoes recirculation, agitation and oxygen transfer through the two-phase flow generated by recirculation loop 50, via the inlet conduits 62a-c.

[0183] The two-phase flow in the recirculation loop is expelled as jets from outlets of the inlet conduits 62a-c. The jets penetrate the fermentation broth inducing flows which circulate substantially throughout the volume of the fermentation broth in the vessel. This may minimise the build-up of un-mixed stagnant zones. Put alternatively, the jets achieve effective agitation. Oxygen transfer occurs through diffusion from entrained bubbles in the two-phase flow and the effective agitation causes convective flows of the resulting oxygen-rich fermentation broth within the fermentation vessel.

[0184] 15216541-1A feed system is provided. The feed system comprises the ammonia source 42, a buffer reservoir 44 and a carbon source reservoir 46. The buffer reservoir 44 and carbon source reservoir 46 may each comprise agitators to ensure homogeneity of the contents of the reservoirs prior to being fed into the recirculation loop 50. The recirculation loop is configured to compositionally condition the contents of the vessel 12. It will be appreciated that more or less feed reservoirs may be provided according to the requirements of the microorganism strain and the biological material being produced. Each reservoir may comprise an automated valve which is opened or closed based on process conditions detected by the process control system.

[0185] In some embodiments, the nitrogen source may be liquid, not gaseous. For example, the nitrogen source could comprise urea and / or amino acid solutions.

[0186] The process control system comprises one or more sensors 70. The one or more sensors may comprise at least one of: a dissolved oxygen sensor, a thermal sensor, a pH meter, a flow meter, a biological material concentration measurement device, an alcoholmeter, a hydrostatic pressure meter, an exhaust gas monitoring system, a foam sensor, an in-process compositional analysis system, a foam control system. At least one of these sensors may be provided within the re-circulation loop 50 and / or within the vessel 12. Sensors 70 are shown located upstream to the inline air diffuser 27 in the recirculation loop 50, but it will be appreciated that these may be located throughout the fermentation system as required. The same applies for other process control components.

[0187] A cleaning system 30 is also provided. The cleaning system is a clean in place (CIP) system 30 configured to supply a (chemical) cleaning solution to be circulated throughout the fermentation system via recirculation loop 50 and / or via rotary jet head 28, as required to reduce the risk of process contamination. The cleaning solution may comprise, for example at least one of ortho-phosphoric acid, sodium hydroxide, chlorine dioxide, hydrogen peroxide, ozone, hypochlorites, iodophors, peroxyacetic acid, quaternary ammonium compositions. The CIP protocol used may comprise circulating 1-2% ortho-phosphoric acid, followed by 2-4 % sodium hydroxide at 85 °C, followed by rinsing the system with clean water. Additionally, or alternatively, a steam in place protocol may be followed. This may comprise circulating chlorine dioxide, oxone or hydrogen peroxide, followed by hypochlorites, iodophors, peroxyacetic acid and quaternary ammonium compositions, followed by steam at 90 -105 °C for between about 60 to 120 minutes. Parameters of the CIP protocol may be varied depending on a number of factors, including roughness of the internal surface of the vessel 12.

[0188] The temperature of a partial-aerobic fed batch fermentation process using the fermentation system 10 of Figure 1 may be controlled within a range of about 15 to 40 °C. The heat exchanger 16 of recirculation loop 50 is provided as part of a cooling system. The cooling system may also include a cooling jacket (omitted from Figure 1 for clarity). The pH of the fermentation broth may be maintained in the range of about 2.5 to 9.5. The pH may be maintained by, for example, controlling supply from buffer reservoir

[0189] 15216541-144 of the feed system. The typical processing cycle for the fermentation systems of the present disclosure can be from about 48 to about 300 hours.

[0190] Alternatively, or additionally, the fermentation system may comprise a heating system. The heating system may comprise a heating jacket. The heating system may assist increasing the metabolic rate of the microorganisms and achieving an optimal temperature for biological material (e.g. protein) production.

[0191] By using in-line diffusers 27 in the recirculation loop 50, good oxygen transfer rates to the fermentation broth can be reached without using high-power agitation (e.g. more than 2.5kWper 1000L vessel volume) and high pressure sparging. The injection of a gas into a flow of liquid (i.e. fermentation broth) by inline air diffuser 27 in the recirculation loop 50 disrupts the linear flow of liquid, causing mixing and thermal / compositional homogenisation of the liquid.

[0192] Although the rotary jet head 28 contributes to aeration of, and oxygen transfer into the fermentation broth, its contribution is relatively smaller. The rotary head 28 contributes to high efficiency of CIP and agitation within the fermentation broth. In some embodiments, the rotary jet head 28 may not be present.

[0193] Use of oxygen-enriched gas may further reduce the need for high-power agitation and high pressure sparging.

[0194] Agitation power of 2.5kW per 1000L vessel volume or less may be used. In some embodiments, the agitation power may be 1.2kW per 1000L vessel volume or more. In an example, the agitation power for a 500.000L vessel may be 600 kW or more (e.g.

[0195] 700kW). In other embodiments, the agitation power may be 1.5kW per 1000L vessel volume or more. In an example, the agitation power for a 500.000L vessel may be 750 kW or more.

[0196] In conventional fully-aerobic aseptic fed batch systems, high power agitation and high gas flowrates in the fermentation broth (e.g. by sparging) may be required to reduce bubble sizes, increasing interfacial area between the gas phase and the fermentation broth. High gas flowrates can result in high pressure within the reaction vessel containing the gas flow. In an example, a reaction vessel for a conventional fed batch system may be pressure rated up to 3 bar, and may operate at around 1.4 to 1.8 bar internal pressure. By increasing the oxygen concentration of the gas, the gas-liquid interfacial area can be reduced for a given oxygen transfer rate, allowing a reduction in the flowrate of introduced gas and agitation power at a reduced internal pressure. For example, a low pressure vessel according to the present disclosure may be pressure rated up to 1 bar or 1.5 bar (e.g. 1.3 bar). The internal pressure of the lower pressure vessel may be 0.5 bar or more. The internal pressure of the lower pressure vessel may be 0.8 bar or less. The internal pressure of the lower pressure vessel may be 1 bar or less. In general, the operating pressure of the fermentation system corresponds to the pressure rating of the vessel and safety margins.

[0197] 15216541-1In use, a starter culture is grown externally to a selected cell density (e.g. 100g / L). Once the selected cell density has been reached the starter culture is introduced to the partial-aerobic fed batch fermentation system 10, as part of a fermentation broth.

[0198] Initially, the system 10 is operated so as to provide conditions under which the culture can grow further (in cell density). In this initial phase, the demand for nutrients (e.g. carbon and nitrogen from the feed system) and oxygen is very high. As a result, the system 10 may be run at a higher gas flow rate from the aeration system 20. The feed rate of the carbon source and ammonia from the feed system may also be increased. Once a target cell density for biological material production has been reached, the gas flow rate may be reduced from its initial level.

[0199] The fermentation system of the present disclosure may be configured to yield biological material at a rate more than 50 % but less than 75% of a fully aerobic fed batch fermentation system. The total yield of biological material (per batch) may be equal to the yield of a fully aerobic fed batch fermentation system. The cell density yielded may be in the range of about 50 g / l to about 70 g / l, or to about 100g / L.

[0200] The inlet conduits 62a, 62b, 62c are shown in more detail in Figure 2, which is a perspective illustration of the inlet conduits within the vessel 12. The inlet conduits are configured to induce helical fluid flows.

[0201] Situating the inlet conduits 62a-c through the adapted top plate 60 may allow an existing brewing fermenter to be retrofitted into partial aerobic fed batch system 400 (e.g. during a retrofitting procedure) more easily, by minimising modifications to the rest of the brewing fermenter. In addition, the uprated pressure release valve 18 may be situated through the adapted top plate 60, further easing the retrofitting procedure.

[0202] The above-described inlet conduits 62a-c can be all served by the same inline air diffuser 27, heat exchanger 16 and pump 17 of the recirculation loop 50. In some alternative embodiments separate inline air diffusers, and / or heat exchangers and / or pumps may be provided for each inlet conduit. Put alternatively, the system may comprise more than one recirculation loop.

[0203] In particular, provision of multiple inline air diffusers reduces the volume of air injected at any one point, reducing the pressure shock on the fluid. In addition, adjusting the amount of air injected at each inline air diffuser may allow for pressure shocks on the fluid to be modulated. Reducing and / or modulating the pressure shock from the inline air diffuser may reduce the impact of hydrostatic pressure changes on the physiological condition of the biological material.

[0204] The entrained air or gas in the two-phase jet from the inlet conduits 62a-c may cause buoyancy-induced upward flow promoting mixing through the vertical extent of the fermentation broth. Enhanced mixing and agitation increase oxygen transfer rates.

[0205] 15216541-1Each inlet conduit 62a-c comprises a respective downwardly extending portion 64a-c and distal portion 63a-c, which is angled relative to the downwardly extending portion. Each distal portion 63a-c terminates at an outlet 65a-c from which the recirculation flow is expelled. This is shown most clearly in Figure 3, which shows a plan view of conduit 62a in isolation. The other conduits 62b, 62c are of a substantially similar arrangement.

[0206] In use, two-phase flow (e.g. air and fermentation broth) from the recirculation loop 50 is expelled as a jet into the interior of the vessel 12 through openings or outlets 65a-c of the distal portions 63a-c. The distal portions 63a-c are generally tangentially oriented relative to the vessel walls 13. This orientation and the downward direction of the distal portions 63a-c means that the recirculation flow is expelled as a jet in a direction which is generally tangential and downwards, inducing a helical flow pattern (indicated by arrows in Figure 2) within the vessel 12.

[0207] The downward angle of the distal portions 63a-c means that initially the jets of the recirculation flow travel downward and penetrate below the outlet level (i.e. the level along the height of the vessel at which outlets of the conduits are situated). As such, the jets are directed toward the bottom of the vessel to allow gas bubbles to penetrate there. This may avoid an oxygen-deficient ‘dead zone’ at the bottom of the vessel. The downward momentum of the jets dissipates, because the gas bubbles entrained in the expelled recirculation flow are buoyant, and the gas bubbles then travel upwards, resulting in buoyancy-induced upward flow. The tangential orientation of distal portions causes the expelled recirculation flow to travel circumferentially around the vessel walls. As a result, the recirculation flow and / or the gas bubbles follow a generally helical path as they are directed towards the bottom of the vessels and also as they rise through the vessel. This helical flow drives a general rotation of the fermentation broth in vessel, aiding mixing and oxygen transfer.

[0208] The downwardly extending portions 64a-c of the inlet conduits 62a-c may create some local turbulence in the fermentation broth, aiding mixing. The downwardly extending portions 64a-c may reduce a velocity of the general rotation of the fermentation broth. It may be desirable that the reduction of general rotation velocity is not significant (e.g. such that mixing of the fermentation broth is not significantly reduced). The combined effect of the increased mixing provided by the local turbulence, and the reduced mixing caused by the reduced general rotation velocity, may result in a substantially unchanged amount of mixing.

[0209] The distal portion 63a is angled relative its respective downwardly extending portion 64a such that it is also angled downward at 45° to the horizontal when the vessel is oriented upright in operation. Other downward angles are possible. In other embodiments, the distal portion may be angled at 25 degrees or more to the horizontal. Angles less than 25 degrees to the horizontal may result in sub-optimal penetration of the recirculation flow below the outlet level and may allow formation of an oxygen-deficient ‘dead zone’. In addition, the air bubbles may have reduced contact time with the fermentation broth due to reduced recirculation flow penetration downward (and thus a reduced upward

[0210] 15216541-1distance to rise through), reducing oxygen transfer. As the angle of the distal portion increases, the downward velocity component of the jet increases relative to the horizontal (tangential) velocity component, increasing penetration of the recirculation flow below the outlet level.

[0211] The distal portion is oriented such that it is also angled downward at 75° to the horizontal, or less. Angles above 75 degrees may result in jets with insufficient horizontal velocity and an excessive downward velocity. Preferably the distal portion is oriented such that it is also angled downward at 60°, or less. Insufficient horizontal velocity will contribute to poor mixing performance because the horizontal velocity drives the rotation of the fermentation broth. In addition, the excessive downward velocity may result in direct suction of the jets back into the recirculation loop via outlet 14 without circulating substantially throughout the radial cross-section of the fermentation broth in the vessel. The upward tendency of the two-phase flow means that it is advantageous to situate the fluid outlets of the inlet conduits at a level within the vessel corresponding to 30% or less of the vessel height in order to promote jet penetration to the bottom portion of the vessel. In general, reducing the level at which the fluid outlets of the inlet conduits are situated correspondingly increases the penetration of the recirculation flow towards the vessel bottom. In combination with the buoyancy-induced upward tendency of the two-phase flow, this allows circulation and mixing throughout the height of the vessel. This is shown in Figure 2, which shows the height, Ho, of the fluid outlets 65a-c which is around 20% of the vessel height, H.

[0212] Computational fluid dynamics (CFD) simulations indicate that the oxygen transfer efficiency (the rate of oxygen transfer into a liquid relative to total oxygen supply rate) of a two-inlet system with the outlets of the conduits situated at a level corresponding to around 6% of the vessel height is 148% higher than the oxygen transfer efficiency of an otherwise-equivalent system with outlets of the conduits situated at a level corresponding to 30% of the vessel height. Situating outlets of the conduits at levels greater than 30% vessel height may result in an even lower oxygen transfer efficiency, which may not be adequate in many applications.

[0213] Because the system is operated as a fed-batch fermentation system the liquid volume is dynamic and grows over each batch. In general, the vessel is around 30% of the total vessel volume at the beginning of a batch and grows to around 60% or more of total vessel volume by the end. The outlets are situated such that they are below the initial liquid level. It will be appreciated that achieving acceptable mixing and aeration performance at the end of a batch increases in difficulty due to the growth in volume of the fermentation broth in the tank. The CFD results referred to here relate to systems at their final fermentation broth volumes.

[0214] Because the recirculation loop draws fluid from the outlet 14 at the bottom of the vessel 12, situating the fluid outlets of the conduits at a level very close to the vessel bottom can result in the jets being directly drawn into the recirculation loop 50 without significant interaction with the fermentation broth inside vessel 12. As such, the fluid outlets of the

[0215] 15216541-1inlet conduits are preferably situated at a level corresponding to 5% or more of the vessel height.

[0216] It has been found that the provision of three inlet conduits advantageously allows higher (local) velocities across the vessel (compared with two inlet conduits). Higher local velocities across the vessel result in more effective, uniform mixing and a relatively uniform oxygen transfer rate across the vessel. CFD Simulations indicate that a three-inlet conduit arrangement can achieve an oxygen transfer efficiency 77% higher than an otherwise equivalent two-inlet conduit arrangement.

[0217] The fluid velocities across the vessel can be increased by further increasing the number of inlet conduits (e.g. to four, five or more). However, arrangements with three inlet conduits deliver sufficient oxygen transfer, and the addition of more inlet conduits would increase the cost and complexity of the overall system (e.g. because of a need to provide more recirculation loops or a larger recirculation loop).

[0218] Turning to Figure 4, which shows a top view through the top plate 60 into the vessel 12, vertical portions of the inlet conduits 62a-c lie on a common (virtual) circular line 66 concentric with the vessel. Each distal portion 63a-c extends in a generally tangential direction relative to the circular line 66. The generally tangential direction of the distal portions 63 define fluid outlets which are configured to expel fluid jets tangentially into the vessel 12, inducing a helical fluid flow pattern within the vessel 12.

[0219] The fluid outlets are all disposed in a circular pattern. This circular pattern has a radius around two thirds of the total vessel inner radius. Put alternatively, with reference to figure 4, the ratio of Ri to R is 1 / 3 - the fluid outlets are disposed around a third of a vessel radius from the vessel walls.

[0220] In an alternative, the fluid outlets may be disposed up to half the vessel radius from the vessel wall. Fluid outlets further from the vessel wall may result in formation of helical flows which do not agitate peripheral portions of the fermentation broth in the vessel. It has been found that the mixing performance is acceptable for fluid outlets substantially at the vessel wall but increases towards an optimum one third of a vessel radius from the walls. From the optimum around one third of the vessel radius from the walls, the mixing performance decreases, although the mixing performance for fluid outlets at half the vessel radius from the walls may be acceptable in practice.

[0221] The above-described embodiments introduce gaseous ammonia into the fermentation broth via the aeration system. In an alternative, gaseous ammonia could be introduced into the fermentation broth separately, via a dedicated system.

[0222] The embodiments described above comprise three inlet conduits (e.g. 62a, 62b, 62c.) However, in an alternative any number of inlet conduits may be provided.

[0223] It will be appreciated that the layout of the inlet conduits and vessel 12 described above is a non-limiting example. Figure 5 shows a top view through an alternative low-pressure

[0224] 15216541-1vessel 112 with three straight inlet conduits 162a-c. Distal ends 163a-c are not angled with respect to the rest of the conduits 162a-c. The inlet conduits 162a-c have a sideentry layout. Unlike the above-described embodiment where the inlet conduits are mounted through a top plate, the inlet conduits 162a-c enter the vessel 112 through the cylindrical side wall 113. The fluid conduits 162a-c are configured to recirculate fluid into the vessel and to induce helical fluid flows. The fluid outlets of distal portions 163a-c of the side-entry inlet conduits have substantially the same position and downward, tangential orientation as those of the previous embodiment described with reference to Figures 1-4. As such, the positioning and direction of the jets will be substantially similar, resulting in the substantially the same flow behaviour described in connection with the previous embodiment and similar performance. A helical flow is provided which drives a general rotation of the fermentation broth in vessel, aiding mixing and oxygen transfer. Other functionally equivalent layouts are possible.

[0225] Although the inlet conduits 162a-c of Figure 5 are straight, in some embodiments with a side entry layout, the inlet conduits may include distal ends angled with respect to the rest of the conduits.

[0226] Particularly where the vessel has a double side wall (e.g. for a heating / cooling jacket) boring the holes in the cylindrical wall 113 for side entry inlet conduits may be challenging. On the other hand, the side entry layout reduces the material (e.g. stainless steel) needed to construct the inlet conduits. The reduced internal structure of the sideentry layout also increases the effectiveness of the CIP system by reducing obstructions to cleaning solution (and the sterilisation agents therein) during cleaning. In addition, the side-entry layout reduces the height to which the fluids must be pumped to, reducing power used for pumping.

[0227] Although, the rotary jet head 28 contributes to oxygen transfer in the system described above with respect to figures 1-4 (and described below with respect to figure 5), it is not essential. The inlet conduits alone can provide sufficient agitation and oxygen transfer performance. As such, connection of the rotary jet head to the aeration system is optional. When the rotary jet head is connected to the aeration system, the aeration drives the rotor to rotate. The rotation of the rotary jet head provides agitation that complements the agitation provided by the inlets.

[0228] The rotary jet head may be provided purely for the CIP system (cleaning system 30) to disperse cleaning solution within a vessel. In some embodiments, the rotary jet head may not be present.

[0229] Although both cylindroconical and dished head vessels are suitable for use a vessel, it has been found that using cylindroconical fermenters results in slightly higher performance.

[0230] As a further remark, the above-discussed in-line air diffuser 27 may comprise a nanobubble generator. Alternatively, in-line air diffuser 27 may be supplemented by a nanobubble generator. Nanobubble generators (as available, for example, from Molaer

[0231] 15216541-1M&C PC931794W0

[0232] 26

[0233] Inc. USA) generate nano-bubbles having a diameter of less than 200 nanometres as well as larger conventional bubbles. The presence of nanobubbles can increase the efficiency of oxygen transfer to micro-organisms (e.g. yeast) in fermentation broth.

[0234] 15216541-1

Claims

27CLAIMS1. A fermentation system, the system comprising:a vessel;a recirculation loop, connected to the vessel, comprising:three or more inlet conduits, provided inside the vessel, configured to recirculate fluid into the vessel and to induce helical fluid flows within the vessel; an aeration system, comprising an in-line diffuser provided in the recirculation loop;a feed system, anda process control system,wherein, in use, the fermentation system is configured for the production of biological material.

2. The system of claim 1 , wherein distal portions of the inlet conduits are angled at 25 degrees or more below the horizontal.

3. The system of claims 1 or 2, wherein distal potions of the inlet conduits are angled at 75 degrees or less below the horizontal.

4. The system of any preceding claim, wherein distal potions of the inlet conduits are tangentially oriented relative to sides of the vessel.

5. The system of any preceding claim, wherein fluid outlets of the inlet conduits are positioned at a level corresponding to 5% or more of the vessel height.

6. The system of any preceding claim, wherein fluid outlets of the inlet conduits are positioned at a level corresponding to 30% or less of the vessel height.

7. The system of any preceding claim, wherein the distal portion of each inlet conduit is angled relative to the rest of the inlet conduit.

8. The system of claim any preceding claim, wherein the fluid outlets of each inlet conduit are disposed up to half the vessel radius from the vessel wall.

9. The system of any preceding claim, wherein the inlet conduits are mounted through a top plate for the vessel.

10. The system of any preceding claim, wherein the inlet conduits are mounted through a side wall of the vessel.

11. The system of any preceding claim, wherein the vessel is a brewing fermenter.

12. The system of any preceding claim wherein the aeration system further comprises a rotary jet head, configured to agitate contents of the vessel.15216541-113. The system of any preceding claim, further comprising a clean in place (CIP) system configured to control the risk of contamination within the fermentation system.

14. A method of retrofitting a brewing fermenter, the method comprising:modifying an brewing fermenter to comprise:a recirculation loop, connected to the vessel, comprising:three or more inlet conduits, provided inside the vessel, configured to recirculate fluid into the vessel and to induce helical fluid flows within the vessel;an aeration system, comprising an in-line diffuser provided in the recirculation loop;a feed system, anda process control system,wherein, in use, the fermentation system is configured for the production of biological material.

15. The method of claim 14, wherein the inlet conduits are positioned such that distal portions of the inlet conduits are angled at 25 degrees or more below the horizontal.

16. The method of claim 14 or 15, wherein fluid outlets of the inlet conduits are positioned at a level corresponding to 30% or less of the vessel height.

17. The method of any of claims 14-16, wherein modifying the brewing fermenter comprises mounting the inlet conduits through a top plate for the vessel.

18. The method of any of claims 14-17, wherein modifying the brewing fermenter comprises mounting inlet conduits through a side wall of the vessel.

19. A method of producing biological material, the method comprising:providing a fermentation system, the system comprising:a vessel;a recirculation loop, connected to the vessel, comprising:three or more inlet conduits, provided inside the vessel, configured to recirculate fluid into the vessel and to induce helical fluid flows within the vessel;an aeration system, comprising an in-line diffuser provided in the recirculation loop;a feed system; anda process control system;controlling the aeration system and feed system to produce biological material using a partial-aerobic fed batch process.15216541-1M&C PC931794W02920. The method of claim 19 comprising supplying oxygen-enriched air with the aeration system to the vessel at up to around 1 VVM (volume air per volume of vessel per minute).15216541-1

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