Bioreactor system with integrated porous membrane sparger for enhanced biological production

Abstract

This invention relates to a bioreactor system that generates gaseous nanobubbles directly in the presence of viable cells or cell-free expression systems using a porous membrane and forced medium flow, improving the growth or activity of viable cells and cell-free expression systems for enhanced biologically derived product formation The resulting high-density gaseous nanobubbles in the medium improve gas transfer rates (GTR) and homogeneity of GTR throughout the medium without harming cell viability or the integrity of cell-free expression systems. Additionally, the invention provides a novel bioreactor architecture based on the internal integration of an in situ nanobubble generation section working in synergy an internally placed coarse bubble sparger, providing an efficient, multipurpose solution for modern bioprocessing.

Description

[0001] BIOREACTOR SYSTEM WITH INTEGRATED POROUS MEMBRANE SPARGER FOR ENHANCED BIOLOGICAL PRODUCTION FIELD OF INVENTION

[0002] The present invention generally relates to bioreactors, specifically to nanobubble bioreactors comprising components configured to increase the gas transfer rate and its homogeneity within a medium containing either viable cells or active cell-free expression systems, for the production of biomass or biological products. The invention further relates to systems comprising said bioreactors, methods of providing a bioreactor system, and methods for using said bioreactor systems.

[0003] The present invention also describes an in situ method for generating gaseous nanobubble(s) using a porous membrane inside gas-driven bioreactors, such as bubble column or air-lift bioreactors, designed to enhance gas transfer rate and its homogeneity in media containing viable cells or active cell-free expression systems.

[0004] BACKGROUND

[0005] Bioreactors are vessels used to culture cells, microorganisms or cell-free expression systems in vitro under tightly controlled conditions to provide optimal productivity, efficiency, and product quality. Bioreactors have traditionally been used to culture viable prokaryotic and eukaryotic cells, or a combination thereof, in vitro — such as yeast, bacterial, plant, animal, and human cells — (hereinafter referred to as “cells”), or to culture active cell- free expression systems. The term “biological production systems” as used hereinafter encompasses both cells and cell-free expression systems, or a combination thereof. These biological production systems are employed for the cultivation of cellular biomass and / or the production of biologically derived compounds, such as, but not limited to, proteins, lipids, vitamins, enzymes, primary or secondary metabolites, or other bioproducts, or a combination thereof (hereinafter referred to as “biological product(s)”). Various types of bioreactors include stirred-tank, rocker, bubble columns, air lift and fixed-bed. For optimal conditions of bioreactors, there is need for nutrients (e.g., substrates for the biological production systems, such as sugars, amino acids and vitamins), gases such as oxygen for aerobic cells to perform cellular respiration, and stable temperatures (e.g., 37°C) for optimum cell growth, proliferation or biological products production. In bioreactors, gas is supplied by bubbling gas from a sparger and, optionally, stirring with impellers to break up bubbles into smaller bubbles and disperse them throughout the medium. The utilized gases may be any gases thatfunction as a substrate for cell growth, proliferation or biological product production, for aiding in the stripping of gasses from the medium, or for aiding hydrodynamic circulation of the medium inside the bioreactor (hereinafter referred to as “gas”). Gas may be composed of air, oxygen, carbon dioxide, nitrogen, methane, hydrogen, any other gas, or a mixture thereof. For example, air or oxygen may be used in highly aerobic processes; carbon dioxide may be used in aerobic phototrophic processes with organisms such as microalgae or cyanobacteria; nitrogen may be used in anaerobic fermentation processes for products such as organic acids and alcohols; and methane may be used in methanotrophic processes for products such as single-cell protein. However, there are limitations to transfer sufficient gas into the medium with biological production systems present within (hereinafter referred to as “medium”) for any of biomass or biologically derived compound production, and limitations to achieve homogeneous gas concentrations throughout the medium volume. When gas concentrations are insufficient, biological production systems can have limited growth or cell proliferation, limited cell densities, limited production of biologically derived compounds, or increased production of byproducts, and consequently it can limit the productivity of the bioprocess as a whole. Another factor to consider is that appropriate and homogeneous gas transfer rate (hereinafter referred to as “GTR”) decreases as the scale and volume of the bioreactor vessel increases, meaning that at bioreactor scales larger than around 100 liters of volume, gas transfer rates, such as oxygen transfer rate for aerobic processes, become an increasingly limiting factor during production. Moreover, in conventional bioreactor systems (e.g. stirred-tank reactors), gas utilization efficiency is often suboptimal, leading to waste of gas and energy used for gas compression and sparging. Typically, a majority of the gas introduced into the bioreactor medium via sparging, exits the system through the exhaust gas without being effectively utilized by the biological production systems in the medium. As a result, amongst other remedies, excess gas input is often used to maintain sufficient dissolved gas (hereinafter referred to as “DG”) levels throughout the medium, leading to increased operational costs, higher energy consumption, and suboptimal process performance.

[0006] Several strategies to improve GTR and its homogeneity throughout the medium include pressurizing the vessel headspace to increase gas solubility, increasing the agitation rate of the impellers, increasing the gas flow through the spargers, adding additional spargers, changing the ratio between height and width of the vessel, and enriching the sparged gas with, for example, pure oxygen. However, these strategies typically fail to deliver sufficient gas to the medium inside the vessel at large scales (such as mentioned above). Instead, these strategies are limited by their feasible scalability to industrial settings, the potential risk they have overthe structural integrity of the equipment, the integrity of biological production systems inside the bioreactor, the cost feasibility of their implementation, and the increase of energy consumption.

[0007] Furthermore, the higher the agitation rate of the impellers in stirred-tank reactors, the more energy is spent, and the higher the damage through shear stress (such as the one caused by the impeller tips) is made to the cells or cell-free expression systems inside the bioreactor. Additionally, the higher the gas flow through the spargers or implementation of additional spargers, the higher the equipment costs and energy costs, and the higher the negative effects on cell viability or the integrity of biological production systems due to elevated turbulence, bubble-induced shear, and gas bubble collapse at the medium surface. Lastly, changing the ratio between height and width can represent a challenge for mixing, standardization, and construction. Various bioreactor designs implement combinations of these strategies to improve GTR within the medium. However, achieving consistent and scalable GTR performance remains challenging and complex.

[0008] In recent years, nanobubbles or ultrafine bubbles (hereinafter may be referred to as “nanobubbles”), i.e., bubbles with diameters between 10 nm and 1,000 nm, have gained increasing attention as a method for improving gas-liquid mass transfer efficiency in various industries. Their unique properties, including high gas retention, large interfacial surface area, and high internal gas pressure, have led to implementations in, for example, wastewater treatment, greenhouse irrigation, and vertical farming.

[0009] Currently, the dominating commercial technology for generating bulk nanobubbles relies on hydrodynamic cavitation. In this method, liquid is typically forced under pressure through a high-velocity constriction or nozzle or vortex generator, creating localized pressure drops that induce the formation and collapse of cavitation bubbles. As these bubbles violently collapse, nanobubbles are formed through a combination of shock waves, gas dissolution, turbulence, and often high shear forces.

[0010] However, the extreme pressure fluctuations, high local heat production, shear stress and shock waves generated in these hydrodynamic cavitation systems can damage or lyse viable cells, or degrade cell-free expression systems, limiting the practical use of such systems in scenarios where biological integrity is required. Accordingly, the destructive characteristics of hydrodynamic cavitation have in fact been exploited in use cases requiring pathogen elimination or cellular disruption. Furthermore, the liquid or medium itself being pumped under pressure, results in high energy consumption and large pumps needed to achieve the required head height. As a result, when nanobubbles might be used in bioprocessing, anattempted approach involves a two-step method: first, the medium is separated from the biological production systems, then nanobubbles are generated, and finally, the gassed medium is reintroduced into the bioreactor. This additional processing increases complexity, capital expenditure, energy consumption, and the risk of contamination.

[0011] Both WO2016117024 Al to Mitsubishi Chemical Engineering Corporation (2015), and W02020121025 Al to Suez Groupe (2018) discloses aerobic bioreactors using micro / nanobubbles where the micro / nanobubble generators are externally placed in a separate unit or tank, where culture medium flows in and out after cells have been separated through a filtration unit before the sparging step. In both bioreactor designs, a cell separation step is required prior to micro- or nanobubble generation, due to the potentially damaging effects of their bubble generation on biological integrity or cell viability. The described systems generate nanobubbles in a separate tank, requiring prior removal or filtration of cells from the culture medium. This configuration increases system complexity, cost, energy consumption, and contamination risk.

[0012] CN119144683A to Xuankai Biotechnology Shandong Co ltd (2024), discloses a stirred tank bioreactor system for rhamnolipid production using Pseudomonas aureginosa wherein an ultramicro-nanobubble generating device is externally placed in a separate unit and connected to the stirred reactor via a constant-flow pump. The bioreactor system relies on conventional mechanical agitation using a motor-driven impeller for mixing and gas dispersion, with the nanobubble device operated at low ventilation volumes primarily to mitigate foam overflow during fermentation. The reliance on a stirred-tank architecture with mechanical agitation inherently limits scalable gas transfer performance, as gas transfer rates in such systems degrade significantly at bioreactor volumes above approximately 100 liters. As such, it describes a conventional stirred-tank bioreactor with an external nanobubble device used for foam mitigation, the bioreactor described remains complex, energy-intensive, and may present a high risk of compromising biological integrity through the stirred-tank architecture.

[0013] W02017017830 Al to Mitsubishi Chemical Engineering Corporation (2015), discloses an aerobic bioreactor system utilizing oxygen-enriched micro- and nanobubbles, generated in a separate external tank. The system may include or omit a step of separating cells from the medium prior to sparging with the micro- or nanobubbles. The use of oxygen-enriched gas is described as a means to reduce the required medium flow rate through the generator, in an effort to mitigate stress on the biological material that could affect cell viability or integrity. The system therefore remains complex, energy-intensive, and maypresent a higher risk of compromising biological integrity.

[0014] US20190083945 Al to New Jersey Institute of Technology (2018), discloses and describes a system, device, and method to manufacture nanobubbles. The document describes a method for generating nanobubbles in an open tank using a ceramic membrane coated in a hydrophobic coating layer connected to a gas supply to generate nanobubbles in a static liquid or semi-liquid medium. The membrane is static and submersed in a static medium, wherein the medium is a liquid or semi-liquid medium, such as water, ethanol, isopropyl alcohol, and oil / water mixtures, and where the gas bubbles spontaneously detach (solely by the bubble buoyancy-driven force) from the membrane surface. The membrane requires a hydrophobic surface coating for nanobubble generation. The system described does not disclose any enhancement of GTR in bioreactor systems, nor does it address gas delivery into medium containing active biological production systems. As such, it describes nanobubble generation by spontaneous detachment in static or semi-static liquids, typically within open tanks, without addressing the requirements of biologically active bioprocessing environments.

[0015] Gas-driven bioreactors, of the types such as bubble columns, airlift reactors, and similar vessels in which coarse or micro bubble sparging serves as the primary mechanism of mixing the liquid contained in them are used for culturing biological production systems in processes generally classified under the fields of fermentation, cell culture, and precision fermentation.

[0016] The current capacity of large-scale bioreactors, that rely on gas-driven mixing, to maintain sufficient and homogeneous GTR is limited. In said bioreactors of the type bubble column and airlift reactors, the medium is gassed and circulated by typically introducing coarse bubbles at or near the base of the vessel; these bubbles rise through the medium and induce circulation patterns, mixing the medium and providing the medium with gas. In airlift bioreactors, the medium flow is structured with upward flow in one region (hereinafter referred to as “riser”) and downward flow in another region (hereinafter referred to as “downcomer”). Although this arrangement and strategy can change (e.g., reduce or eliminate) the need for mechanical agitators and can reduce shear forces typically associated with impellers, gas transfer in airlift bioreactors is inherently constrained by the short residence time of bubbles in the riser, where coarse bubbles rise rapidly through the medium and exit the system before a substantial portion of the gas is transferred into the liquid phase, often resulting in zones of poor mixing with low GTR, and therefore, low DG concentrations. As an example of these limitations, in airlift bioreactors, the downcomer region may experience insufficient DG levels, causing biological production systems in that region toexperience suboptimal growth, biological product formation, or to shift into different metabolic pathways. Another similar example, in bubble columns, the peripheral outer regions or bottom of the vessel can develop poor mixing or dead zones with limited gasliquid mass transfer. Furthermore, as these bubbles ascend through the riser, they tend to coalesce into progressively larger bubbles, reducing the total gas-liquid interfacial surface area available for mass transfer and thereby further diminishing GTR. The downcomer region remains substantially devoid of gas bubbles and deficient in dissolved gas, as the buoyancy of the bubbles prevents them from being entrained into the downward liquid flow, resulting in zones with insufficient DG levels where biological production systems may experience suboptimal growth, limited biological product formation, or shifts into alternative metabolic pathways. In bubble columns, the peripheral outer regions or bottom of the vessel can similarly develop poor mixing or dead zones with limited gas-liquid mass transfer.

[0017] Current solutions have attempted to remedy or improve said limitations within the bioreactors by using some, or a combination of, strategies such as increasing gas flow rates, changing vessel geometry, enriching the sparged gas with, for example, pure oxygen, pressurizing the headspace, among others. However, these strategies do not fully address the identified limitations and may introduce or cause additional challenges, such as increased energy consumption, elevated stress on the biological production systems, and greater engineering complexity.

[0018] One derivation of gas-driven bioreactors has been described in US11884909B2 to Ark Biotech Inc (2023), where a cluster airlift bioreactor is described. The document describes an airlift bioreactor with a plurality of vertical circulation loops and a controller, the loops are composed of a circulation channel and a sparger. Said attempts to increase the mass transfer coefficient and mixing of the airlift bioreactor with the use of multiple circulation loops with annulus or tube spargers. Additionally this bioreactor features a controller to control the cumulative mass transfer coefficient associated with the first vertical circulation loop. US 11884909B2 discloses the use of multiple traditional spargers in a cluster airlift configuration to enhance mixing and DG levels within the bioreactor. This approach relies on the injection of a high volume of gas through coarse bubbles, and does not address methods that may be required in certain sensitive or high-efficiency applications.

[0019] Despite various prior developments, there remains a need for bioreactor systems that enable higher and more uniform GTR, particularly bioreactor systems that do not harm or degrade biological production systems present in the medium, and rather enhance their viability, productivity, or metabolic activity, without requiring prior separation of saidbiological production systems from the medium or the use of external tanks. Additionally, there is a need for bioreactor designs that offer improved energy efficiency and reduced operational costs.

[0020] In summary, the prior art fails to provide a bioreactor system that simultaneously achieves sufficient and homogeneous gas transfer rates, preserves the integrity of biological production systems, and maintains scalable performance at industrial bioreactor volumes. Existing approaches either introduce additional system complexity, energy consumption, and contamination risk, or remain constrained by the inherent gas transfer limitations of conventional sparging and agitation strategies. There remains a clear and unmet need in the art for bioreactor systems that address these combined limitations.

[0021] SUMMARY OF THE INVENTION

[0022] An object of the present invention is to overcome the limitations of the prior art by providing a bioreactor system comprising an internally placed nanobubble generator, such as a nanobubble generation section, that operates in conjunction with a gaseous coarse bubble sparger, which serves as the primary mixing device, thereby providing high gas retention and homogeneous dispersion of gas throughout the medium, thereby increasing and homogenizing gas transfer rates throughout the medium, increasing gas utilization, as well as providing a bioreactor design that reduces cost and energy consumption.

[0023] Thus, an aspect of the invention is the generation of nanobubbles within a medium for culturing biological production systems, such as generating nanobubbles within the medium comprising viable cells or cell-free expression systems without separating the viable cells or cell-free expression systems from the medium to an external tank of any type or filtering the biological production systems from the medium in any way prior to generating nanobubbles within the medium.

[0024] In an embodiment of the invention, the medium is suitable for culturing prokaryotic cells and / or eukaryotic cells and / or cell-free expression systems in vitro to the end of producing cellular biomass and / or biologically derived compounds.

[0025] The term “culturing” refers to maintaining in vitro conditions suitable for the survival, growth, metabolism, and / or activity of prokaryotic or eukaryotic cells, or for the proper functioning and activity of cell-free expression systems, such that they are capable of synthesizing cellular biomass and / or biologically derived products, including but not limited to fermenting and cell cultivating.

[0026] An embodiment of the invention is to provide a porous membrane-based nanobubblesparger, which allows for in situ nanobubble generation directly within the bioreactor medium containing biological production systems without damaging or lysing viable cells, or degrading cell-free expression systems, but rather, on the contrary, promote and enhance their growth, proliferation and biological product production. This embodiment of the invention maintains gentle gas transfer conditions, reduces shear stress, and improves gas transfer efficiency while preserving the integrity of biological production systems.

[0027] Yet an embodiment of the present invention is providing a bioreactor system comprising a treatment tank (may also be referred to as a “bioreactor vessel” or a “closed-loop circulation vessel”), wherein the tank comprises a medium for culturing biological production systems, wherein the system further comprises biological production systems. The system further comprises a nanobubble generator, such as a nanobubble generation section, configured for generating nanobubbles within the medium comprising biological production systems, such as in the presence of viable prokaryotic cells, viable eukaryotic cells, active cell-free expression systems or a combination thereof, wherein the nanobubble generator comprises at least one porous membrane. The system comprises a treatment tank comprising a medium for culturing prokaryotic cells and / or eukaryotic cells and / or cell-free expression systems in vitro for the purpose of producing cellular biomass and / or biologically derived compounds. The at least one porous membrane comprises an outer surface and an inner surface. In a specific embodiment, the at least one channel is arranged internally within the inner surface of the membrane, wherein the at least one channel is extending longitudinally at least partially along the length of the porous membrane, wherein the porous membrane further comprises a plurality of pores arranged on the porous membrane outer surface, such as arranged to achieve fluid contact between the porous membrane outer surface and inner surface of the membrane. The pores may be of a diameter of 1 to 1,000 nm. The nanobubble generator, such as a nanobubble generation section, is further configured to receive a crossflow of medium along at least a portion of a primary surface, said primary surface being either the outer surface or the inner surface of the porous membrane, thereby enabling forced detachment of generated nanobubbles from said membrane surface. While the porous membrane can be of any geometry, such as a flat sheet or a hollow fiber, the following description refers to a specific embodiment where the membrane is tubular and comprises one or more internal channels. The nanobubble generator, such as a nanobubble generation section, further comprises a gas inlet in fluid contact with the nanobubble generator, and configured to flow gas for generating nanobubbles.

[0028] According to the invention there is disclosed a bioreactor system comprising a treatmenttank, wherein said treatment tank comprises a medium for culturing prokaryotic cells, eukaryotic cells, and / or cell-free expression systems in vitro for the purpose of producing cellular biomass and / or biologically derived compounds. The bioreactor system further comprises of viable prokaryotic cells, viable eukaryotic cells, active cell-free expression systems or a combination thereof. The system further comprises of a nanobubble generation section configured to generate nanobubbles in the medium in the presence of said viable prokaryotic cells, viable eukaryotic cells, active cell-free expression systems or a combination thereof, wherein the nanobubble generation section is further configured to receive a crossflow of medium along at least a portion of a primary surface, said primary surface being either the outer surface or the inner surface of the porous membrane, thereby enabling forced detachment of generated nanobubbles from said membrane surface, said nanobubble generation section comprising: i) at least one porous membrane comprising an outer surface and an inner surface, wherein at least one channel is formed by at least a part of the inner surface of the membrane, wherein the at least one channel is extending longitudinally at least partially along a length of the porous membrane, wherein the porous membrane comprises a plurality of pores arranged to achieve fluid contact between the outer surface of the membrane, and the inner surface of the membrane, wherein the pores are of a diameter of 1 to 1,000 nm, ii) a gas inlet in fluid contact with the nanobubble generator and configured to flow gas for generating nanobubbles. The system is characterized by the nanobubble generator being placed internally within the treatment tank, and wherein the treatment tank further comprises at least one gaseous coarse bubble or micro bubble sparger.

[0029] The term "closed-loop circulation vessel" refers to a bioreactor vessel comprising a single integrated unit in which the medium circulates continuously through one or more distinct functional sections and returns to its point of origin within the same vessel. Said functional sections may include, but are not limited to, a nanobubble generation section, a gas-liquid separation section, a mixing section, a riser, and a downcomer, depending on the bioreactor configuration and embodiment. Any conduits, passages, or structural sections connecting said functional sections are integral parts of the vessel and do not constitute separate or disconnected equipment. The term "closed-loop" refers both to the continuous circulation path of the medium through the functional sections and to the containment of said circulation within a single integrated vessel assembly.

[0030] In one or more embodiments of the invention, the bioreactor system comprises a closed-loop circulation vessel defining a circulation path for medium. The vessel may comprise a medium suitable for culturing biological production systems (as defined herein) in vitro for thepurpose of producing cellular biomass and / or biologically derived compounds, said biological production systems being present in said medium. The vessel may comprise at least one nanobubble generation section integrated within said vessel, comprising at least one porous membrane having a pore diameter of 1 to 1,000 nm, said membrane having a primary surface exposed to said medium and a secondary surface, wherein gas flows from said secondary surface through said pores to form gas protrusions at said primary surface, and wherein said medium containing said biological production systems circulates in crossflow across said primary surface to forcibly detach said gas protrusions while said protrusions are in the nanoscale, thereby generating nanobubbles directly within said medium without prior separation of said biological production systems from said medium. The vessel also comprises at least one coarse bubble sparger integrated within said vessel and configured to introduce gas into said medium independently of said nanobubble generation section; wherein medium circulation through said nanobubble generation section is driven by a circulation pump integrated within said vessel or by airlift effect generated by said coarse bubble sparger, or a combination thereof.

[0031] In an embodiment, the treatment tank is an airlift bioreactor, and wherein the circulation of the medium is driven by airlift generated by the at least one coarse bubble sparger, said airlift-driven circulation providing the crossflow of medium required by the nanobubble generations section.

[0032] In an embodiment, the treatment tank further comprises a circulation pump configured to circulate the medium, and wherein said circulation provides the crossflow of medium required by the nanobubble generation section.

[0033] In an embodiment, the nanobubble generator, such as a nanobubble generation section, is adapted to create nanobubbles with a diameter of between 1 to 1,000 nm, such as 10 to 1,000 nm, for example such as between 200 to 400 nm.

[0034] In an embodiment of the invention, the treatment tank may be a closed vessel, which may be suitable for a controlled reaction, such as those used to culture cells in-vitro under tightly controlled conditions. For example, the treatment tank may be a vessel or container designed to hold biological materials, or other substances where biological, chemical, or physical treatment processes occur as part of a bioreactor system. Said treatment tank can be built of, and not limited to, reusable materials such as stainless steel or borosilicate glass, or single-use materials such as plastic polymers, or any other material suitable to hold biological materials, such as biological production systems and / or biological products. Said treatment tank can have a total volume of, but not limited to, 500 L and up to 150,000 L.The nanobubble generator, such as a nanobubble generation section, further comprises the at least one porous membrane and a gas inlet for generating bubbles, where the generator, such as a nanobubble generation section, is configured to receive a cross-flow of medium over the membrane surface(s). In certain embodiments the porous membrane is housed in an external nanobubble sparger and the medium is driven through the sparger by a dedicated conduit, entraining nanobubbles into the medium return stream. In preferred embodiments the membrane is mounted directly inside the treatment tank and medium flows across the porous membrane surface via the vessel’s own mixing or sparging and no separate housing or external medium loop is required. In the system of the invention, the nanobubbles are generated in the presence of biological production systems in the medium without the need of separating the biological production systems from the medium to, for example, an external tank prior to generating the nanobubbles, or the need of filtrating the biological production systems prior to the generation of the nanobubbles in the medium. In an embodiment, the at least one porous membrane is placed within the housing in a co-linear or perpendicular manner to the flow direction of the medium. In an embodiment, the at least one porous membrane comprises a functional surface modification. In one or more embodiments, the functional surface modification comprises a hydrophobic surface layer applied to at least one of the first surface, the second surface, and / or the walls of the pores of the porous membrane.

[0035] In a further embodiment, the nanobubble generator is placed internally within the treatment tank.

[0036] In an embodiment of the invention, the medium circulates through the nanobubble generator, such as a nanobubble generation section, via the at least one channel in the inner surface of the at least one porous membrane. In another embodiment, the medium circulates through the nanobubble generator, such as a nanobubble generation section, past the outer surface of the at least one porous membrane. In an embodiment, the gas flows via the at least one channel in the inner surface of the at least one porous membrane.

[0037] In an embodiment of the invention, the treatment tank (or bioreactor tank / vessel) may comprise of a mixing device for promoting the mixing of nutrients or gas within the medium. For example, the treatment tank may comprise agitators and / or impellers as mixing devices. In some examples, a buoyant gas bubble generator (such as a micro or coarse bubble sparger), an axial-flow pump, a low-shear positive displacement pump, or other devices may be the mixing device. In other embodiments of the invention, the treatment tank does not comprise a mixing device as the forced flow of the medium (as will be discussed further below) provides sufficient mixing within the system.In one or more embodiments, the system further comprises a gas supply system having a single gas source, wherein said single gas source is fluidly connected to both the gas inlet of the nanobubble generation section and a gas supply conduit of the at least one coarse bubble sparger.

[0038] In one or more embodiments, the treatment tank is an internal loop airlift bioreactor comprising a riser and a downcomer, wherein the nanobubble generation section is located in said downcomer, and wherein the at least one coarse bubble sparger is located to introduce gas at the base of said riser.

[0039] In one or more embodiments, the riser and the downcomer define a closed-loop fluid circulation path entirely within the treatment tank, wherein the system is arranged such that the introduction of gas from the at least one coarse bubble sparger into the riser induces an upward flow of the medium in the riser and a corresponding downward flow of the medium in the downcomer, said downward flow constituting the crossflow of medium received by the nanobubble generation section.

[0040] In one or more embodiments, the nanobubble generation section comprises a plurality of porous membranes arranged in a vertical or horizontal ladder-like configuration.

[0041] In one or more embodiments, the nanobubble generation section comprises a plurality of porous membranes arranged in a helix-like configuration.

[0042] In one or more embodiments, the nanobubble generation section comprises a plurality of porous membranes arranged in a circular pattern along the perimeter of a section of the treatment tank.

[0043] In an embodiment of the invention, the system further comprises at least one system selected from the following group consisting of a gas supply system, an exhaust system, a pH adjustment system, a feed system, a temperature control system, and an automated control system.

[0044] Yet another object of the present invention is providing a method for culturing biological production systems and producing biological products in a bioreactor system as defined herein, the method comprising the steps of :

[0045] (a) filling a treatment tank with a medium;

[0046] (b) introducing biological production systems into the medium;

[0047] (c) operating the bioreactor system by simultaneously:

[0048] (i) introducing a first gas into the medium via the at least one coarse bubble sparger to induce a circulating flow of the medium and to strip unwanted dissolved gases; and(ii) introducing a second gas into the medium via the internally placed nanobubble generation section, wherein said circulating flow induced in step (c)(i) provides the crossflow for said nanobubble generation section, to entrain the medium with nanobubbles of said second gas.

[0049] In one or more embodiments, the first gas and the second gas have the same composition.

[0050] In one or mor embodiments, the first gas and the second gas both comprise air.

[0051] Yet another object of the present invention is providing an improved method to generate nanobubbles within a bioreactor system via a forced flow of medium (such as liquid, growth media, culture broth, or a combination thereof), that passes over a porous membrane surface.

[0052] The cross-flow of medium over the porous membrane inner or outer surface actively intercepts and pinches off the gas protrusions emanating from the membrane pores while the protrusions are still in the nanoscale, resulting in the generation of nanobubbles with a diameter between 1 to 1,000 nm, such as 10 to 1,000 nm within the medium. This mechanism contrasts with passive or spontaneous detachment, where bubbles may continue growing beyond nanobubble size (e.g., exceeding 1 pm in diameter) before detaching due to buoyant forces or instability. The dimensions of the medium flow volumes, flow rate of medium through the nanobubble generator, such as a nanobubble generation section, and the corresponding medium flow velocity may be selected to maintain wall shear stresses well below critical thresholds known to compromise the performance or integrity of biological production systems. These parameters are tailored in accordance with medium-specific variables such as viscosity, density, and flow behavior. Furthermore, the mechanism results in stable nanobubbles that are formed without relying on any form of cavitation or shockwaves, and therefore mitigates any harm to the viability or activity of the biological production systems present in the medium in the process.

[0053] The nanobubble diameter might be dependent, amongst other parameters, on the pore size, gas injection pressure, membrane surface water contact angle, medium crossflow velocity, medium viscosity, and medium temperature. By changing these parameters, nanobubbles with a precise diameter distribution can be generated to tailor to applications where variables, e.g. GTR, longevity of the bubbles, or internal pressure are balanced in different ways. The forced flow of medium may in conjunction also be intended to create a certain degree of turbulence that can allow improved mixing in a vessel with or without a stirring system and baffles, such as those provided in stirred tank bioreactors.In a yet an aspect of the invention, there is provided the use of a bioreactor system as defined herein for in situ generation of nanobubbles in a medium containing viable prokaryotic cells, eukaryotic cells, or cell-free expression systems for the production of cellular biomass and / or biologically derived products.

[0054] In one or more embodiments, said use is performed as a bioprocess for the simultaneous and decoupled control of: (i) the supply of a process gas to a medium via the nanobubble generation section, and (ii) the removal of an unwanted gas from the medium via the coarse bubble sparger. In one or more embodiments, the process gas is oxygen. In one or more embodiments, the unwanted gas is carbon dioxide, such as metabolically produced carbon dioxide.

[0055] Accordingly, several advantages over existing bioreactors of similar size, such as those mentioned in the prior art, involve increasing GTR within the medium without compromising the viability or state of the biological production systems present in the medium, increasing the homogeneity of GTR and nutrient concentration throughout the entire medium, increasing the gas-liquid mass transfer limit (hereinafter referred to as “kLa”') within the bioreactor, to potentially eliminate dependence on a mixing system based on an agitator / stirrer, impellers and baffles, increasing the freedom for the design geometry of the bioreactor, to improve growth, proliferation of the cells present in the medium, to enable cells or biological production systems present in the medium to improve their production of biological products (proteins / compounds).

[0056] The term "airlift bioreactor" refers to a type of bioreactor vessel in which mixing and medium circulation are driven by gas introduction rather than by mechanical agitation, wherein the medium circulates between at least one riser and at least one downcomer within a single integrated vessel or vessel assembly. Airlift bioreactors are classified into two principal configurations: an "internal loop airlift bioreactor," in which the riser and downcomer regions are defined within a single vessel by internal structural elements such as baffles, draft tubes, or concentric arrangements; and an "external loop airlift bioreactor," in which the riser and downcomer are separate vertical conduits connected at the top and bottom to form one closed, integrated unit. In both configurations, the terms "internal" and "external" refer exclusively to the arrangement of the medium circulation path relative to the vessel geometry, and do not denote the presence of any separate or disconnected equipment external to the bioreactor.

[0057] In an aspect of the invention where there is described a porous membrane or a multitude of porous membranes placed inside a bubble column, airlift reactor, or other gas-driven bioreactor, in different configurations and placements, the coarse or micro bubble sparger, typically, but not exclusively, located close to the bottom of the vessel, continues to serve as the primary mixing mechanism, while the porous membranes introduce nanobubbles into the medium in other sections, such as, for example, the downcomer of an airlift reactor or the outer perimeter of a bubble column. The invention and technology describe improved GTR and its homogeneity, thereby enhancing cell or biological production systems viability and product yields.

[0058] Nanobubbles effectively address the limitations of existing bioreactors (e.g. stirred- tank reactors) and gas-driven bioreactors by providing higher GTR, improved DG levels and homogenous dispersion and diffusion of gas throughout the bioreactor volume, due to their high surface to volume ratio, high internal gas pressure, and their neutral buoyancy, among other beneficial characteristics, allowing nanobubbles to stay suspended within the medium in regions of the bioreactor which normally are considered zones of poor mixing or dead zones.

[0059] The present invention provides a system where gaseous nanobubbles are generated in situ in the medium with biological production systems inside a bioreactor using medium flow across the surface of a porous membrane, while not harming the biological production systems and in fact promoting the cell growth, proliferation and / or biological product production of the cells or biological production systems via an increase in GTR and dissolved gas available for metabolism, as well as improved homogeneity of both within the medium, without the need of external tanks for nanobubble generation purposes, and without the need for separation of the cells or biological production systems prior to the introduction of nanobubbles into the medium.

[0060] DRAWINGS

[0061] The invention is described with reference to the following drawings in which:

[0062] FIG. 1 shows a perspective side view of a single-channel tubular porous membrane 8a according to an embodiment of the invention.

[0063] FIG. 2 shows a perspective side view of a multi-channel tubular porous membrane 8b according to an embodiment of the invention.

[0064] FIG. 3 shows a lateral cross-sectional view of an external nanobubble sparger according to an embodiment of the invention wherein tubular membranes 8a, 8b are positioned co-linear to the medium flow direction.

[0065] FIG. 4 shows a lateral cross-sectional view of an external nanobubble spargeraccording to an embodiment of the invention wherein tubular membranes 8a, 8b are positioned tangent to the medium flow direction.

[0066] FIG. 5 shows a process diagram view of a system according to an embodiment of the invention comprising a stirred tank bioreactor (STR) vessel 11 connected to a pump and subsequently to an external nanobubble sparger 13 where the medium flows through the external nanobubble sparger, for example, as described in one of the external nanobubble sparger embodiments in FIG. 3 or FIG. 4.

[0067] FIG. 6 shows a process diagram view of a system according to an embodiment of the invention comprising a bioreactor vessel without agitator / impeller connected to a pump 10 and subsequently to an external nanobubble sparger 13 where the medium flows through the external nanobubble sparger, for example, as described in one of the external nanobubble sparger embodiments in FIG. 3 or FIG. 4.

[0068] FIG. 7 shows a transverse cross-sectional view of a tubular porous membrane according to an embodiment of the invention, where the gas flows from the internal channel(s) through the pores and the outer surface into the medium which is flowing past the outer surface.

[0069] FIG. 8 shows a transverse cross-sectional view of a tubular porous membrane according to an embodiment of the invention, where the gas flows from the outside of the porous membrane through the pores and the inner surface into the medium which is flowing through the internal channel(s).

[0070] FIG. 9 shows a longitudinal cross-sectional view of a tubular porous membrane according to an embodiment of the invention such as that depicted and described in FIG. 8.

[0071] FIG. 10 shows a longitudinal cross-sectional view of the tubular porous membrane according to an embodiment of the invention such as that depicted and described in FIG. 7.

[0072] FIG. 11 shows a close-up cross-sectional schematic view of gaseous nanobubble formation where medium flowing past the membrane surface picks up the nanobubbles from the membrane pores.

[0073] FIG. 12 shows a schematic view of a bubble column bioreactor with an internal porous membrane nanobubble generation section according to an embodiment of the invention.

[0074] FIG. 13 shows a cross-sectional view of an external loop airlift bioreactor with an internal porous membrane nanobubble generation section according to an embodiment of the invention.

[0075] FIG. 14 shows a schematic view of an external loop airlift bioreactor with an internalporous membrane nanobubble generation section in the downcomer according to an embodiment of the invention.

[0076] FIG. 15 shows a schematic view of an external loop airlift bioreactor with an internal porous membrane nanobubble generation section in the riser according to an embodiment of the invention.

[0077] FIG. 16A shows a schematic side view of an internal loop airlift bioreactor with an internal porous membrane nanobubble generation section in the downcomer according to an embodiment of the invention. Where the porous membrane nanobubble generation section is located in the outer section, such as the side section, of the bioreactor and the sparger is located in the concentric section of the bioreactor.

[0078] FIG. 16B shows a schematic side view of an internal loop airlift bioreactor with an internal porous membrane nanobubble generation section in the riser according to an embodiment of the invention. Where the porous membrane nanobubble generation section and the spargers are located in the outer section, such as the side section, of the bioreactor.

[0079] FIG. 17A shows a schematic side view of an internal loop airlift bioreactor with an internal porous membrane nanobubble generation section in the riser according to an embodiment of the invention. Where the porous membrane nanobubble generation section and the sparger are located in the concentric section of the bioreactor.

[0080] FIG. 17B shows a schematic side view of an internal loop airlift bioreactor with an internal porous membrane nanobubble generation section in the downcomer according to an embodiment of the invention. Where the porous membrane nanobubble generation section is located in the concentric section of the bioreactor and the spargers are located in the outer section, such as the side section, of the bioreactor.

[0081] FIG. 18 shows a cross-sectional top view of the nanobubble generation section configuration described in FIG. 16A and FIG. 16B.

[0082] FIG. 19A shows an embodiment of the nanobubble generation section configuration described in FIG. 16A and FIG. 18, where the porous membranes are located vertically in the bioreactor around the perimeter of the vessel. The inside volume of the draft tube functions as the riser, and the annulus functions as the downcomer.

[0083] FIG. 19B shows an embodiment of the configuration described in FIG. 16B and FIG.

[0084] 18, where the porous membranes are located vertically in the bioreactor around the perimeter of the vessel. The inside volume of the draft tube functions as the downcomer, and the annulus functions as the riser.

[0085] FIG. 20A shows a schematic diagram of a porous membrane nanobubble generationsection 23 according to an embodiment of the invention where one or more porous membranes 8a, 8b are located horizontally along the center line of the circular section, configured parallel to one another in a vertical ladder-like configuration.

[0086] FIG. 20B shows a cross sectional side view of the configuration described in FIG.

[0087] 20A.

[0088] FIG. 21 A shows a schematic diagram of a porous membrane nanobubble generation section 23 according to an embodiment of the invention where one or more porous membranes 8a, 8b are located vertically and parallel to one another in the section, configured in a row along the center line of the circular section into a sideways ladder-like configuration pattern, where two gas manifolds extend along the center line of the circular section to support the top and bottom of the porous membranes and supply them with gas.

[0089] FIG. 2 IB shows a cross sectional side view of the configuration described in FIG.

[0090] 21A.

[0091] FIG. 22A shows a schematic diagram of a porous membrane nanobubble generation section 23 according to an embodiment of the invention where one or more porous membranes 8a, 8b are located vertically in the section, configured in a circular pattern along the perimeter of the section, with the porous membrane(s) being supported and supplied with gas via gas manifolds mounted to the perimeter of the section.

[0092] FIG. 22B shows a cross sectional side view of the configuration described in FIG.

[0093] 22A.

[0094] FIG. 23 A shows a schematic diagram of a porous membrane nanobubble generation section 23 according to an embodiment of the invention where one or more porous membranes 8a, 8b are located horizontally along one or more cross-sectional horizontal planes, configured parallel to one another in a horizontal ladder-like configuration pattern.

[0095] FIG. 23B shows a cross sectional side view of the configuration described in FIG.

[0096] 23 A.

[0097] FIG. 24A shows a schematic diagram of a porous membrane nanobubble generation section 23 according to an embodiment of the invention where one or more porous membranes 8a, 8b are located horizontally along the center line of the circular section, configured such that for the membrane along the vertical dimension, the consecutive membrane is rotated by a fixed or varying number of degrees in the horizontal plane with respect to the previous membrane, to form a helix-like configuration pattern.

[0098] FIG. 24B shows a cross sectional side view of the configuration described in FIG.FIG. 25 shows an embodiment of the invention illustrating the disposable culture vessel bag of a single-use bioreactor system, as commonly used in pharmaceutical or cell therapy manufacturing environments.

[0099] FIG. 26A shows a transverse cross-sectional view of an embodiment of a singlechannel tubular porous membrane 1 in which a functional surface modification 25, e.g. a hydrophobic surface layer, is applied to the membrane’s surfaces.

[0100] FIG. 26B shows a longitudinal cross-sectional view of the tubular porous membrane embodiment as depicted and described in FIG. 26A.

[0101] It is to be understood that these drawings serve as a representation and are not in any way limiting of the embodiments of the invention.

[0102] DETAILED DESCRIPTION

[0103] Before the invention is disclosed and described in detail, it is to be understood that this invention is not limited to particular devices, systems, configurations, method steps, disclosed herein as such devices, systems, configurations, method steps may somewhat vary. It is also to be understood that the terminology employed herein is used for the purpose of describing particular embodiments and is not intended to be limiting since the scope of the present invention is limited by the appended claims and equivalents thereof.

[0104] It is to be noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0105] If nothing else is defined, any terms and scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains.

[0106] As mentioned above, the term “nanobubbles” includes both nano- and ultrafine gaseous bubbles, such as bubbles with a diameter of 1 to 1,000 nm, such as 10 to 1,000 nm. In an example, the nanobubbles have a diameter of between 200 nm to 400 nm. Various methods for measuring the bubble size are available including dynamic light scattering and nanoparticle tracking analysis techniques. Various factors affect the size of the nanobubbles including the crossflow velocity of the medium, the membrane pore size, the pressure of the gas injected into the system, the properties (e.g. hydrophobicity) of the surfaces of the porous membrane, medium viscosity, and other factors.

[0107] For the avoidance of doubt, the term “porous membrane" according to the present invention includes materials comprising pores with a size of 1 to 1000 nm allowing certainsubstances (such as gas, medium, liquid, fluid, or particles) to pass through while blocking others based on properties like size, charge, chemical affinity, etc. The porous membrane of the present invention may be configured in various geometries, including but not limited to, tubular membranes, hollow fibers, or flat-sheet membranes. It is characterized by having a first surface and an opposing second surface, with pores extending therethrough. The following description often refers to a tubular embodiment with an 'inner' and 'outer' surface and internal channels, but it is to be understood that this is a non-limiting example of the broader inventive concept.

[0108] The term “bioreactor” includes a vessel or system designed to support biologically active environments, where biological reactions involving organisms or biochemically active substances are carried out under controlled conditions, typically for purposes such as cell culture, fermentation, or biomanufacturing.

[0109] Bioreactors have traditionally been used to culture viable prokaryotic and eukaryotic cells, or a combination thereof, in vitro — such as yeast, bacterial, plant, animal, and human cells — (hereinafter referred to as “cells”), or to culture active cell-free expression systems.

[0110] The term “biological production systems” as used hereinafter encompasses both cells and cell-free expression systems, or a combination thereof. These biological production systems are employed for the cultivation of cellular biomass and / or the production of biologically derived compounds, such as, but not limited to, proteins, lipids, vitamins, enzymes, primary or secondary metabolites, or other bioproducts, or a combination thereof (hereinafter referred to as “biological product(s)”).

[0111] The term “medium” refers to any liquid or semi-liquid containing living cells or biological production systems present within the medium for the culturing of said living cells or biological production systems, such as those often used in biological, industrial, medical, or research purposes. In some embodiments, the biological production systems may be present in suspension, adhered to carriers in the medium, or immobilized on fixed substrates or support matrices within the medium.

[0112] The terms "coarse / micro bubble sparger", "coarse or micro bubble sparger", and “gaseous coarse / micro bubble sparger” as used herein refers to any gas-sparging device configured to produce bubbles with a diameter of approximately 0.1 mm (100 pm) or larger. This definition is intended to encompass what may be referred to in the art as both 'microbubbles' (typically 1 pm to 1 mm) and 'coarse bubbles' (typically >1 mm). For the sake of clarity and simplicity throughout this specification and the appended claims, the singular term "coarse bubble sparger" may be used to denote any sparger producingbubbles in this range, distinguishing them from the "nanobubbles" (diameter < 1,000 nm) generated by the nanobubble generation section.

[0113] The term “functional surface modification” includes those modifications applied to specific surfaces of the at least one porous membrane or all surfaces of the at least one porous membrane to impart specific, enhanced properties, such as gas flux, hydrophobicity, antifouling behavior, or chemical resistance, without blocking or altering the fundamental porosity of the membrane.

[0114] The term "treatment tank", as used herein, refers to the primary vessel of the bioreactor system. For the purposes of the present invention, this vessel is to be understood as a "closed-loop circulation vessel", wherein the medium is continuously circulated within a defined, closed path. This circulation is a key feature of the invention and can be driven either internally by pneumatic action (e.g., via the coarse bubble sparger in gas-driven embodiments) or via a pump in alternative embodiments. While the term "treatment tank" is retained for consistency, its functional role in all described embodiments is that of a closed-loop circulation vessel.

[0115] In a first aspect, there is provided a bioreactor system 1 comprising a treatment tank 2, such as a closed-loop circulation vessel 2, (which may also be referred to as a bioreactor vessel) wherein the treatment tank comprises a medium 100 suitable for culturing biological production systems. In an example, the medium comprises viable cells, wherein the cells may be prokaryote cells, eukaryote cells, or a combination of the cells, or comprises cell-free expression systems. The treatment tank 2 further comprises a nanobubble generator which is configured to generate nanobubbles within the medium, such as in the presence of biological production systems. In one example, the nanobubble generator is an external nanobubble sparger 13. In one example, the nanobubble generator is a nanobubble generation section 23, 30. In some embodiments the nanobubble sparger 13 is placed externally of the treatment tank 2. In some embodiments, the nanobubble generator 23, 30 is placed internally within the treatment tank 2, such as fixed internally within the treatment tank 2. The treatment tank 2 may be a stirred tank bioreactor (STR) vessel 11 with an agitator / impeller 27 or a bioreactor vessel without agitator / impeller 12. In a further embodiment, the treatment tank 2 may be a disposable vessel for single-use bioreactor systems.

[0116] In the embodiments where the nanobubble generator is an external nanobubble sparger, the external nanobubble sparger consists of a housing in which one or more porous membranes are mounted, with one or more internal channels per porous membrane. Themembranes are placed such that they separate the inner volume of the housing into two distinct sub-volumes, one being defined by the space between the inner housing walls and the outer surface of the membranes, and one being defined by internal volumes of the membrane(s) which are in fhiid / gaseous connection with one another within the housing. The ends of the porous membranes are held in place in the housing by seals around the perimeter of the porous membranes that create a sterile boundary between the two described sub-volumes. The housing comprises at least one, such as two, connections for medium to flow through the external nanobubble sparger, and at least one, such as two, connections for gas to flow in, and optionally out of, the external nanobubble sparger. The positioning of the porous membranes inside the housing may either be in the co-linear direction with respect to the medium flow direction inside the housing, or alternatively, placed tangent to the medium flow direction inside the housing.

[0117] In an embodiment of the invention, the nanobubble generator is positioned externally from the treatment tank and connected via a fluid circulation loop, such that it remains an integral component of the closed bioreactor system.

[0118] The external placement of the nanobubble generator with respect to the treatment tank may be used in, for example, embodiments where the flow rate of medium circulation through the nanobubble generator must be tightly controlled, or where the treatment tank is a disposable vessel such as those for single-use bioreactor systems.

[0119] The treatment tank according to the present invention includes configurations in which a sterile barrier is maintained between the interior of the tank and the surrounding environment. It is to be understood that although the treatment tank maintains a sterile boundary with the surrounding environment, it is connected to the various outlets, inlets, and conduits in a manner as discussed below. Examples of such vessels include those comprising a vessel, a lid and optionally a mixing device such as a sparger, stirrer, impeller or agitator. In a preferred embodiment of the invention, the treatment tank does not comprise any stirrer, impeller or agitator. In a preferred embodiment of the invention, the mixing device is a sparger, e.g., a coarse or microbubble sparger as defined herein. In an example, the treatment tank is suitable for a gas-driven bioreactor, such as that comprising a cylindrical vessel which is configured to allow compressed air or gas mixture to be introduced, for example, at the bottom of the vessel through nozzles, perforated plates, ring spargers, etc. for gas stripping or aeration, mixing and fluid circulation, in the absence of moving mechanical parts. Thus, in some embodiments, the treatment tank 2 may be a bubble column bioreactor vessel 20a, an external loop airlift bioreactor vessel 20b, aninternal loop airlift bioreactor vessel 20c, etc.

[0120] The bioreactor system may further comprise the at least a gas supply system, an exhaust system, a pH adjustment system, a feed system, a temperature control system, or an automated control system. In an example, the treatment tank (i.e., bioreactor vessel) is connected to the at least a gas supply system, an exhaust system, a pH adjustment system, a feed system, a temperature control system, or an automated control system through a pipeline. The gas supply system may be configured to supply gas to, for example, the nanobubble generator. The nanobubble generator comprises generating nanobubbles, which are supplied to the medium, such as that comprising biological production systems. The exhaust system may be configured to discharge gas out of the treatment tank when, for example, the gas pressure exceeds the set pressure. The pH adjustment system may be configured to adjust the pH value of, for example, the medium in the treatment tank. The feeding system may be configured to dynamically replenish medium substrate and nutrients in a treatment tank in real time. The temperature control system may be configured to adjust the temperature of the medium, for example, in the treatment tank. The defoaming system may be configured to control accidental foam in, for example, the treatment tank.

[0121] The nanobubble generator 13, 23, 30 comprises a nanobubble generator gas supply conduit 26 to flow gas for the generation of nanobubbles. The inlet pressure of the gas supplied to the nanobubble generator 13, 23, 30 via gas supply conduit 26 may be between 0.05 MPa to 1.50 MPa, such as between 0.2 MPa to 0.4 MPa.

[0122] The nanobubble generator comprises the at least one porous membrane 8. In an embodiment, the at least one porous membrane is a single-channel porous membrane 8a, FIG. 1. In another embodiment, the at least one porous membrane is a multi-channel porous membrane 8b, FIG. 2.

[0123] The at least one porous membrane 8a, 8b may have different shapes and sizes. Suitable cross-sectional shapes may, for example, be circular, rectangular, hexagonal, star-shaped, or any other suitable geometric shape. In one embodiment, the at least one porous membrane 8a, 8b is a tubular porous membrane. In another embodiment, the at least one porous membrane 8a, 8b is a flat sheet porous membrane. The porous membrane 8a, 8b may be composed of different materials like inorganic (ceramic, metals, etc.), organic (polymers), composite materials, or combinations thereof. It is to be understood that the porous membrane 8a, 8b may comprise a single material throughout its entire body or a combination of materials. In the embodiment where the membrane comprises one channel, i.e., porous membrane 8aaccording to FIG. 1, then the distance between the outer 16 and inner 17 surface of the membrane (i.e., the thickness of the wall of the membrane) may be in the range of, for example, about 0.1 mm to 50 mm. In the embodiment where the membrane comprises more than one channel, e.g., porous membrane 8b according to FIG. 2, then the distance between the outer 16 and inner 17 surface of the membrane (i.e., the thickness of the wall of the membrane) will depend on the number of channels incorporated in the membrane.

[0124] Thickness, diameter and width of the membrane can be adjusted to generate nanobubbles on different scales.

[0125] The at least one porous membrane 8a, 8b comprises an outer surface 16 and an inner surface 17, as illustrated in FIG. 7. The at least one porous membrane 8a, 8b comprise at least one pore 15, such as a plurality of pores 15a, 15b, wherein the pores have pore size (e.g. diameter) being between 1 nm and 1,000 nm, such as those configured to produce nanobubbles. In one example, the pore size is between 200 nm to 400 nm. The at least one pore 15, such as a plurality of pores 15a, 15b, may be arranged to achieve fluid contact between the outer surface 16 and inner surface 17 of the membrane, such as arranged on the outer surface 16 of the at least one porous membrane 8a, 8b and extending through the outer surface 16 to the inner surface 17.

[0126] For the avoidance of doubt, the term “fluid contact” refers to the fluid (e.g., gas, medium) being able to penetrate through the pores enabling transfer of the fluid through the wall of the membrane.

[0127] The at least one pore 15, such as plurality of pores 15a, 15b, include those wherein the walls of the pores are the same width throughout the pore (such as cylindrical) or those wherein the pores have different width extending throughout the pore (such as conical or triangular). It is to be understood that when more than one pore 15 is present, then the pores might not be of the same shape. Moreover, when more than one pore 15 is present on the outer surface 16 of the membrane, then the pores may have different sizes (e.g., diameter) from one another and all pores are not necessarily of the same size.

[0128] As a non-limiting example of the membrane structure, the at least one porous membrane 8a, 8b comprises at least one channel 18, such as multiple channels 18a, 18b, wherein the at least one channel 18, may be formed by at least a part of the inner surface of the membrane 17. In an embodiment, the at least one channel 18 may be extending longitudinally at least partially along the length of the porous membrane, such as that the channel(s) 18 run through the entire length of the membrane (as illustrated in FIG. 1 and FIG.

[0129] 2) and can vary in number depending on, for example, the membrane area, the medium flow,turbulence, amongst others. The at least one channel 18 may, for example, have a cross-sectional shape that is circular, rectangular, hexagonal, star-shaped, or any other suitable geometric shape.

[0130] The at least one channel 18 may comprise the same material as the porous membrane (as discussed herein) or be treated with a functional surface modification that does not obstruct the fundamental porosity of the membrane. The cross-sectional width and height of the channel(s) 18 may be, for example, between approximately 0.1 to 100 mm.

[0131] According to an embodiment, with reference to FIG. 3, there is described a nanobubble sparger 13a according to an embodiment of the invention, wherein the porous membrane(s) is a tubular membrane(s). In this embodiment, a porous membrane 8a, 8b is positioned in a colinear manner to the medium flow direction. As illustrated in FIG. 3, the porous membrane 8a, 8b, resides in a membrane housing 7 in an air-tight and liquid-tight manner using seals such as o-rings and sanitary gaskets, such that no leakage of any fluid (liquid or gas) happens between the inside and outside of the membrane, except through the membrane pores themselves. In other words, the porous membrane(s) 8a, 8b resides in a membrane housing 7 in a fluidly and / or gaseous sealable manner. In an embodiment, the membrane housing 7 is configured to receive the at least one porous membrane 8a, 8b. The membrane housing 7 may be a cylindrical vessel that encloses the at least one porous membrane 8a, 8b. The membrane housing 7 comprises a fluid inlet, such as a nanobubble sparger utility inlet 5 and an optional fluid outlet, such as nanobubble sparger utility outlet 6. Additionally, the membrane housing 7 comprises a medium inlet 3 where medium 100 with biological production systems coming from the bioreactor vessel, such as that coming from the treatment tank 2, flows inside the membrane housing 7 and through the membrane internal channel(s) 18 of the membrane 8a, 8b, such that the medium 100 flows past the porous membrane inner surface 17 of the membrane 8a, 8b and picks up the nanobubbles generated at the surface 17. The medium 100 comprising biological production systems and nanobubbles flows out of the housing 7 back into the treatment tank 2 through the medium outlet 4. When the membrane 8a, 8b is placed inside of the housing 7, then a sub-volume Vsa is created in the space between the inner housing walls and the outer surface 16 of the membrane 8a, 8b, as illustrated in FIG. 3.

[0132] According to an embodiment, with reference to FIG. 4, there is described an external nanobubble sparger 13b according to another embodiment of the invention, wherein the porous membrane(s) is a tubular membrane(s). In this embodiment, a plurality of porous membranes 8a, 8b are positioned in a tangent / perpendicular manner to the medium flow direction. As illustrated in FIG. 4, the porous membranes 8a, 8b, reside in a membrane housing7 in an air-tight and liquid-tight manner using seals such as o-rings and sanitary gaskets, such that no leakage of any fluid (liquid or gas) happens between the inside and outside of the membranes, except through the membrane pores themselves. In other words, the porous membrane(s) 8a, 8b resides in a membrane housing 7 in a fluidly and / or gaseous sealable manner. In an embodiment, the membrane housing 7 is configured to receive the at least one porous membrane 8a, 8b. The membrane housing 7 may be a cylindrical vessel that encloses the at least one porous membrane 8a, 8b. The membrane housing 7 comprises a fluid inlet, such as a utility inlet 5 and an optional fluid, such as gas, utility outlet 6. The membranes 8a, 8b are placed such that they separate the inner volume of the housing 7 into two distinct subvolumes, one Vsc being defined by the space between the inner housing walls and the outer surface 16 of the membranes, and one Vsb being defined by internal volumes of the membranes 8a, 8b which are in fluid / gaseous connection with one another within the housing 7. Additionally, the membrane housing 7 comprises a medium inlet 3 where medium 100 with biological production systems coming from the bioreactor vessel, such as that coming from the treatment tank 2, flows inside the membrane housing 7 through the sub-volume Vsc being defined by the space between the inner housing walls and the outer surface 16 of the membranes, such that the medium 100 flows past the porous membrane outer surface 16 of the membrane 8a, 8b and picks up the nanobubbles generated at the surface 16. The medium 100 comprising biological production systems and nanobubbles flows out of the housing 7 back into the treatment tank 2 through the medium outlet 4.

[0133] According to an embodiment, with reference to FIG. 3 and FIG. 4, the nanobubble sparger utility inlet 5 is connected to a 3-way valve 39a, which switches between the gas supply conduit 26 during nanobubble generation operation and CIP / SIP supply conduit 40 during cleaning-in-place (hereinafter referred to as “CIP”) or sterilization-in-place (hereinafter referred to as “SIP”) of the external nanobubble sparger 13. The utility outlet 6 is connected to a 3-way valve 39b, which switches between an optional gas exhaust conduit 21 during nanobubble generation operation and CIP / SIP drain conduit 41 during cleaning and sterilization. In an embodiment, the gas exhaust conduit 21 is closed or omitted to enable a dead-end sparging configuration, wherein all gas introduced via the utility inlet 5 is forced through the membrane and into the liquid medium. The conduits 3, 4, 5, 6 are constructed from materials compatible with the biochemical processes conducted within the bioreactor. Suitable materials include, for example, food-grade and pharmaceutical-grade metals such as 316L stainless steel and other corrosion-resistant steel alloys. In certain embodiments, high-performance polymers may be used, including PTFE, PFA and PVDF, depending on thechemical compatibility, sterility, and pressure conditions of the application. The conduits 3, 4, 5, 6 may be connected to the housing using industry-standard sanitary fittings such as triclamp connections, compression fittings, hygienic flanges, or aseptic welding techniques. These connection methods ensure a secure, leak-proof seal compliant with any clean-in-place (CIP) and steam-in-place (SIP) procedures, thereby preventing ingress or egress of fluids or gases during operation. Additionally, one or more optional sensor ports 25 may be integrated into the membrane housing 7 for interfacing with sensors capable of measuring temperature, pressure, conductivity, or other relevant process and safety parameters during operation or cleaning cycles.

[0134] In an embodiment, there is described a method for generating nanobubbles 19, wherein the method comprises the steps of flowing the medium 100 through the external nanobubble sparger 13 via the inside of the porous membrane 8a, 8b through the internal channels 18. In another embodiment, there is described a method for generating nanobubbles 19, wherein the method comprises the step of flowing the medium 100 through the external nanobubble sparger 13 via the outside of the porous membrane, such as past the outer surface 16. Such as that the nanobubbles are generated in the medium within the housing 7 of the nanobubbles sparger 13.

[0135] According to an embodiment, there is described a bioreactor system for generating nanobubbles within a medium comprising biological production systems, wherein the bioreactor vessel further comprises one or more auxiliary internal components such as baffles, an agitator and / or impeller, a sparger, an internal heat exchanger, or probes. The one or more auxiliary internal components may be used for purposes including, but not limited to, nutrient distribution, medium homogeneity, or process monitoring within the bioreactor vessel.

[0136] According to an alternative embodiment, such as for certain applications (e.g., retrofitting existing tanks), an external loop may be utilized. With reference to FIG. 5, there is described a process comprising a stirred tank bioreactor (STR) vessel 11 connected to a pump 10 and subsequently to an external nanobubble sparger 13, where the medium 100 flows through the external nanobubble sparger 13, for example, as described in one of the external nanobubble sparger 13 embodiments in FIG. 3 or FIG. 4. The stirred tank bioreactor (STR) vessel 11 comprises a medium outlet conduit configured to release the medium 100 with biological production systems into the pump 10, where the outlet conduit acts as the pump medium inlet conduit 9. The stirred tank bioreactor (STR) vessel 11 comprises a medium inlet conduit configured to flow the medium 100 with biological production systems and nanobubbles back into the stirred tank bioreactor (STR) vessel 11, wherein the medium inlet conduit acts as the nanobubble sparger medium outlet 4. The medium conduits 3, 4, 9, aremade of materials suitable for the process carried inside the bioreactor like food grade stainless steel metal, or other materials. Such conduits may be attached to the STR vessel 11 using industry-standard sanitary fittings such as tri-clamp connections, compression fittings, hygienic flanges, or aseptic welding techniques. These connection methods ensure a secure, leak-proof seal compliant with any clean-in-place (CIP) and steam-in-place (SIP) procedures, thereby preventing ingress or egress of fluids or gases during operation inside or outside STR vessel 11. The STR vessel 11 might be constructed from materials compatible with the biochemical processes conducted within the bioreactor. Suitable materials include, for example, food-grade and pharmaceutical -grade metals such as 316L stainless steel and other corrosion-resistant steel alloys. In certain embodiments, high-performance polymers may be used, including PTFE, PFA and PVDF, depending on the chemical compatibility, sterility, and pressure conditions of the application. The STR vessel 11 might contain any quantity of ports, inlets or outlets according to the process being carried out inside the bioreactor. Such ports / inlets / outlets can be used for process sensors of any kind, valves, additional conduits for medium and other compounds. The STR vessel 11 can have any type of device to control the temperature inside the bioreactor; for example, a cooling / heating jacket around the bioreactor, coils to control the medium temperature. The STR vessel 11 can be equipped with baffles, and multiple impellers of any kind, as well as traditional air sparging devices; for example, sparger rings.

[0137] According to an alternative embodiment, such as for certain applications (e.g., retrofitting existing tanks), an external loop may be utilized. With reference to FIG. 6, there is described a process comprising a bioreactor vessel without agitator / impeller 12 connected to a pump 10 and subsequently to an external nanobubble sparger 13, where the medium 100 flows through, for example, as described in one of the external nanobubble sparger 13 embodiments in FIG. 3 or FIG. 4. The bioreactor vessel without agitator / impeller 12 comprises a medium outlet conduit configured to release the medium 100 with biological production systems into the pump 10, where the outlet conduit acts as the pump medium inlet conduit 9. The bioreactor vessel without agitator / impeller 12 comprises a medium inlet conduit configured to flow the medium 100 with biological production systems and nanobubbles back into the bioreactor vessel without agitator / impeller 12, wherein the medium inlet conduit acts as the nanobubble sparger medium outlet 4. The medium conduits 3, 4, 9, are constructed from materials compatible with the biochemical processes conducted within the bioreactor. Suitable materials include food-grade and pharmaceutical-grade metals such as 316L stainless steel and other corrosion-resistant steel alloys. In certainembodiments, high-performance polymers may be used, including PTFE, PFA and PVDF, depending on the chemical compatibility, sterility, and pressure conditions of the application. Such conduits may be attached to the bioreactor vessel without agitator / impeller 12 using industry-standard sanitary fittings such as tri-clamp connections, compression fittings, hygienic flanges, or aseptic welding techniques. These connection methods ensure a secure, leak-proof seal compliant with any clean-in-place (CIP) and steam-in-place (SIP) procedures, thereby preventing ingress or egress of fluids or gases during operation inside or outside the bioreactor vessel without agitator / impeller 12. The bioreactor vessel without agitator / impeller 12 might be constructed from materials compatible with the biochemical processes conducted within the bioreactor. Suitable materials include food-grade and pharmaceutical -grade metals such as 316L stainless steel and other corrosion-resistant steel alloys. In certain embodiments, high-performance polymers may be used, including PTFE, PFA and PVDF, depending on the chemical compatibility, sterility, and pressure conditions of the application. The bioreactor vessel without agitator / impeller 12 might contain any quantity of ports, inlets or outlets according to the process being carried out inside the bioreactor. Such ports / inlets / outlets can be used for process sensors of any kind, valves, additional conduit for medium and other compounds. The bioreactor vessel without agitator / impeller 12 can have any type of device to control the temperature inside the bioreactor; for example, a cooling / heating jacket around the bioreactor, coils to control the medium temperature. The bioreactor vessel without agitator / impeller 12 can be equipped with baffles, and multiple impellers of any kind, as well as traditional air sparging devices; for example, ring spargers.

[0138] The nanobubble sparger 13a, 13b consists of a housing 7 in which at least one or more porous membrane, such as porous membranes, 8a, 8b are mounted. The membranes are placed such that they separate the inner volume of the housing 7 into two distinct sub-volumes, one Vsa, Vsb being defined by the space between the inner housing walls and the outer surface of the porous membranes, and one V3c being defined by internal volumes of the porous membrane(s) which are in fluid / gaseous connection with one another within the housing 7. In some embodiments, the ratio V3c / Vib may be between 3:1 and 10:1 such that the medium, which typically exhibits higher viscosity and lower flowability than gas, is provided with increased relative volume to facilitate its flow and minimize its pressure drop, whereas the gas can traverse the smaller volume with minimal pressure loss. The ends of the at least one porous membrane, such as the porous membranes, 8a, 8b are held in place in the housing 7 by seals around the perimeter of the membrane(s) that create a sterile boundary between thetwo described sub-volumes. The housing comprises at least one, such as two, connections 3, 4 for medium to flow through the external nanobubble sparger 13a, 13b. The housing further comprises at least one, such as two, connections for gas or CIP / SIP fluid to flow in, such as a nanobubble sparger utility inlet 5, and optionally for gas or CIP / SIP fluid to flow out, such as a nanobubble sparger utility outlet 6, of the external nanobubble sparger 13a, 13b. The positioning of the porous membranes inside the housing may, for example, be in the co-linear direction with respect to the medium flow direction inside the housing as described in relation to FIG. 3, or placed tangent to the medium flow direction inside the housing as described in relation to FIG. 4. Furthermore, the external nanobubble sparger may be configured such that the medium flows either along the inner surface 17 or along the outer surface 16 of the porous membrane(s) 8a, 8b.

[0139] According to an embodiment, with reference to FIG. 7 and FIG. 10, there is described a tubular porous membrane 8a, 8b, where the gas flows from the internal channel(s) 18 through the pores 15 and the outer surface 16 into the medium 100 which is flowing past the outer surface 16, entraining the medium with nanobubbles. The pores 15 extend through the porous membrane material 14 from the inner surface 17 to the outer surface 16. In other words, the pores are in fluid contact with the inner surface of the membrane.

[0140] According to an embodiment, with reference to FIG. 8 and FIG. 9, there is described a porous membrane 8a, 8b, where the gas flows from the outside of the porous membrane through the pores 15 and into the internal channel(s) 18 through which medium 100 is flowing past the inner surface 17, entraining the medium with nanobubbles. The pores 15 extend through the porous membrane material 14 from the inner surface 17 to the outer surface 16. In other words, the pores are in fluid contact with the inner surface of the membrane.

[0141] In an embodiment of the invention, there is provided at least one porous membrane 8a, 8b comprising a functional surface modification, such as a functional coating. As defined earlier and for the avoidance of doubt, the term “functional surface modification” includes those applied to specific surfaces of the at least one porous membrane or all surfaces of the at least one porous membrane to impart specific, enhanced properties, such as gas flux, hydrophobicity, antifouling behavior, or chemical resistance, without blocking or altering the fundamental porosity of the membrane.

[0142] According to an embodiment, with reference to FIG. 11, there is described a gaseous nanobubble 19 formation where medium flowing past the outer 16 or inner surface 17 of the porous membrane 8a, 8b picks up the nanobubbles 19 from the porous membrane pores 15.Since the figure is a close-up cross sectional view of both the membrane embodiments described in FIG. 9 and FIG. 10, the pores 15 extend from the outer surface 16 to inner surface 17.

[0143] One embodiment of the invention includes a stirred tank bioreactor (STR) vessel 11 comprising a pump circulation loop that circulates medium over the bioreactor volume and passes it through a stationary porous membrane nanobubble sparger 13a, 13b, placed externally with respect to the treatment tank 2. The medium may be pumped by an aseptic low-shear pump 10 through a housing 7 containing one or multiple porous membranes 8a, 8b, where the medium flows past along at least a portion of either the outer 16 or inner 17 primary surface of the membrane(s) 8a, 8b, and a gas is being forced into the medium from the opposite secondary surface of the membrane through the membrane pores, and entraining gaseous nanobubbles in the medium flowing past the at least a portion of the primary surface. The medium circulation flow arising from the pump operation may be the primary driver in this embodiment of the liquid flow over the porous membrane surfaces to pick up nanobubbles from its surface.

[0144] As used herein, the term "primary surface" refers to a first surface of the porous membrane in contact with the pressurized gas, and the term "secondary surface" refers to the opposing surface of the porous membrane through which the gas exits into the medium. The designation of primary and secondary surfaces may vary depending on system configuration, but is consistently defined by the gas flow direction. For example, in one embodiment, the pressurized gas may contact the porous membrane inner surface 17 of a tubular membrane (primary surface) and exit through the porous membrane outer surface 16 (secondary surface) into the medium 100. In one or more embodiments, the term "primary surface" refers to the surface of the porous membrane through which the gas exits into the medium, and the term "secondary surface" refers to the opposing surface of the porous membrane in contact with the pressurized gas. The designation of primary and secondary surfaces may vary depending on system configuration, but is consistently defined by the gas flow direction.

[0145] Yet another embodiment of the invention includes a system comprising a bioreactor vessel 12 which does not contain an impeller nor an agitator, instead in which an external aseptic pump 10 may be the sole device responsible for providing medium circulation and mixing inside the bioreactor, while at the same time circulating the medium through nanobubble sparger 13a, 13b, placed externally with respect to the treatment tank 2, to entrain the medium with nanobubbles. In this embodiment the intake of the pump 10 may be fluidly connected to the bioreactor via the pump medium inlet conduit 9, and connected via ananobubble sparger medium inlet 3 to the intake of an external nanobubble sparger 13a, 13b on the outlet side of the pump, from where a nanobubble sparger medium outlet 4 fluidly connects the outlet of the external nanobubble sparger 13 a, 13b back into the internal volume of the bioreactor. The pump medium inlet conduit 9 may either be placed at the location of the internal wall of the bioreactor, or extend closer to the middle of the internal bioreactor volume. Likewise, the medium outlet 4 may either be placed at the location of the internal wall of the bioreactor, or extend closer to the middle of the internal bioreactor volume. In this embodiment the pump 10 may act to create a circulation flow of the medium inside the bioreactor and mix the medium to maintain homogeneous concentrations of nutrients, biological production systems and gases, while at the same time providing the medium flow over the porous membrane(s) 8 in the external nanobubble sparger 13a, 13b. The bioreactor vessel 12 in this embodiment may either have a conical shaped bottom, or a rounded bottom, or any other shape as described herein.

[0146] For the avoidance of doubt, the term “fluidly connected” includes that fluids are allowed to pass through the entities that are fluidly connected, such as that the pump is configured to allow the fluids to flow to the bioreactor.

[0147] In addition, another embodiment of the invention includes a system comprising a bioreactor vessel 12 which does not contain an impeller nor an agitator, but instead relies on the buoyancy-driven flow generated by gas introduced through a microbubble or coarse bubble gas sparger (ring sparger, open pipe sparger, fritted plate sparger, or any other sparger) as a primary means for providing medium circulation and mixing inside the bioreactor, while an external aseptic pump 10 circulates some of the medium through nanobubble sparger 13 a, 13b, placed externally with respect to the treatment tank 2, to entrain the medium with nanobubbles, from where the medium flows back inside the bioreactor. In this embodiment the intake of the pump 10 may be fluidly connected to the bioreactor via a pump inlet conduit 9, and connected via a medium inlet 3 to the intake of an external nanobubble sparger 13a, 13b on the outlet side of the pump, from where a medium outlet 4 fluidly connects the outlet of the external nanobubble sparger 13a, 13b back into the internal volume of the bioreactor. The pump inlet conduit 9 may either be placed at the location of the internal wall of the bioreactor, or extend closer to the middle of the internal bioreactor volume. Likewise, the nanobubble sparger medium outlet 4 may either be placed at the location of the internal wall of the bioreactor, or extend closer to the middle of the internal bioreactor volume. Two examples of such a bioreactor system, and not limited thereto, are a bubble column with the pumped nanobubble sparger circulation loop and an airlift bioreactor with the pumpednanobubble sparger circulation loop.

[0148] It is known that bubble column and airlift reactors face inherent limitations in achieving high GTRs, as medium volume per bioreactor increases beyond a certain threshold it raises the superficial gas velocity. However, higher velocities lead to unstable flow regimes such as slug flow and gas channeling, which counteract the intended goal by in fact reducing mass transfer efficiency and destabilizing the process. Without wishing to be bound by theory, it is believed that by using the nanobubble generation section in conjunction with the gaseous coarse / micro bubble sparger, the control over GTR levels can for a large part be decoupled from controlling the rate of off-gas(es) stripping from the medium. Since the nanobubbles will be the primary actor to maintain GTR at a set target level, coarse bubble gas sparging can be dialed up and down to control dissolved gas(es) levels by stripping the metabolically produced off-gas(es) from the medium. Inversely, for a fixed coarse bubble gas sparging rate that maintains a fixed level of dissolved gas(es), independently from this the GTR can be dialed up or down by increasing or decreasing the gas flow rate to the nanobubble generation section without disturbing dissolved gas(es) levels. This allows, for example, a more precise control of the process parameters, further improving the process optimization that can be achieved.

[0149] According to preferred embodiments of the invention, with reference to FIG. 12, there is provided a bubble column bioreactor vessel 20a which is equipped externally with a bioreactor off gas exhaust conduit 21, and internally with a gaseous coarse / micro bubble sparger 22 and a porous membrane nanobubble generation section 23. The gaseous coarse / micro bubble sparger 22 comprises a gaseous coarse / micro bubble sparger gas supply conduit 24 as an inlet conduit to generate gaseous coarse / micro bubble 34 inside the bioreactor to generate medium circulation and mix the medium, supply gas to the medium, and strip the medium of metabolically produced off-gas. The porous membrane nanobubble generation section 23 comprises a nanobubble generator gas supply conduit 26 as an inlet conduit to generate nanobubbles 19 in the medium contained in the bioreactor. The porous membrane nanobubble generation section 23 may, for example, be any of the types described and shown in FIG. 20A to FIG. 24B, which will be discussed further below.

[0150] As a general principle underlying the embodiments shown in FIG. 12 to FIG. 17B, the system can be understood as a closed-loop circulation vessel. This vessel defines a circulation path for medium, wherein said vessel comprises: a) a medium suitable for culturing viable prokaryotic cells, eukaryotic cells, active cell-free expression systems, or a combination thereof (hereinafter collectively "biological production systems"), in vitro for the purpose of producing cellular biomass and / or biologically derived compounds, said biological productionsystems being present in said medium; b) at least one nanobubble generation section integrated within said vessel, comprising at least one porous membrane having a pore diameter of 1 to 1,000 nm, said membrane having a primary surface exposed to said medium and a secondary surface, wherein gas flows from said secondary surface through said pores to form gas protrusions at said primary surface, and wherein said medium containing said biological production systems circulates in crossflow across said primary surface to forcibly detach said gas protrusions while said protrusions are in the nanoscale, thereby generating nanobubbles directly within said medium without prior separation of said biological production systems from said medium; c) at least one coarse bubble sparger integrated within said vessel and configured to introduce gas into said medium independently of said nanobubble generation section; wherein medium circulation through said nanobubble generation section is driven by a circulation pump integrated within said vessel or by airlift effect generated by said coarse bubble sparger, or a combination thereof. The medium circulation can be driven by the airlift effect generated by the coarse bubble sparger 22. In other embodiments, a circulation pump may be used to assist or drive the circulation.

[0151] According to a preferred embodiment, with reference to FIG. 13, there is described a cross-sectional view of an external loop airlift bioreactor as shown in FIG. 14 which is equipped with an internal porous membrane nanobubble generation section 23, wherein the porous membrane nanobubble generation section 23 comprises a nanobubble generator gas supply conduit 26 as an inlet conduit to generate nanobubbles 19 in the medium contained in the bioreactor. The porous membrane nanobubble generation section 23 may, for example, be any of the types described and shown from FIG. 20A to FIG. 24B, which will be discussed further below.

[0152] According to a preferred embodiment, with reference to FIG. 14, there is provided an external loop airlift bioreactor vessel 20b which is equipped externally with a bioreactor off gas exhaust conduit 21, and internally with a gaseous coarse / micro bubble sparger 22 located in the external loop airlift bioreactor riser 28, and a porous membrane nanobubble generation section 23 located in the external loop airlift bioreactor downcomer 29. The gaseous coarse / micro bubble sparger 22 comprises a gaseous coarse / micro bubble sparger gas supply conduit 24 as an inlet conduit to generate gaseous coarse / micro bubble 34 inside the bioreactor vessel to generate medium circulation and mix the medium, supply gas to the medium, and strip the medium of metabolically produced off-gas. The porous membrane nanobubble generation section 23 comprises a nanobubble generator gas supply conduit 26 as an inlet conduit to generate nanobubbles 19 in the medium contained in the bioreactor vessel. Theporous membrane nanobubble generation section 23 may, for example, be any of the types described and shown from FIG. 20A to FIG. 24B, which will be discussed further below.

[0153] According to a preferred embodiment, with reference to FIG. 15, there is provided an external loop airlift bioreactor vessel 20b which is equipped externally with a bioreactor off gas exhaust conduit 21, and internally with a gaseous coarse / micro bubble sparger 22 and a porous membrane nanobubble generation section 23 that are located in the external loop airlift bioreactor riser 28. The gaseous coarse / micro bubble sparger 22 comprises a gaseous coarse / micro bubble sparger gas supply conduit 24 as an inlet conduit to generate gaseous coarse / micro bubble 34 inside the bioreactor vessel to generate medium circulation and mix the medium, supply gas to the medium, and strip the medium of metabolically produced offgas. The porous membrane nanobubble generation section 23 comprises a nanobubble generator gas supply conduit 26 as an inlet conduit to generate nanobubbles 19 in the medium contained in the bioreactor vessel. The porous membrane nanobubble generation section 23 may, for example, be any of the types described and shown from FIG. 20A to FIG. 24B, which will be discussed further below.

[0154] According to a preferred embodiment, with reference to FIG. 16A, there is provided an internal loop airlift bioreactor vessel 20c which is equipped externally with a bioreactor off gas exhaust conduit 21. Internally the bioreactor vessel is equipped with an annular porous membrane nanobubble generation section 30 in the shape of a cylindrical annulus which is located in the internal loop airlift bioreactor downcomer 31 in the annulus between the internal loop airlift reactor draft tube 32 and the internal loop airlift bioreactor vessel 20c, and a gaseous coarse / micro bubble sparger 22 that is located beneath the internal loop airlift bioreactor riser 33. The internal loop airlift bioreactor riser 33 in this embodiment is defined by the cylindrical volume inside the draft tube 32. The gaseous coarse / micro bubble sparger 22 comprises a gaseous coarse / micro bubble sparger gas supply conduit 24 as an inlet conduit to generate gaseous coarse / micro bubble 34 inside the bioreactor vessel to generate medium circulation and mix the medium, supply gas to the medium, and strip the medium of metabolically produced off-gas. Nanobubble generator gas supply conduit 26 supplies the main gas manifold 35 located along the perimeter of the annular porous membrane nanobubble generation section 30 with gas to generate nanobubbles 19 in the medium contained in the bioreactor vessel.

[0155] According to a preferred embodiment, with reference to FIG. 16B, there is provided an internal loop airlift bioreactor vessel 20c which is equipped externally with a bioreactor off gas exhaust conduit 21. Internally the bioreactor vessel is equipped with an annular porousmembrane nanobubble generation section 30 in the shape of a cylindrical annulus which is located in the internal loop airlift bioreactor riser 33 in the annulus between the draft tube 32 and the internal loop airlift bioreactor vessel 20c, and a gaseous coarse / micro bubble sparger 22 that is located beneath the internal loop airlift bioreactor riser 33. The downcomer 31 in this embodiment is defined by the cylindrical volume inside the draft tube 32. The gaseous coarse / micro bubble sparger 22 comprises a gaseous coarse / micro bubble sparger gas supply conduit 24 as an inlet conduit to generate gaseous coarse / micro bubble 34 inside the bioreactor vessel to generate medium circulation and mix the medium, supply gas to the medium, and strip the medium of metabolically produced off-gas. Nanobubble generator gas supply conduit 26 supplies the main gas manifold 35 located along the perimeter of the annular porous membrane nanobubble generation section 30 with gas to generate nanobubbles 19 in the medium contained in the bioreactor vessel.

[0156] According to a preferred embodiment, with reference to FIG. 17A, there is provided an internal loop airlift bioreactor vessel 20c which is equipped externally with a bioreactor off gas exhaust conduit 21. Internally the bioreactor vessel is equipped with a porous membrane nanobubble generation section 23 in the shape of a cylinder which is located in the internal loop airlift bioreactor riser 33 inside the draft tube 32, and a gaseous coarse / micro bubble sparger 22 that is located beneath internal loop airlift bioreactor riser 33. The downcomer 31 in this embodiment is defined by the annulus between the draft tube 32 and the internal loop airlift bioreactor vessel 20c. The gaseous coarse / micro bubble sparger 22 comprises a gaseous coarse / micro bubble sparger gas supply conduit 24 as an inlet conduit to generate gaseous coarse / micro bubble 34 inside the bioreactor vessel to generate medium circulation and mix the medium, supply gas to the medium, and strip the medium of metabolically produced offgas. The porous membrane nanobubble generation section 23 comprises a nanobubble generator gas supply conduit 26 as an inlet conduit to generate nanobubbles 19 in the medium contained in the bioreactor vessel. The porous membrane nanobubble generation section 23 may, for example, be any of the types described and shown from FIG. 20A to FIG. 24B, which will be discussed further below.

[0157] According to a preferred embodiment, with reference to FIG. 17B, there is provided an internal loop airlift bioreactor vessel 20c which is equipped externally with a bioreactor off gas exhaust conduit 21. Internally the bioreactor vessel is equipped with a porous membrane nanobubble generation section 23 in the shape of a cylinder which is located in the internal loop airlift bioreactor downcomer 31 in the internal volume of the draft tube 32, and a gaseous coarse / micro bubble sparger 22 that is located beneath internal loop airlift bioreactor riser 33.The internal loop airlift bioreactor riser 33 in this embodiment is defined by the annulus between the draft tube 32 and the internal loop airlift bioreactor vessel 20c. The gaseous coarse / micro bubble sparger 22 comprises a gaseous coarse / micro bubble sparger gas supply conduit 24 as an inlet conduit to generate gaseous coarse / micro bubble 34 inside the bioreactor vessel to generate medium circulation and mix the medium, supply gas to the medium, and strip the medium of metabolically produced off-gas. The porous membrane nanobubble generation section 23 comprises a nanobubble generator gas supply conduit 26 as an inlet conduit to generate nanobubbles 19 in the medium contained in the bioreactor vessel. The porous membrane nanobubble generation section 23 may, for example, be any of the types described and shown from FIG. 20A to FIG. 24B, which will be discussed further below.

[0158] A key aspect of the invention, particularly in the embodiments shown in FIG. 12 to FIG. 17B, is the synergistic relationship between the gaseous coarse / micro bubble sparger 22 and the internal nanobubble generation section 23, 30. The coarse / micro bubble sparger 22 is not only used for conventional aeration or gas stripping, but it also serves as the primary engine for medium circulation within the vessel via the airlift effect. This induced medium circulation creates a continuous crossflow across the surface of the porous membranes of the internal nanobubble generation section 23, 30. This crossflow is essential for the forced detachment mechanism that shears off gas protrusions to form nanobubbles. Consequently, the system cleverly potentially eliminates the need for an external pump to create said crossflow, thereby reducing system complexity, capital cost, and potential shear damage to the cells. The two internal components thus form a self-contained and highly efficient gas transfer and mixing system.

[0159] With reference to FIG. 18, there is an embodiment of the porous membrane nanobubble generation section 30 described in FIG. 16A and FIG. 16B, with a porous membrane nanobubble generation section 30, in the shape of a cylindrical annulus, which is located in between the draft tube 32 and the internal loop airlift bioreactor vessel 20c. Nanobubble generator gas supply conduit 26 supplies the main gas manifold 35 located along the perimeter of the porous membrane nanobubble generation section 23 with gas to generate nanobubbles 19 in the medium contained in the bioreactor. The annular porous membrane nanobubble generation section 30 in this configuration may, for example, be any of the types described and shown in FIG. 19A or FIG. 19B, which will be discussed further below.

[0160] With reference to FIG. 19A, there is an embodiment of the porous membrane nanobubble generation section 30 as described in FIG. 16A and FIG. 18, where one or more porous membranes 8a, 8b are located vertically inside the section, configured in a circularpattern along the perimeter of the internal loop airlift bioreactor vessel 20c, with the porous membrane(s) 8a, 8b being supported and supplied with gas via gas manifolds mounted to the perimeter wall of the internal loop airlift bioreactor vessel 20c, being in fluid connection with the main gas manifold 35. The inside volume of the draft tube 32 functions as the internal loop airlift bioreactor riser 33, and the annulus functions as the internal loop airlift bioreactor downcomer 31. Nanobubble generator gas supply conduit 26 supplies the main gas manifold 35 located along the perimeter of the porous membrane nanobubble generation section 23 with gas to generate nanobubbles 19 in the medium contained in the bioreactor vessel.

[0161] With reference to FIG. 19B, there is an embodiment of the annular porous membrane nanobubble generation section 30 described in FIG. 16B and FIG. 18, where one or more porous membranes 8a, 8b are located vertically inside the section, configured in a circular pattern along the perimeter of the internal loop airlift bioreactor vessel 20c, with the porous membrane(s) 8a, 8b being supported and supplied with gas via gas manifolds mounted to the perimeter wall of the internal loop airlift bioreactor vessel 20c, being in fluid connection with the main gas manifold 35. The inside volume of the draft tube 32 functions as the internal loop airlift bioreactor downcomer 31, and the annulus functions as the internal loop airlift bioreactor riser 33. Nanobubble generator gas supply conduit 26 supplies the main gas manifold 35 located along the perimeter of the porous membrane nanobubble generation section 23 with gas to generate nanobubbles 19 in the medium contained in the bioreactor vessel.

[0162] With reference to FIG. 20A, there is described an embodiment of the porous membrane nanobubble generation section 23 where one or more porous membranes 8a, 8b are located horizontally along the center line of the circular section, configured parallel to one another in a vertical ladder-like configuration. Nanobubble generator gas supply conduit 26 supplies the main gas manifold 35 located along the perimeter of the porous membrane nanobubble generation section 23 with gas to generate nanobubbles 19 in the medium contained in the bioreactor vessel. FIG. 20B shows a cross sectional side view of the embodiment described in FIG. 20A.

[0163] With reference to FIG. 21A, there is described an embodiment of the porous membrane nanobubble generation section 23 in which one or more porous membranes are located vertically and parallel to one another, configured in a row along the center line of the porous membrane nanobubble generation section 23 into a sideways ladder-like configuration pattern, where two cross beam gas manifolds 36 extend along the center line to support the top and bottom of the porous membranes 8a, 8b and supply them with gas. Nanobubblegenerator gas supply conduit 26 supplies the main gas manifold 35 located along the perimeter of the porous membrane nanobubble generation section 23 with gas to generate nanobubbles 19 in the medium contained in the bioreactor vessel. FIG. 21B shows a cross sectional side view of the embodiment described in FIG. 21 A.

[0164] With reference to FIG. 22A, there is described an embodiment of the porous membrane nanobubble generation section 23 in which one or more porous membranes 8a, 8b are located vertically, configured in a circular pattern along the perimeter of the section, with the porous membrane(s) 8a, 8b being supported and supplied with gas via wall support gas manifolds 37 mounted to the perimeter wall of the section, being in fluid connection with the main gas manifold 35. Nanobubble generator gas supply conduit 26 supplies the main gas manifold 35 located along the perimeter of the porous membrane nanobubble generation section 23 with gas to generate nanobubbles 19 in the medium contained in the bioreactor vessel. FIG. 22B shows a cross sectional side view of the embodiment described in FIG. 22A.

[0165] With reference to FIG. 23A, there is described an embodiment of the porous membrane nanobubble generation section 23 where one or more porous membranes 8a, 8b are located horizontally along one or more cross-sectional horizontal planes, configured parallel to one another in a horizontal ladder-like configuration pattern. The nanobubble generator gas supply conduit 26 supplies the main gas manifold 35 located along the perimeter of the porous membrane nanobubble generation section 23 with gas to generate nanobubbles 19 in the medium contained in the bioreactor vessel. FIG. 23B shows a cross sectional side view of the embodiment described in FIG. 23 A.

[0166] With reference to FIG. 24A, there is described an embodiment of the porous membrane nanobubble generation section 23 where one or more porous membranes 8a, 8b are located horizontally along its center line, configured such that for the membrane(s) along the vertical dimension, the consecutive membrane is rotated by a fixed number of degrees in the horizontal plane with respect to the previous membrane, to form a helix-like configuration pattern. The nanobubble generator gas supply conduit 26 supplies the main gas manifold 35 located along the perimeter of the porous membrane nanobubble generation section 23 with gas to generate nanobubbles 19 in the medium contained in the bioreactor vessel. FIG. 24B shows a cross sectional side view of the embodiment described in FIG. 24A.

[0167] In an aspect of the invention is disclosed a gas-driven bioreactor system with a nanobubble sparging system, designed to improve gas-liquid mass transfer, GTR and more homogeneous DG, within a medium containing biological production systems compared to existing similarly sized bioreactors. Said aspect of the invention centers around one or moreporous membranes integrated directly within the bioreactor vessel. These membranes receive pressurized gas through their internal channels or outer surface, which permeate through the membrane walls into the surrounding medium, forming gaseous nanobubbles in situ. The nanobubble formation process occurs as the medium, as a result of gas-driven mixing (via coarse / micro bubble spargers), flows across the outer or inner surface of the porous membranes, cutting off emerging gas protrusions into stable sub-micron bubbles.

[0168] Unlike conventional nanobubble generators, the gas-driven bioreactor system has no external recirculation loops, cavitation, or separation of the biological production systems from the medium, and instead introduces nanobubbles directly into the medium where biological production systems are producing biomass or biological products. The membrane section can be installed in multiple orientations and reactor geometries (such as cylindrical, annular, vertical, horizontal, etc.), depending on the bioreactor design and flow conditions.

[0169] Yet another embodiment of the invention includes an internal loop airlift bioreactor vessel 20c in which an external aseptic pump is placed (and configured) to aid medium circulation and mixing inside the bioreactor, while at the same time circulating the medium through the porous membrane nanobubble generation section 23 or the annular porous membrane nanobubble generation section 30 to entrain the medium with gaseous nanobubbles 19. The pump may be connected with an inlet drawing medium from the top of the bioreactor and an outlet in the lower portion of the bioreactor which may be before or after either nanobubble generation section.

[0170] In airlift bioreactors, the addition of nanobubbles to the medium is an effective way to reach more homogeneous DG and higher GTR in the downcomer and poor-mixing or dead zones, without disturbing the mass density-difference between the riser and downcomer due to the inherently high gas mass density of nanobubbles compared to coarse / micro bubbles, therefore not disturbing the airlift medium circulation loop. This can, for example, be used for biological production systems that are sensitive to a periodic lack of gases, or that shift their metabolic pathways in the case of low gas availability, and consequently, this invention allows for using large scale airlift reactors at high performance for biological production systems culture and / or cell growth, proliferation and / or biological product production.

[0171] In a further embodiment of the invention, there is provided a bioreactor system comprising a disposable culture vessel bag for a single-use bioreactor system, such as that commonly used, but not limited to, in pharmaceutical or cell therapy manufacturing environments. In an embodiment, with reference to FIG. 25, the medium 100 contained within the treatment tank 2 of the disposable vessel is circulated via an external loop using a low-shear, sterile peristaltic pump 10, which drives fluid flow without contaminating the medium. The medium 100 exits the bioreactor through a sterile port, flows through a disposable pump medium inlet conduit 9, flows through peristaltic pump 10, and then flows through nanobubble sparger medium inlet 3 and enters the external nanobubble sparger 13, which houses a multitude of porous membranes, for example disposable polymeric hollow fibers, amongst others. A gas such as air or oxygen is introduced into the outer chamber of the sparger (not explicitly shown in this figure but described in prior figures), allowing gas to flow inward through the hollow fiber pores into the lumen where the medium is flowing. This generates nanobubbles within the circulating medium without requiring any internal gas sparging or bubble-forming agitation inside the vessel itself. The nanobubble-enriched medium then reenters the bioreactor vessel through a second sterile port at a different height or orientation, enabling gentle recirculation and efficient gas transfer — a feature, for example, for sensitive cell cultures such as CHO cells, stem cells, or viral vectors.

[0172] In a further and preferred embodiment relating to the single-use bioreactor system shown in FIG. 25, the nanobubble generation and medium circulation are achieved entirely through internally integrated components, thereby eliminating the need for the external circulation loop 9, 10, 13.

[0173] In this configuration, the disposable culture vessel bag 2 is supplied with a presterilized, internally mounted nanobubble generation section 23, 30, for example comprising one or more polymeric hollow fiber membranes integrated into an internal support structure within the bag. Furthermore, the bag is equipped with an integrated gaseous coarse / micro bubble sparger 22, such as a ring sparger or a drilled-tube sparger, typically located at the bottom of the vessel.

[0174] During operation, the coarse / micro bubble sparger 22 generates a buoyant flow of larger bubbles, which induces gentle but effective medium circulation throughout the vessel (i.e., via the airlift principle or general bubble column mixing). This internally generated medium circulation provides the necessary crossflow over the surfaces of the internal nanobubble generation section 23, 30, enabling the forced detachment of nanobubbles directly into the culture medium.

[0175] This fully integrated arrangement provides significant advantages for single-use applications, as it simplifies the setup, minimizes the number of sterile connections, eliminates the risk of tubing failure or contamination associated with an external pump loop, and provides a completely disposable flow path, all while achieving highly efficient gas transfer and gentle mixing.In a preferred and more integrated embodiment for single-use bioreactor systems, as depicted in FIG. 25, the nanobubble generation and medium circulation are achieved entirely through internally integrated components.

[0176] In this configuration, the disposable culture vessel bag 2 is supplied with a presterilized, internally mounted nanobubble generation section 23, 30, for example comprising polymeric hollow fiber membranes, and an integrated gaseous coarse / micro bubble sparger 22. The coarse / micro bubble sparger 22 induces medium circulation via the airlift principle, which in turn creates the necessary crossflow over the internal nanobubble generation section 23, 30 for forced detachment of nanobubbles. This fully integrated arrangement provides significant advantages for single-use applications by simplifying the setup, minimizing sterile connections, and eliminating the risks associated with an external pump loop.

[0177] According to an embodiment, with reference to FIG. 26A, there is described a singlechannel porous membrane 8a in which a functional surface modification 38, e.g. a hydrophobic surface layer, is applied to (such as coating) the surfaces of the membrane, such as at least one of the outer membrane surface 16, the inner membrane surface 17, and / or the walls of pores 15. It should be noted that the functional surface modification 38 may also be applied to one or more of these surfaces selectively, or a mix of different types of functional surface modification for each surface. For example, a hydrophobic surface layer may be applied to either the outer surface 16, to the inner surface 17, or to the walls of pores 15, in order to tailor specific functional properties such as, for example, improved bubble detachment, increased fouling resistance, or lowered transmembrane pressure by preventing wetting of the pores and thereby reducing the capillary pressure that needs to be overcome by the sparged gas. The functional surface modification is illustrated further as a longitudinal cross-section in FIG. 26B.

[0178] The porous membrane utilized in the present system may be composed of any material with pore sizes ranging from 1 nm to 1,000 nm, allowing for effective gas transfer while maintaining structural integrity. While ceramic materials may be used due to their high durability, temperature resistance, and chemical stability, other suitable materials include, but are not limited to, polystyrene, polypropylene, polyethylene, polyethersulfone (PES), polyvinylidene fluoride (PVDF), or other polymeric, metallic, or composite materials exhibiting certain porosity and permeability characteristics. The choice of material may depend, but not limited to, on the specific application, compatibility with the operating environment, cost considerations, ease of manufacturing and single-use or reusable applications.Furthermore, the term “porous membrane” as used herein also encompasses membranes that are treated with a hydrophobic, hydrophilic, or other functional surface modification that modifies the surface properties while maintaining the fundamental porosity of the membrane. Such functional surface modification may be applied for various purposes, including but not limited to, decreasing wettability of the pores to reduce transmembrane pressure for gas, preventing fouling, improving bubble detachment and hence nanobubble formation, increasing chemical resistance, or altering surface energy properties. Examples of suitable functional surface modification include silicone-based hydrophobic grafts, fluoropolymer coatings, plasma-treated hydrophilic layers, biofilm-resistant coatings, or other functionalized surface treatments that do not obstruct the membrane pores but improve performance in specific applications.

[0179] The present invention further comprises methods for culturing biological production systems comprising generating nanobubbles within the medium 100, such as a method for culturing biological production systems, such as cell cultivation and fermentation, in any mode of operation (batch, fed-batch, draw-and-fill, perfusion, continuous, or any other mode of operation), where the goal is to culture biological production systems and improve cell growth, cell proliferation and / or biological product production.

[0180] Thus, in an aspect of the invention, there are described methods for generating nanobubbles within the medium comprising entraining the medium with nanobubbles in the presence of biological production systems and biological products for the duration of the production run, such as for the duration of the entire production run.

[0181] In some methods, the nanobubbles are generated using an external nanobubble sparger consisting of a porous membrane through which gas flows from one side of the porous membrane, through the membrane material and to the other side of the membrane material, where a crossflow of medium cuts off the gaseous protrusions from the membrane pores at the membrane surface such that nanobubbles are formed, and such that they become entrained in the medium. Said forced flow of the medium may be produced by pumping using any type or mechanism suitable for circulating the liquid medium (hereinafter referred to as “pump”) or mixing device for generating a flow of the medium.

[0182] In an alternative embodiment, such as for pump-driven systems, there is provided a method for culturing biological production systems and producing biological products comprising generating nanobubbles comprising the steps of:

[0183] (a) filling 1001 a treatment tank partially or completely with previously sterilized or pasteurized medium at the start of a production run;(b) circulating 1002 the medium through the nanobubble generator, for example by means of a pump 10;

[0184] (c) activating 1003 the nanobubble generator to entrain the medium with nanobubbles;

[0185] (d) introducing 1004 biological production systems into the medium;

[0186] (e) continuing 1005 the circulation of the medium through the active nanobubble generator, entraining the medium with nanobubbles in the presence of biological production systems and biological products for the duration of the production run. In a further embodiment, there is provided a method for culturing biological production systems and producing biological products comprising generating nanobubbles comprising the steps of:

[0187] (a) filling 2001 a treatment tank partially or completely with previously sterilized or pasteurized medium at the start of a production run;

[0188] (b) activating 2002 the gaseous coarse / micro bubble sparger to circulate the medium, inducing medium flow through the nanobubble generator, such as a nanobubble generation section;

[0189] (c) activating 2003 the nanobubble generator, such as a nanobubble generation section, to entrain the medium with nanobubbles;

[0190] (d) introducing 2004 biological production systems into the medium;

[0191] (e) continuing 2005 the circulation of the medium through the active nanobubble generator, entraining the medium with nanobubbles in the presence of biological production systems and biological products for the duration of the production run. In a preferred embodiment, there is provided a method for culturing biological production systems and producing biological products in a bioreactor system as defined herein, the method comprising the steps of:

[0192] (a) filling a treatment tank 2 with a medium 100;

[0193] (b) introducing biological production systems into the medium 100;

[0194] (c) operating the bioreactor system by simultaneously:

[0195] (i) introducing a first gas into the medium 100 via the gaseous coarse or micro bubble sparger 22 to induce a circulating flow of the medium and to strip unwanted dissolved gases; and

[0196] (ii) introducing a second gas into the medium 100 via the internally placed nanobubble generation section 23, 30, wherein said circulating flow induced in step (c)(i) provides the crossflow for said nanobubble generation section, toentrain the medium with nanobubbles of said second gas.

[0197] This preferred method provides a significant process advantage by decoupling the function of supplying essential gases (e.g., oxygen via the nanobubble generation section) from the function of removing unwanted gases (e.g., CO2 stripping via the coarse / micro bubble sparger). This allows an operator to independently control the dissolved oxygen (DO) concentration and the dissolved carbon dioxide (dCO?) concentration, enabling a more precise and optimized control of the cell culture environment compared to prior art systems where these functions are inherently linked. A particular advantage of this decoupled functionality is the ability to achieve independent control over two or more dissolved gas concentrations even when using the same gas type, such as ambient air, for both the nanobubble generation section 23, 30 and the gaseous coarse / micro bubble sparger 22. This is possible due to the fundamentally different physical properties of nanobubbles versus coarse / micro bubbles. For example, nanobubbles with their high surface area and long residence time, are highly efficient at dissolving the supplied gas (e.g., oxygen from the air) into the medium. In contrast, coarse / micro bubbles, with their rapid buoyancy and short residence time, are highly efficient at stripping already dissolved gases (e.g., metabolic CO2) from the medium with minimal dissolution of the sparged gas itself. This enables a simplified and cost-effective system design that, for example, can use a single compressed air source to both supply oxygen and strip carbon dioxide, as opposed to prior art approaches that would require two different gas sources (e.g., pure oxygen and a separate stripping gas like nitrogen) to achieve a similar decoupled control.

[0198] In one or more embodiments, the methods defined herein are characterized in that the first gas and the second gas have the same composition. In one or more examples, the first gas and the second gas both comprise air.

[0199] According to an embodiment of the invention, the methods are suitable for the production of cellular biomass and / or biologically derived compounds by culturing biological production systems in vitro, including but not limited to culturing, fermenting, and facilitating activity of cell-free expression systems.

[0200] In an example, the method comprises flowing gas within the at least one internal channel 18, wherein the gas flows through the at least one pore 15 to the outer surface 16 into the medium, which is flowing past the outer surface 16, and entraining the medium with nanobubbles.

[0201] In another example, the method comprises flowing gas from the outside of the porous membrane 8a, 8b through the at least one pore 15 and into the at least one internal channel18 through which medium is flowing past the inner surface 17, and entraining the medium with nanobubbles.

[0202] According to an embodiment of the invention, there is described a method for generating nanobubbles within the medium wherein the step of entraining the medium with nanobubbles comprises the step of allowing the medium flowing past the inner or outer surface, cutting of the gas protrusions originating from the at least one pore 15, such as plurality of pores 15a, 15b, such that nanobubbles are generated.

[0203] In an embodiment, the bioreactor tank / vessel 11 or 12 is partially or completely filled with previously sterilized / pasteurized medium at the start of a production run (batch, fed-batch, draw-and-fill, continuous, perfusion, or any other mode of operation). The medium is circulated via pump 10 where it is run through the external nanobubble sparger, inside of which the medium flows past the porous membrane 7 surfaces through which a gas is pushed through from the opposite side of the membrane(s), and consequently generating gaseous nanobubbles in the liquid medium. From there the medium entrained with nanobubbles flows back into the bioreactor vessel 11 or 12. A cell starter culture or cell-free expression system is then introduced to the medium. The generation of nanobubbles in the medium containing biological production systems is then continued for the duration of the production process. In the configuration where the bioreactor does not contain any agitators and impellers 12, the pump 10 can also act to provide the primary mechanism of mixing of the medium inside the bioreactor.

[0204] In an embodiment, the medium circulation is maintained either for the complete duration of the production process, or solely for a part of the total duration, for example (though not exclusively) by activating the external nanobubble sparger and medium circulation loop during the exponential phase of the production process where gas demand is highest.

[0205] In a further embodiment, the treatment tank / vessel 20a, 20b, 20c is filled completely with previously sterilized or pasteurized medium at the start of a production run (batch, draw-and-fill, continuous, perfusion, or any other mode of operation). The gaseous coarse / micro bubble sparger 22 is then activated to circulate the medium of the bioreactor through buoyancy forces and pneumatic action, and thereby inducing the medium flow through the porous membrane nanobubble generation section 23 or the annular porous membrane nanobubble generation section 30. Inside these sections the medium flows past the porous membrane(s) 8a, 8b, inside which a gas is inserted into the internal channel(s) 18 of the porous membrane(s) 8a, 8b, thereafter permeating through the membrane material 14 towards the porous membrane outer surface 16 creating gas protrusions at the membrane pores 15, where the crossflow ofmedium over the outer membrane surface cuts off the gaseous bubble protrusions such that nanobubbles 19 are formed, and such that they become entrained in the medium. A cell starter culture or cell-free expression system is then introduced to the medium. The generation of nanobubbles in the medium containing biological production systems is then continued for the duration of the production process.

[0206] In an embodiment, the medium circulation through pneumatically induced motion is maintained for the complete duration of the production run in order to keep circulating the medium and stripping off-gases, such as those produced by the biological production systems in the medium.

[0207] In an embodiment, with reference to the embodiment shown in FIG. 3, following a production cycle, the method further comprises switching 3 -way valve 39a from the nanobubble generator gas supply conduit 26 to the CIP / SIP supply conduit 40, and closing 3- way valve 39b to isolate the nanobubble sparger utility outlet 6. Cleaning fluid, such as a CIP medium, is then introduced under pressure into CIP / SIP supply conduit 40, thereby entering the nanobubble generator housing 7 and filling sub-volume Vsa created in the space between the inner housing walls and the outer surface 16 of the membrane 8a, 8b. The cleaning fluid flows across the porous membrane outer surfaces 16 of the at least one porous membrane 8a or 8b and is forced through the membrane pores 15 into the porous membrane internal channel 18, contacting and removing any accumulated biofilm, fouling, and residues along all wetted surfaces. The spent cleaning fluid exits via nanobubble sparger medium outlet 4, whereafter it may be discarded via a standard CIP / SIP drainage system connected to the bioreactor. After the cleaning fluid has been flushed, sterilization may be performed by introducing steam at elevated temperature into the nanobubble sparger via the same CIP / SIP supply conduit 40 and nanobubble sparger utility inlet 5. The steam is forced through membranes 8a and 8b, thereby sterilizing both the membrane interiors and the internal volume of housing 7. Sterilization may be performed for a pre-configured amount of time based on the specific application. Once sterilization is complete, 3-way valve 39b and CIP / SIP drain conduit 41 may be opened to evacuate any remaining condensate or residual cleaning fluid, after which all valves are returned to their production configuration, and the nanobubble generator is ready for subsequent production runs.

[0208] The present invention is advantageous over known techniques at least by providing an increased GTR in the medium without compromising the viability or alive state of the cells or the activity of biological production systems present in the medium, providing improved homogeneity of GTR throughout the medium, providing increased the gas-liquid mass transferlimit within the bioreactor, optionally eliminating the need to depend on a mixing system comprised by agitator / stirrer, impellers and baffles, for example those provided in stirred-tank bioreactors, for proper mixing and gas diffusion, providing freedom in the design geometry of the bioreactor, providing an improved proliferation or growth of the cells or activity of the biological production systems present in the medium, enabling cells or biological production systems present in the medium to improve their production of biological products.

[0209] The disclosed system and method are suitable for both aerobic and anaerobic bioprocesses. In aerobic applications, the nanobubble generation section is typically used to efficiently supply oxygen to achieve a high Oxygen Transfer Rate (OTR), while the coarse bubble sparger is used for bulk mixing and stripping of metabolically produced CO2. In anaerobic applications, the nanobubble generation section can be used to sparge an inert gas like nitrogen, while the coarse bubble sparger can be used for mixing and stripping of potentially inhibitory gaseous byproducts. This independent control over gas supply and stripping offers superior process control for a wide range of biological production systems.

[0210] Further embodiments

[0211] In an embodiment, there is disclosed a bioreactor system 1 comprising:

[0212] a) a treatment tank 2, wherein said treatment tank 2 comprises a medium 100 for culturing prokaryotic cells, eukaryotic cells, and / or cell-free expression systems in vitro for the purpose of producing cellular biomass and / or biologically derived compounds;

[0213] b) viable prokaryotic cells, viable eukaryotic cells, active cell-free expression systems or a combination thereof;

[0214] c) a nanobubble generator 13, 23, 30 configured to generate nanobubbles in the medium 100 in the presence of said viable prokaryotic cells, viable eukaryotic cells, active cell-free expression systems or a combination thereof, wherein the nanobubble generator 13, 23, 30 is further configured to receive a crossflow of medium 100 along at least a portion of a primary surface, said primary surface being either the outer surface 16 or the inner surface 17 of the porous membrane 8a, 8b, thereby enabling forced detachment of generated nanobubbles from said membrane surface, said nanobubble generator 13, 23, 30 comprising:

[0215] i) at least one porous membrane 8a, 8b comprising an outer surface 16 and an inner surface 17, wherein at least one channel 18 is formed by at least a part of the inner surface 17 of the membrane 8a, 8b, wherein the at least one channel 18 is extending longitudinally at least partially along a length of the porous membrane 8a, 8b, wherein the porous membrane 8a, 8b comprises a plurality of pores 15a, 15b arranged to achieve fluid contact between the outersurface 16 of the membrane, and the inner surface 17 of the membrane, wherein the pores 15a, 15b are of a diameter of 1 to 1,000 nm,

[0216] ii) a gas inlet 26 in fluid contact with the nanobubble generator 13, 23, 30 and configured to flow gas for generating nanobubbles.

[0217] In an embodiment, the nanobubble generator 13, 23, 30 is adapted to create nanobubbles with a diameter of 10 to 1,000 nm.

[0218] In an embodiment, the nanobubble generator 13 comprises a housing 7 wherein the at least one porous membrane 8a, 8b is placed within the housing 7 in a co-linear or perpendicular manner to the flow direction of the medium 100.

[0219] In an embodiment, the nanobubble generator 13 is configured to receive the medium 100 via a medium conduit configured to circulate the medium 100 through the nanobubble generator 13 which is adapted to entrain nanobubbles within the medium inside of the nanobubble generator 13.

[0220] In an embodiment, the at least one porous membrane 8a, 8b comprises a functional surface modification.

[0221] In an embodiment, the nanobubble generator 13 is placed externally to the treatment tank 2. In an embodiment, the nanobubble generator 23, 30 is placed internally within the treatment tank 2.

[0222] In an embodiment, the medium 100 circulates through the nanobubble generator 13 via the at least one channel 18 in the inner surface 17 of the at least one porous membrane 8a, 8b.

[0223] In an embodiment, the medium 100 circulates through the nanobubble generator 13, 23, 30 past the outer surface 16 of the at least one porous membrane 8a, 8b.

[0224] In an embodiment, the gas flows via the at least one channel 18 in the inner surface 17 of the at least one porous membrane 8a, 8b.

[0225] In an embodiment, the treatment tank 2 further comprises a mixing device.

[0226] In an embodiment, the mixing device is a gaseous coarse / micro bubble sparger 22.

[0227] In an embodiment, the system further comprises at least one system selected from the group consisting of a gas supply system, an exhaust system, a pH adjustment system, a feed system, a temperature control system, and an automated control system.

[0228] In an embodiment, there is described a method for culturing biological production systems and producing biological products comprising generating nanobubbles within a system as defined herein, wherein the method comprises the steps of

[0229] filling 1001 a treatment tank / vessel 11, 12 partially or completely with previously sterilized or pasteurized medium at the start of a production run;circulate 1002 the medium 100 through the nanobubble generator 13;

[0230] activating 1003 the nanobubble generator 13 to entrain the medium 100 with nanobubbles; introducing 1004 a cell starter culture or cell-free expression systems into the medium 100, whereafter the medium comprises viable prokaryote cells, eukaryote cells, cell-free expression systems or a combination thereof;

[0231] continuing 1005 the circulation of the medium 100 through the active nanobubble generator 13, entraining the medium 100 with nanobubbles in the presence of prokaryote cells, eukaryote cells, cell-free expression systems or a combination thereof, for the total or the partial duration of the production run.

[0232] In an embodiment, there is described a method for culturing biological production systems and producing biological products comprising generating nanobubbles within a system as defined herein, wherein the method comprises the steps of:

[0233] filling 2001 a treatment tank / vessel 20a, 20b, 20c completely with previously sterilized or pasteurized medium at the start of a fermentation or cultivation run;

[0234] activating 2002 the gaseous coarse / micro bubble sparger 22 to circulate the medium, inducing medium flow through the nanobubble generator 23, 30;

[0235] activating 2003 the nanobubble generator 23, 30 to entrain the medium 100 with nanobubbles; introducing 2004 a cell starter culture or cell-free expression systems into the medium 100, whereafter the medium comprises viable prokaryote cells, eukaryote cells, cell-free expression systems or a combination thereof;

[0236] continuing 2005 the circulation of the medium 100 through the active nanobubble generator 23, 30, entraining the medium 100 with nanobubbles in the presence of prokaryote cells, eukaryote cells, cell-free expression systems or a combination thereof, for the total or the partial duration of the production run.REFERENCE NUMERALS

[0237] 1 - bioreactor system

[0238] 2 - treatment tank

[0239] 3 - nanobubble sparger medium inlet

[0240] 4 - nanobubble sparger medium outlet

[0241] 5 - nanobubble sparger utility inlet

[0242] 6 - nanobubble sparger utility outlet

[0243] 7 - porous membrane(s) housing

[0244] 8, 8a, 8b - porous membrane(s)

[0245] 9 - pump medium inlet conduit

[0246] 10 - pump

[0247] 11 - stirred tank bioreactor (STR) vessel

[0248] 12 - bioreactor vessel without agitator / impeller

[0249] 13, 13a, 13b - external nanobubble sparger

[0250] 14 - porous membrane material

[0251] 15, 15a, 15b - membrane pore

[0252] 16 - porous membrane outer surface

[0253] 17 - porous membrane inner surface

[0254] 18, 18a, 18b - porous membrane internal channel

[0255] 19 - nanobubble

[0256] 20a - bubble column bioreactor vessel

[0257] 20b - external loop airlift bioreactor vessel

[0258] 20c - internal loop airlift bioreactor vessel

[0259] 21 - off gas exhaust conduit

[0260] 22 - gaseous coarse / micro bubble sparger

[0261] 23 - porous membrane nanobubble generation section

[0262] 24 - gaseous coarse / micro bubble sparger gas supply conduit 25 - external nanobubble sparger sensor port

[0263] 26 - nanobubble generator gas supply conduit

[0264] 27 - agitator / impeller

[0265] 28 - external loop airlift bioreactor riser

[0266] 29 - external loop airlift bioreactor downcomer

[0267] 30 - annular porous membrane nanobubble generation section 31 - internal loop airlift bioreactor downcomer32 - internal loop airlift reactor draft tube 33 - internal loop airlift bioreactor riser 34 - gaseous coarse / micro bubble

[0268] 35 - main gas manifold

[0269] 36 - cross beam gas manifold

[0270] 37 - wall support gas manifolds

[0271] 38 - functional surface modification 39a, 39b - 3 -way valve

[0272] 40 - CIP / SIP supply conduit

[0273] 41 - CIP / SIP drain conduit

[0274] 100 - medium

Claims

CLAIMS1. A bioreactor system (1) comprising:a) a treatment tank (2), wherein said treatment tank (2) comprises a medium (100) for culturing prokaryotic cells, eukaryotic cells, and / or cell-free expression systems in vitro for the purpose of producing cellular biomass and / or biologically derived compounds; b) viable prokaryotic cells, viable eukaryotic cells, active cell-free expression systems or a combination thereof;c) a nanobubble generation section (23, 30) configured to generate nanobubbles in the medium (100) in the presence of said viable prokaryotic cells, viable eukaryotic cells, active cell-free expression systems or a combination thereof, wherein the nanobubble generation section (23, 30) is further configured to receive a crossflow of medium (100) along at least a portion of a primary surface, said primary surface being one of the first and second surfaces of the porous membrane (8a, 8b), thereby enabling forced detachment of generated nanobubbles from said membrane surface, said nanobubble generation section (23, 30) comprising:i) at least one porous membrane (8a, 8b) comprising a first surface and an opposing second surface, and a plurality of pores (15a, 15b) extending through the membrane to provide fluid contact between said first surface and said second surface, wherein the pores (15a, 15b) are of a diameter of 1 to 1,000 nm, ii) a gas inlet (26) in fluid contact with the nanobubble generation section (23, 30) and configured to flow gas for generating nanobubbles,wherein the nanobubble generation section (23, 30) is placed internally within the treatment tank (2), and wherein the treatment tank (2) further comprises at least one coarse bubble sparger (22).

2. The system according to claim 1, wherein the treatment tank (2) is an airlift bioreactor, and wherein the circulation of the medium (100) is driven by airlift generated by the at least one coarse bubble sparger (22), said airlift-driven circulation providing the crossflow of medium required by the nanobubble generations section (23, 30).

3. The system according to claim 1, wherein the treatment tank (2) further comprises a circulation pump configured to circulate the medium (100), and wherein said circulation provides the crossflow of medium required by the nanobubble generation section (23, 30).

534. The system according to any of claims 1 to 3, wherein the nanobubble generation section (23, 30) is adapted to create nanobubbles with a diameter of 10 to 1,000 nm.

5. The system according to any one of the preceding claims, wherein the at least one porous membrane (8a, 8b) comprises a functional surface modification.

6. The system according to claim 5, wherein the functional surface modification comprises a hydrophobic surface layer applied to at least one of the first surface (16), the second surface (17), and / or the walls of the pores (15) of the porous membrane (8a, 8b).

7. The system according to any one of the preceding claims, further comprising a gas supply system having a single gas source, wherein said single gas source is fluidly connected to both the gas inlet (26) of the nanobubble generation section (23, 30) and a gas supply conduit (24) of the at least one coarse bubble sparger (22).

8. The system according to any one of the preceding claims, wherein the treatment tank (2) is an internal loop airlift bioreactor (20c) comprising a riser (33) and a downcomer (31), wherein the nanobubble generation section (23, 30) is located in said downcomer (31), and wherein the at least one coarse bubble sparger (22) is located to introduce gas at the base of said riser (33).

9. The system according to claim 8, wherein the riser (33) and the downcomer (31) define a closed-loop fluid circulation path entirely within the treatment tank (2), wherein the system is arranged such that the introduction of gas from the at least one coarse bubble sparger (22) into the riser (33) induces an upward flow of the medium (100) in the riser and a corresponding downward flow of the medium (100) in the downcomer (31), said downward flow constituting the crossflow of medium received by the nanobubble generation section (23, 30).

10. The system according to any one of the preceding claims, wherein the nanobubble generation section (23, 30) comprises a plurality of porous membranes (8a, 8b) arranged in a vertical or horizontal ladder-like configuration.

11. The system according to any one of the preceding claims, wherein the nanobubblegeneration section (23, 30) comprises a plurality of porous membranes (8a, 8b) arranged in a helix-like configuration.

11. The system according to any one of the preceding claims, wherein the nanobubble generation section (23, 30) comprises a plurality of porous membranes (8a, 8b) arranged in a circular pattern along the perimeter of a section of the treatment tank (2).

12. The system according to any one of the preceding claims further comprising at least one system selected from the group consisting of a gas supply system, an exhaust system, a pH adjustment system, a feed system, a temperature control system, and an automated control system.

13. A method for culturing biological production systems and producing biological products in a bioreactor system as defined in any one of claims 1 to 12, the method comprising the steps of:(a) filling a treatment tank (2) with a medium (100);(b) introducing biological production systems into the medium (100);(c) operating the bioreactor system by simultaneously:(i) introducing a first gas into the medium (100) via the at least one coarse bubble sparger (22) to induce a circulating flow of the medium and to strip unwanted dissolved gases; and(ii) introducing a second gas into the medium (100) via the internally placed nanobubble generation section (23, 30), wherein said circulating flow induced in step (c)(i) provides the crossflow for said nanobubble generation section, to entrain the medium with nanobubbles of said second gas.

14. The method according to claim 13, wherein the first gas and the second gas have the same composition.

15. The method according to claims 13 or 14, wherein the first gas and the second gas both comprise air.

16. Use of a bioreactor system according to any one of claims 1 to 12 for in situ generation of nanobubbles in a medium (100) containing viable prokaryotic cells, eukaryotic55cells, or cell-free expression systems for the production of cellular biomass and / or biologically derived products.

17. The use according to claim 16, wherein said use is performed as a bioprocess for the simultaneous and decoupled control of: (i) the supply of a process gas to a medium (100) via the nanobubble generation section (23, 30), and (ii) the removal of an unwanted gas from the medium (100) via the coarse bubble sparger (22).

18. The use according to claim 17, wherein the process gas is oxygen.

19. The use according to claims 17 or 18, wherein the unwanted gas is carbon dioxide.

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