NL2034413B1

NL2034413B1 - An elongated separation device for multiphase biocatalytic reactor

Info

Publication number: NL2034413B1
Application number: NL2034413A
Authority: NL
Inventors: Johanna Maria Van Beersum Ellen; Marinus Nelisse Pieter; Jacoba Andriana Van Den Berg Emily; Van Lotringen Marion; Oudshoorn Arjan
Original assignee: Delft Advanced Biofuels B V
Priority date: 2023-03-23
Filing date: 2023-03-23
Publication date: 2024-09-30

Abstract

The invention relates to a bioreactor system (1), comprising a bioreaction compartment (2) for holding a ﬂuid reaction mixture comprising a biocatalyst and a substrate for the biocatalyst, the bioreaction compartment (2) comprising - an agitator (3); - an inlet or inlets for feeding liquid reactor contents; and - a gravitational liquid-liquid separation device (5), the separation device (5) comprising a liquid phase inlet opening (6) into an internal holding space (7) inside the device (5), which liquid phase inlet opening (6) has a surface area (A1) smaller than the inner cross sectional area (As) of said holding space, the separation device further comprising a gas outlet and a liquid phase withdrawal conduit (9) extending into said space (7) of the separation device (5), the withdrawal conduit (9) having an inlet (10) for withdrawing the liquid phase located in the space (7) at a position between the liquid phase inlet opening (6) and the gas outlet (8) of the separation device (5). {Figure 1}

Description

P134311NL00

Title: AN ELONGATED SEPARATION DEVICE FOR MULTIPHASE

BIOCATALYTIC REACTOR

The present invention relates to multiphase bioreactor system for biocatalytically producing a substance. The invention further relates to a gravitational liquid-liquid separation device. The invention further relates the use of the separation device for the recovery of a biocatalytically produced substance and to a method for obtaining a substance, comprising the use of the separation device or the bioreactor system.

There is a strong world-wide effort to provide more sustainable production routes, e.g. transition from fossil based substances to bio-based substances, for production of various organic substances, e.g. alcohols, esters, amino acids, carbohydrates, ethers, lipids, ketones, aldehydes, organic acids, pyridines. These substances can be used in a variety of applications for example as flavours, fragrances, cosmetic or food ingredients, nutraceutical products, bioinsecticides, specialty or commodity chemicals, or as biofuels, all as either sustainable replacement or as novel more sustainable product.

These more sustainable production routes include amongst others biocatalytic processes. It is known in the art to produce organic substances by biocatalytic processes such as fermentation and cell free conversions. In such a fermentation process, micro-organisms are used to convert a suitable substrate into an organic substance of interest. In a cell free conversion, enzymes convert a substrate into an organic substance of interest. As is generally known in the art, various micro-organisms and enzymes are known that can be used on an industrial scale for the production of a wide variety of organic substances. Generally known fermentative processes include the use of natural micro-organisms and/or genetically modified organisms, e.g. yeasts or bacteria, for the production of various organic substances, e.g. alcohols, esters, amino acids, carbohydrates, lipids, ketones, aldehydes, organic acids, ethers, pyridines, imines.

Organisms may be genetically modified to increase the product titre of a naturally produced organic substance and/or to enable a micro-organism to produce an organic substance that it does not produce naturally. E.g. a micro-organism may be modified by incorporating genes from a plant responsible for the production of an organic substance, e.g. a terpene, a terpenoid, not naturally produced by the micro-organism.

More specifically in the world wide effort to produce substances more sustainably there is a need to make more lipophilic products via bio-catalysis. Most lipophilic products typically have limited solubility in water due to their ‘oily’ nature and some are toxic/inhibiting to micro-organisms. These lipophilic products and/or by-products can be inhibiting or toxic to the micro-organism, the products can be unstable, can be unstable under the reaction conditions in the overall reaction vessel or can stick to the micro-organism or any other surface. These effects create challenges for industrial production of these products and hamper the transition towards more sustainable industrial bio-based production. The production per process volume will be low resulting in high production cost per unit of product, creating competition disadvantage for bio-based production routes.

Additionally different feed stocks may be used in the search for a cheaper carbon source. Second and third generation biomass may contain contaminants, which can be inhibiting and/or toxic to the micro-organism as well. High product, by-product and/or contaminant concentrations can be rate limiting to the conversion. They pose a limit on how efficient a biocatalytic process can be related to the overall production rate possible and also on product concentration being reached. Lower product concentrations lead to large reactor volumes and the diluted product containing phase increased effort needed to perform product recovery and/or purification to end product.

It is known in the art that biocatalytic and or fermentation production systems experiencing diluted product streams, inhibition, equilibrium limited, and/or product stability constrained systems benefit from in situ or integrated product removal. Product removal can occur through a continuous phase in and out flow. Often this has the form of a gas stripping or liquid phase extraction. At lab scale it can also involve membranes to separate the phases in a system.

Reactor systems and reaction mixtures obtained in biocatalytic processes tend to be rather complex, especially when living micro-organisms, cell- mass of non-living micro-organisms, or sensitive enzymes are used. The reaction mixtures comprise typically the biocatalyst, substrate, nutrient (in case living organisms are used), the produced organic substance, and possibly side-products, e.g. a fermentation gas which is optionally produced (e.g. COz2) or other substances secreted by the biocatalyst (in case living organisms are used). The complexity of the reaction mixture, chemical processes, microbiological processes and physical processes taking place in the reactor system can make it difficult to simulate the actual industrial conditions.

WO02021/010822 describes a way to produce these substances in an economically feasible manner by using an integrated multiphase reactor system.

The integrated multiphase bio(catalytic)reactor comprises a bioreactor compartment, a riser, and downcomer configuration above the bioreactor for degassing a multi-phase process flow. In the riser the net flow is upflow as it also is in the bioreactor and in the downcomer the net flow is downward. Downstream of the down-comer a liquid-liquid separator compartment is provided. In this reactor system the inhibiting and/or toxic and/or unstable substance(s) are removed from the reaction mixture by (continuous) substance removal through extraction and separation.

The system disclosed by WO2021/010822 is adding and removing an additional liquid phase, the product recovery liquid phase, allowing for in-situ product removal, which further challenges simulating or approximating at lab scale actual industrial conditions for strain and fermentation process development, because traditional lab scale systems either don’t foresee product removal capability or provide alternative technologies with limited scalability or only have functionality for addition of the product recovery phase and therefore lack the additional degree of operational testing freedom this invention enables.

Bednarz et al. ( Development of a CFD model for the simulation of a novel multiphase counter-current loop reactor, Chemical Engineering

ScienceVolume 161, 6 April 2017, Pages 350-359) describe a multi-phase loop reactor allowing integrated fermentation and extraction, aiming to overcome problems with product yield due to inhibition of the micro-organism by the formed product. It allows simultaneous aeration and extraction of the fermentation product with an organic phase in one unit operation. The reactor has an inner cylinder and an outer cylinder. Air is dispersed in the outer cylinder and causes a loop flow (upward flow of the fermentation medium in the outer cylinder, downward flow in the inner cylinder). The organic phase is dispersed in the inner cylinder and flows in counter current (upward) with the fermentation medium. The organic phase containing the fermentation product forms a top layer above the inner cylinder. For a satisfactory separation of the organic phase containing the fermentation product from the fermentation medium, no stirring or agitation with a gas is applied in the inner cylinder. This may limit oxygen transfer. Further, the position of the liquid-liquid separator in this integrated system relative to the fermentation compartment is critical, whereby flexibility in positioning the separator is lacking.

Teke and Pott (Design and evaluation of a continuous Semi-Partition

Bioreactor (SPB) for in-situ liquid-liquid extractive fermentation DOI: 0.1002/bit.27550, Biotechnology and Bioengineering, 2020; 1-14) describe a stirred reactor in which a tubular settler with an opening to the side and insert tube for in situ product removal by extraction with a product recovery phase (Figure 3), allowing continuous operation. Product recovery phase, containing the product is withdrawn from the tubular settler via a conduit. An abiotic simulation experiment is described, that 1s, chemical extraction experiment, with low aeration rate and at 200 rpm, which provides low turbulence conditions. The design is not suitable for high oxygen demand fermentation because low agitation provides relative large gas bubbles, significantly restricting the oxygen transfer in the system. An expert in the field would know that this mass transfer rate is far below the typical values at industrial aerobic fermentation processes.

Further known in the art are Applikon B.V./Getinge chemostat tube (M18 X 1.5 PORT) and chemostat tube (27 MM PORT) are commercial available and are used to withdraw liquid samples from fermentation systems, however they are not selective for withdrawing organic phase. Also, the non-restrictive opening does not mitigate turbulence.

There is a need for alternatives allowing in situ product removal from a bioreaction system, in particular an alternative that offers satisfactory recovery of the product, also from a system wherein the product is produced under highly turbulent conditions and/or aerobic conditions, wherein air or oxygen is fed to the reaction mixture. It is further desired to provide a liquid-liquid separator that can be easily implemented and/or adjusted in an existing bioreactor system, in particular with respect to its position in the bioreaction compartment of the bioreactor system. Further, it is an object of the invention to provide a separation device that can be used for in situ recovery in existing (multiphase) hiocatalytic 5 reactors, in particular existing lab-scale multiphase biocatalytic reactors (typically having a volume of less than 100 L), also when the biocatalytic production of a substance of interest (product) takes place under (highly) turbulent conditions.

Batch fermentation systems when producing inhibiting (or unstable) compounds have a competitive disadvantages to fossil-based production as overall production rate and/or yield leads to poor process metrics in relation to costs of production.

Continuous extractive removal of these products enhance fermentation performance, however the current R&D infrastructure and methods at lab scale are not suitable for such process development. The current invention enables continuous extractive fermentation process development in existing R&D bioreactors, removing the current limitation.

The inventors have found that one or more of the objects of the present disclosure are achieved by an liquid-liquid separation device making use of gravity (such as by settling), wherein the inlet for a liquid phase comprising a biocatalytically produced substance into the device meets certain requirements.

Accordingly, the present disclosure relates to an liquid-liquid separation device (5), in particular an elongated separation device, the separation device (5) comprising a liquid phase inlet opening (6) into an internal holding space (7) for liquid inside the device (5), said holding space having an inner cross-sectional area (As), which liquid phase inlet opening (6) has a surface area (Ai) smaller than the inner cross sectional area (As) of said holding space, the separation device further comprising a gas outlet (e.g. a gas vent or gas exchange opening) and a liquid phase withdrawal conduit (9) extending into said space (7) of the separation device (5), the withdrawal conduit (9) having an inlet (10) for withdrawing the liquid phase located in the space (7) at a position between the liquid phase inlet opening (6) and the gas outlet (8) of the separation device (5).

The separation device is a gravitational separation device, i.e. configured to allow separation of liquids under the influence of gravity (into a phase with a relatively high density and a phase having a relative low density).

In use in a bioreactor system, the separation device is positioned such that —in use — the liquid phase inlet opening is below the liquid level of the reaction mixture in the bioreaction compartment, the inlet of the conduit for withdrawing the liquid phase inside the separation device is below the liquid level of the liquid phase inside the separation device and the gas outlet is above the liquid level of the liquid phase inside the separation device. The (elongated) separation device is thus adapted for preferential removal of a liquid (typically non- aqueous) phase, comprising a biocatalytically produced substance, from a bioreactor comprising the liquid product phase dispersed in a liquid (typically aqueous) reaction phase, comprising a biocatalyst, which product phase has a lower density than the reaction phase.

The separation device typically has a liquid phase inlet opening (6) at or near a first extremity of the elongated separation device. Thus — in use, when contacted with a reaction mixture in a bioreactor — the liquid phase inlet opening (6) is typically at (e.g. in) the bottom-side of the device or near the bottom-side of the device. The location and/or size of the opening mitigates turbulence, contributing to effective phase separation also under turbulent conditions in the bioreaction compartment. The number of inlet openings (6) into the separation device can be 1, in which case it is preferably at (in particular in) the bottom side (15) of the separation device. Alternatively, or in addition one or more liquid inlet openings can be located along the elongated separation device. It is also possible to have a plurality of openings at (in particular) in the bottom side of the separation device.

The present disclosure further relates to a multiphase bioreactor system (1) comprising a bioreaction compartment (2) and the separation device according to the invention, wherein the elongated separation device (5) is adapted for in situ product recovery from the bioreaction compartment under influence of gravity, allowing the formation of a phase enriched in biocatalytically produced substance as an upper layer in the holding space (7) of the elongated separation device.

Accordingly, in use, the elongated separation device is generally placed in the bioreactor system such that the liquid phase inlet opening (6) is submerged in the reaction mixture inside the bioreaction compartment (2) and lower than the inlet of the withdrawal conduit. Further, the elongated separation device can generally adequately be positions without conflicting unacceptably with other internals, e.g. stirrer, pH probe, dissolved oxygen (DO) sensor, temperature sensor, sparger ete. …; the elongated separation device is preferably positioned — off-centre - but also preferably not against the outer edge.

In particular, a multiphase bioreactor system according to the present disclosure comprises a bioreaction compartment (2) for holding a fluid reaction mixture comprising a biocatalyst, a substrate for the biocatalyst and a liquid carrier, the bioreaction compartment (2) comprising - an agitator (3), e.g. a stirrer or a gas injector, typically configured for turbulently mixing an aqueous fluid; - an inlet or inlets for feeding liquid reactor contents, such as at least one liquid selected from aqueous phases containing a substrate (inlet 4a) and non-aqueous product recovery phases (inlet 4b); and - a gravitational liquid-liquid separation device (5), in particular an elongated gravitational liquid-liquid separation device, the separation device (5) comprising a liquid phase inlet opening (6) into an internal holding space (7) for liquid inside the device (5), said holding space having an inner cross-sectional area (As), which liquid phase inlet opening (6) has a surface area (A) smaller than the inner cross sectional area (A) of said holding space, the separation device further comprising a gas outlet (e.g. a gas vent or gas exchange opening) and a liquid phase withdrawal conduit (9) extending into said space (7) of the separation device (5), the withdrawal conduit (9) having an inlet (10) for withdrawing the liquid phase located in the space (7) at a position between the liquid phase inlet opening (6) and the gas outlet (8) of the separation device (5).

The present disclosure further relates to the use of the gravitational liquid-liquid separation device, in particular the elongated separation device, according to the present disclosure for recovering a biocatalytically produced substance from a fluid reaction mixture, comprising the produced substance and a biocatalyst.

The present disclosure further relates to a method for obtaining a substance (product), comprising the use of the multiphase bioreactor system according to the present disclosure or the liquid-liquid separation device (5) according to the present disclosure, the method comprising - providing reaction mixture comprising a liquid carrier, a biocatalyst and a substrate in a bioreaction compartment (2) of the bioreactor system; - biocatalytically converting the substrate, whilst the reaction mixture is mixed in the bioreaction compartment (2), under formation of the substance; - withdrawing a fluid comprising the produced substance, from the bioreaction compartment (2) using the separation device (5), in particular the elongated separation device (5), wherein the fluid comprising the produced substance enters the internal holding space (7) via the liquid phase inlet opening (6); - subjecting the fluid comprising the produced substance to a liquid-liquid phase separation under the influence of gravitational force, whereby in an upper part of the fluid inside the internal holding space (7) a liquid phase (7b) enriched in the produced substance is formed; and - withdrawing liquid phase (7b) enriched in the produced substance from the separation device (5) via withdrawal conduit (9) of the separation device (5).

The present disclosure further provides a method for revamping a(n existing) bioreactor, in particular a continuously stirred bioreactor, comprising the mounting of the gravitational liquid-liquid separation device according to the present disclosure into the bioreactor. Herein, the liquid phase inlet opening (6) of the separation device 5 1s positioned below the intended reaction mixture contents level of the bioreactor, the inlet (10) of liquid phase withdrawal conduit 9 is positioned below the intended liquid-gas interface level in the internal holding space (7) inside the separation device and the gas outlet (8) is positioned above the intended liquid-gas interface level in the internal holding space (7).

Advantageously, the bioreaction system is modular, i.e. the separation device can be mounted into and out of the system. Advantageously, the location of the separation device (in particular the inlet 6) in the reaction compartment can be changed: up and down, centrally or close to the compartment side wall, depending on availability and use requirement.

The method, use, bioreactor system, respectively separation device in accordance with the present disclosure is in particular of benefit to perform continuous overlay (liquid-liquid extraction) fermentations. The method, use, bioreactor system, respectively separation device in accordance with the present disclosure is particularly useful in fermentation process development in a laboratory environment. More specifically, a tool is offered to enable enhanced strain and process development, in particular regarding inhibiting compounds for the biocatalyst. More specifically, the control of the dosing and removal of product recovery phase (as an extractant) allows control over the aqueous and organic content of the system not possible in batch fermentation and allows investigation of a wider variety of combinations of growth rate, product concentration, nutrient conditions. An expert in the field will understand that the invention allows new study of microbial kinetics (e.g. Monod, Herbert-Pirt) especially in regards to yield and biomass growth, under more continuous and/or steady state conditions, not restricted to the aqueous dilution rate of the system. In batch systems a current limitation is that inhibitory levels are reached which hamper process development.

Identification of process conditions and/or strains suitable for continuous overlay production systems at scale/industrial production systems is limited as the performance in continuous flow systems is not possible when using batch systems.

This is addressed by the method, use, bioreactor system, respectively separation device in accordance with the present disclosure.

Further, the separation device (5) can adequately be used in a bioreactor for in situ product recovery. This is, amongst others beneficial for controlling inhibition of biocatalyst, avoiding substantial product degradation and/or substantial product consumption.

Brief description of the drawings:

These and other features, aspects, and advantages of the device, system use, and method of the present disclosure will become better understood from the following description, appended claims, and accompanying drawings wherein:

Figure 1: bioreactor system according to the present disclosure.

Figure 2: elongated gravitational liquid-liquid separation device according to the present disclosure.

Terminology used for describing particular embodiments is not intended to be limiting of the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. The term “or” is used to mean "and/or", unless the context, clearly indicates otherwise. The terms “or” respectively “and/or” include any and all combinations of one or more of the associated listed items. It will be understood that the terms "comprises" and/or "comprising" specify the presence of stated features but do not preclude the presence or addition of one or more other features.

Any text or reference signs placed between parentheses shall not be construed as limiting, unless the context clearly indicates otherwise.

When referred herein to an area, length, width, height, diameter or the like, the internal area, length, width , height, diameter or the like is meant unless specified otherwise or unless it follows otherwise from context. If an area, length, width, height, diameter or the like is not constant for a specific item, the skilled person will understand that the average value is meant and the skilled person will know how to determine said average value.

It will be further understood that when a particular step of a method is referred to as subsequent to another step, it can directly follow said other step or one or more intermediate steps may be carried out before carrying out the particular step, unless specified otherwise. Likewise it will be understood that when a connection between structures or components is described, this connection may be established directly or through intermediate structures or components unless specified otherwise.

The invention is described more fully hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown. In the drawings, the absolute and relative sizes of systems, components, layers, and regions may be exaggerated for clarity. Embodiments may be described with reference to schematic and/or cross-section illustrations of possibly idealized embodiments and intermediate structures of the invention. In the description and drawings, like numbers refer to like elements throughout. Relative terms as well as derivatives thereof should be construed to refer to the orientation as then described or as shown in the drawing under discussion. These relative terms are for convenience of description and do not require that the system be constructed or operated in a particular orientation unless stated otherwise.

In an embodiment, the (elongated) separation device (5) is a helical or otherwise wound device or at least contains a helical or otherwise wound structure defining the internal holding space Herewith, the wall(s) defining the holding space are not straight, which increases buoyancy due to an effect on the force balance in relation to gravity, having a positive effect on the separation.

In an embodiment, the (elongated) separation device (5), or at least the holding space (7) of the separation device (5) has an at least substantially constant cross sectional area.

In an embodiment, the (elongated) separation device (5), or at least the holding space (7) of the separation device (5) is at least substantially cylindrical, advantageously at least substantially round cylindrical. A cylindrical, in particular a round-cylindrical separation device or holding space can be made for readily available (round-cylindrical) starting-product.

In an embodiment, the (elongated) separation device (5) or at least the internal holding space (7) has a cross sectional area which changes over the length of the device or at least the internal holding space (7), e.g. a conical shape. This shape can also be beneficial to enhance buoyancy and thereby enhance separation.

The (elongated) separation device is in particular advantageous for use in a lab-scale multiphase reactor system, in particular a system having a reaction compartment (2) volume of about 100 L or less, more in particular of 50 L or less.

The presently disclosed separation device can be produced well in various dimension, including small dimensions. Accordingly, a separation device is provided in the present disclosure that is also suitable for use in small bioreactor systems, including systems having a bioreaction compartment (2) volume about 100 ml to about 500 ml. Usually, the reaction compartment (2) volume is at least about 0.5 L, in particular at least about 2 L, more in particular at least about 5 L.

Thus, advantageously, the elongated separation device typically has an internal holding space (7) of less than 100 L, in particular of less than 50 L. Usually, said internal holding space is at least about 0.38 L, in particular at least about 0.76 L,

with the proviso that the cross sectional area of the separation device is smaller than the cross sectional area of the bioreaction compartment.

The separation device (5) or at least the holding space is typically elongated, i.e. having a length or flow path in the holding space exceeding the (average) internal diameter. Herein, the term ‘elongated separation device’ is used for both separation devices that are elongated as a whole and for separation devices wherein at least the internal holding space (7) is elongated. When present in a bioreactor system, the length of the separation device is usually at least long enough to allow the internal holding space to be at about 1 cm or more submerged below the liquid level of the bioreaetion compartment. Preferably the device extends close to the bottom of the reaction compartment, leaving enough space for liquid to be introduced via inlet (6) in the internal holding space. The bottom of the separation device may touch the bottom of the bioreaction compartment if inlet (6) into the internal holding space (7) is in a side of the separation device.

The internal cross sectional area (A) of the liquid inlet opening (6) of the (elongated) separation device (5) is generally less than 99 % of the internal cross sectional area (As) of the holding space (7) for liquid of the elongated separation device (adjacent to the position of the liquid inlet opening). Generally, A: is more than 0.05 % of A.. For a particularly advantageous reduction of adverse effects of turbulence and/or disturbance by gas bubbles, A: preferably is up to 50 % of As, more preferably up to 25% of As, even more preferably up to 16% of As. A very low Ai relative to As may result in a very low flow of product phase into the holding space of the separation device. Accordingly, Ai preferably is at least 1 % of

A, more preferably at least to 2% of As, even more preferably at least 4% of A.

Advantageously, the (elongated) separation device (5) has at least one liquid phase inlet opening (6) at or near an extremity of the (elongated) separation device (5); this is generally the extremity facing the bottom of the bioreaction compartment, when present in the bioreaction system, i.e. at or near the bottom of the separation device. In this context ‘near’ in particular means that the liquid phase inlet opening (6) is situated between an extremity (15) of the (elongated) separation device and the inlet (10) of liquid phase withdrawal conduit (9) and closer to said extremity (15) than to said inlet (10), preferably at said extremity (15).

The presence of a liquid phase inlet opening in a side wall of the separation device is for instance advantageous because it can contribute to advantageously introducing recovery phase into the internal holding space and/or to have reduced introduction of gas bubbles in the internal holding space.

Advantageously such inlet opening is positioned at least substantially opposite to a stirrer to avoid direct disturbance of the internal volume by mechanical agitation, if present. The presence of an additional liquid phase inlet opening in a side wall of the separation device is for instance advantageous in addition to an inlet (6) in the bottom (15) of the holding space (7) as it facilitates return of a relatively dense liquid phase (rich in aqueous phase, lean in product phase, compared to the product phase that is recovered via the withdrawal conduit (9)). Unacceptable inflow of gas bubbles (when gas is injected/sparged) can be avoided by having a relatively small cross sectional area (A: ) of the inlet opening (6).

The bioreaction compartment (2) can in principle be any kind of bioreaction compartment wherein the separation device is operationally mountable. The bioreaction compartment can be a batch reaction compartment.

Advantageously, the bioreaction compartment (2) is configured for continuous or intermittent feed of substrate and/or withdrawal of liquid enriched in the product phase from the separation device (5). In a particularly preferred embodiment the bioreaction compartment is a continuously stirred tank bioreaction compartment.

The multiphase bioreactor system according to the present disclosure comprises one or a plurality (at least two) of the separation devices (5). Typically, for an advantageous recovery rate, the total (average) inner cross sectional area of said separation device(s) is at least 0.05%, preferably at least 0.1 %, more preferably at least 0.2 %, in particular at least 0.3 % of the (average) inner cross sectional area of the internal holding space (7) of bioreaction compartment (2). The higher the cross sectional area of the holding space (As) relative to the cross sectional area (Ap) of the bioreaction compartment, the higher the recovery capacity, but the lower the production capacity. Typically As is up to 25%, preferably up to 15%, more preferably up to 5 %, in particular up to 2.5% of Ar. In practice relatively high As is particularly preferred for a relatively high reaction compartment volume.

The (elongated) separation device (5) typically has a length (Ls) inserted (determined from the top of the bioreactor compartment) into the bioreaction compartment (2) of at least 25 %, preferably at least 50 %, more preferably at least 70 %, even more preferably at least 90 % of the height (Hy) of the bioreaction compartment (2); In use, the length Ls is chosen such that inlet (10) into the withdrawal conduit (9) and the inlet opening (6) into the internal liquid holding space (7) are below liquid level in the bioreactor compartment and that said inlet opening (6) into said space (7) is at a lower position than the inlet (10) of the withdrawal conduit (9). In use, the gas outlet (8) of the separation device (5) is above liquid level inside the internal holding space (7) and can be positioned such that the gas leaves the separation device inside the bioreaction compartment (2) or outside the bioreaction compartment (2) .

The length Ls of the separation device inserted into the bioreaction compartment is generally up to 99 %, in particular up to 97 %, more in particular upto 95 % of the height (Hy) of the bioreaction compartment. Advantageously, the separation device can be positioned such that the inlet opening (6) into holding space (7) is below the gas injection/sparger (if present) and/or below the stirrer (if present). In principle, Ls can be 100 % of Hy, if the inlet opening (6) into holding space (7) is present in a side wall of the separation device (5).

In an advantageous embodiment, the (elongated) separation device has a tilted configuration. This offers enhanced buoyancy and thereby enhanced separation. The angle relative to the vertical axis (when the bioreactor system is placed upright, ready for use) in a tilted configuration is usually at least 10°, preferably at least 30°, more preferably at least 40° . The angle relative to the vertical axis (when the bioreactor system is placed upright, ready for use) in a tilted configuration is usually up to 60°, preferably up to 50°, in particular about 45° angle or less.

Usually, at least when the multiphase bioreactor system is intended for producing a substance under aerobic conditions, the bioreaction compartment (2) comprises a gas inlet (12), for instance a gas sparger, for aerating a liquid reaction mixture in the bioreaction compartment (2). This not only allows the introduction of oxygen, needed for aerobic production, but can also be used for generating or increasing turbulence. Accordingly, a gas inlet can also be advantageous when using the system under oxygen-limited conditions or (when using nitrogen or another essentially oxygen-free gas) even anaerobic conditions.

For the biocatalytic production of a substance of interest use can be made of any biocatalyst. The substance can but does not need to have a density lower than the density of the liquid carrier (usually an aqueous phase); the substance can but does not have to be soluble in the liquid carrier . A product recovery phase - having a lower density than the liquid carrier, usually a non-aqueous phase — can be used to recover the substance from the liquid carrier. Details of the biocatalyst, substrate, product recovery phase, liquid carrier and choices thereof dependent on the substance that is to be produced can be based on what is known in the art, e.g. from the prior art cited herein; in particular this can be based on WO2021/010822 of which the contents are incorporated by reference.

The substance produced in accordance with the invention can in principle be any kind of substance that can be produced using a biocatalyst. Typically it is an organic substance; the substance can be liquid under reaction conditions; the substance it can be a solid under reaction conditions, dissolvable in a product recovery phase; or the substance can be a vapour, dissolvable in a product recovery phase. The organic substance can be a substance having 1 carbon or more carbons.

Usually, the organic substance has at least 3 carbons, in particular at least 4 carbons, more in particular at least 5 carbons. The organic substance can have over a 1000 carbons (such as in case of produced biopolymers. In an embodiment the organic substance has up to 100, up to 50, up to 30 or up to 25 carbons.

Advantageously, the method according to the present disclosure can be used to produce one or more organic substances selected from the group consisting of hydrocarbons, in particular monoterpenes, sesquiterpenes, aromatic hydrocarbons; isoprenoids (terpenoids); organic acids, in particular C5-C24 fatty acids, more in particular C12-C20 fatty acids; alcohols, in particular alcohols having at least 4 carbon atoms; ketones, in particular ketones having at least 5 carbon atoms; aldehydes in particular aldehydes having at least 5 carbon atoms; cyclic carboxylic esters, in particular lactones; non-cyclic esters, in particular non- cyclic esters having at least 5 carbon atoms; lipids in particular glycerides; ethers; pyridines; imines; imides; amines; amino acids; and peptides. For further details of preferred organic substances that can in be produced in accordance with the invention, reference is made to the prior art cited herein, in particular

WO2021/010822 page 23, line 27 till page 27, line 10 of which the contents are incorporated herein by reference.

Advantageously, the biocatalyst comprises a micro-organism selected from the group of bacteria, archaea and fungi, preferably selected from the genera

Pseudomonas, Gluconobacter, Rhodobacter, Clostridium, Escherichia, Paracoccus,

Methanococcus, Methanobacterium, Methanocaldococcus , Methanosarcina,

Aspergillus, Penicillium, Saccharomyces, Kluyveromyces, Pichia, Candida,

Hansenula, Bacillus, Corynebacterium, Blakeslea, Phaffia (Xanthophyllomyces),

Yarrowia, Schizosaccharomyces, Zygosaccharomyces, Saccharopolyspora and

Zymomonas more preferably from the group of Corynebacterium glutamicum,

Escherichia coli, Bacillus subtilis , Bacillus methanolicus, Pseudomonas aeruginosa, Pseudomonas putida, Rhodobacter capsulatus, Rhodobacter sphaeroides, Paracoccus carotinifaciens, Paracoccus zeaxanthinifaciens,

Saccharomyces cerevisiae, Saccharomyces pastorianus, Schizosaccharomyces pombe, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae , Blakeslea trispora, Penicillium chrysogenum, Phaffia rhodozyma (Xanthophyllomyces dendrorhous), Pichia pastoris, Yarrowia hpolytica, Saccharopolyspora spinosa and

Zymomonas mobilis, in particular from the group of Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, Saccharomyces cerevisiae, Saccharopolyspora spinosa and Zvmomonas mobilis. A biocatalyst selected from this group may in particular be used for the production of a substance according to the previous paragraphs.

Advantageously, the biocatalyst comprises a living organism and the produced substance is secreted into the reaction mixture. Thus, the produced substance can efficiently be recovered without lysing the living organism.

In an embodiment, the biocatalyst comprises an isolated enzyme or combination of isolated enzymes. The isolated enzyme(s) can be dispersed or dissolved in the reaction mixture or immobilized on one or more support materials dispersed in the reaction mixture.

The liquid carrier (forming a continuous phase in the bioreaction compartment, wherein the biocatalyst is dispersed) is typically an aqueous liquid suitable for the biocatalyst, in particular when the biocatalyst is a living organism.

Suitable carrier liquids are generally known in the art, dependent on the biocatalyst.

When the produced substance of interest forms a separate phase from the carrier liquid, it can be recovered in accordance with a method according to the present disclosure without using a product recovery phase. Advantageously a product recovery phase is used for efficient recovery of the produced substance of interest. This liquid phase for extracting (recovering) the produced substance of interest forms a phase separate from the biocatalytic liquid phase (the liquid carrier comprising the biocatalyst and substrate).

The product recovery phase is fed into the bioreaction compartment and dispersed in the liquid carrier. The product recovery phase has a lower density than the liquid carrier. Thus, in the internal holding space (7) of the separation device (5), under influence of gravity it will migrate upward. Hereby, liquid phase enriched in the produced substance is formed in the upper part (7b) of the liquid column in the internal holding space, containing the produced substance.

For the product recovery phase typically a compound or mixture of compounds is used that is at least substantially insoluble in water. Typically the aqueous solubility is less than 85 g/l, in particular less than 10 g/l, preferably 0-1 g/l, at least at the temperature(s) at which it is contacted with the fermentation medium and/or at ambient temperature (25 °C). Further, the product recovery phase is usually chosen based on the organic substance that is to be recovered, i.e. it is a phase for which the produced substance has a higher affinity than for the aqueous phase in which the product recovery phase is dispersed. Thus, the organic substance of interest is removed from the aqueous phase by phase affinity difference.

Particularly suitable liquids for the product recovery phase are organic liquids, in particular hydrophobic organic liquids. These may in particular comprise one or more compounds selected from the group of hydrocarbons, organic acids, alcohols, ketones, aldehydes, cyclic carboxylic esters, non-cyclic esters, lipids, amines, including mixtures thereof. Preferred are alkanes, triglycerides and hydrophobic alcohols. Particularly preferred alkanes have 6 carbons (C6) or more, more preferably C7-C25, e.g. C7-C15. Specific examples of particularly suitable

Liquid alkanes are hexanes, heptanes, octanes, nonanes, decanes, dodecanes and pentacosane. The alkane may be linear or branched or cyclic. Good results have, amongst others been achieved with dodecane. Further, liquid hydrophobic alcohols are particularly suitable. Preferably the alcohol is selected from C8 alcohols and higher, more preferably C10-C20 alcohols. The alcohol may be linear or branched or cyclic. Specific examples of particularly suitable alcohols are an octanol, a decanol, a dodecanol, oleyl alcohol and isomeric mixtures thereof. Further, liquid triglycerides are particularly suitable, preferably vegetable oils. Particularly preferred examples are castor oil, sunflower oil and soy(bean) oil. The various examples of liquids for the recovery phase that are given are not or poorly soluble in water. Furthermore they typically show good biocompatibility, which is of interest when using a living cell for the biocatalysis.

The liquid phase enriched in product (7b) can be withdrawn continuously or intermittently via the withdrawal conduit (9), whilst the contents of the bioreaction compartment (2) are withdrawn from the biocatalytic reactor under conditions of low to high intensity mixing and/or low to high intensity aeration of the biocatalytic reactor.

If desired, the liquid phase comprising the product of interest that has been recovered via the withdrawal conduit (9) can be subjected to further downstream processing. This can be done in a manner known per se, e.g. by back extraction to a lower boiling solvent, and/or solvent evaporation as described in

WO2021/010822.

The biocatalytic formation of the substance is usually carried out in the reaction compartment, whilst agitating, in particular mixing the reaction mixture, comprising biocatalyst and substrate in a liquid carrier. Preferably, the flow conditions in the reaction compartment for aerobic biocatalytic conversion are turbulent for a high production rate.

For the purpose of clarity and a concise description, features are described herein as part of the same or separate embodiments, however, it will be appreciated that the scope of the invention may include embodiments having combinations of all or some of the features described. For example, while embodiments were shown for biocatalytic reactions, also alternative ways such as processes using different catalysts or even non-catalytic processes may be envisaged by those skilled in the art having the benefit of the present disclosure for achieving a similar function and result. The various elements of the embodiments as discussed and shown offer certain advantages, such as efficient recovery of a biocatalytically produced substance, even when the concentration of produced substance in the reaction mixture is kept at a relatively low level in the aqueous phase (such as a fermentation broth), while offering reliable control over process conditions. Whereas the recycle flow section 4 in some embodiments is depicted as an external loop section it will be appreciated that recycle flow section 4 may also be provided as being adjacent or adjoining to an external wall of the upflow reaction section (e.g. as described in relation to FIG 7C) or even by a conduit extending partially within the upflow reaction volume. Routing the recycle flow adjacent, adjoining or even within the upflow reaction section can advantageously reduce thermal losses and/or mitigate temperature variations within the system.

Of course, it is to be appreciated that any one of the above embodiments or processes may be combined with one or more other embodiments or processes to provide even further improvements in finding and matching designs and advantages.

EXAMPLE 1 exemplary process description

With reference to Figure 1, the bioreactor system (1) comprises a bioreaction compartment (2), which — in use — is at least for a substantial part filled with a reaction mixture, comprising a liquid carrier (typically an aqueous liquid), biocatalyst and a substrate for the biocatalyst. Nutrients may be present as well (for living biocatalysts). The bioreaction compartment contains an agitator (3), such as a stirrer. In addition or alternatively, it is also possible to use a gas injector, such as a sparger as an agitator, or use another agitation means. The gas injector can also be used to feed oxygen into the compartment. The system contains one or more inlets (4, 4a, 4b) for reactor contents. The inlet for product recovery phase (4b) is optional. The (elongated) separation device (5) is inserted in the bioreaction compartment, such the liquid phase inlet opening (6) is submerged in the reaction mixture. Thus, reaction mixture, containing biocatalytically produced substance can enter the internal holding space (7). The device is placed such that it is not in conflict with other internals, e.g. stirrer, pH probe, dissolved oxygen (DO)

sensor, temperature sensor, etc.; preferably off centre but also preferably not against the bioreactor compartment’s side wall(s).

The composition of the fluid entering the holding space does not have to be exactly the same as the overall composition in the bioreaction compartment (2).

The restrictive inlet opening (6) may limit entry of gas and/or the substance (when forming a separate phase) respectively the product recovery phase containing the substance has a lower density than the liquid carrier, favouring an upward motion.

Inside the internal holding space (7) the flow conditions favour phase separation allowing the formation of a liquid enriched in produced substance in the upper part (7b) of the liquid in the internal holding space, whilst the liquid in the lower part (7a) has a higher density than the liquid enriched in produced substance (7b) and will be returned into the bioreaction compartment (2), assisted by hydrostatic force.

Typically Reynolds number is lower inside the holding space (7) than in the reaction compartment. Flow conditions in the holding space are advantageously non-turbulent, whilst they are turbulent in the bioreaction compartment. The liquid enriched in produced substance is removed (continuously or intermittently) via withdrawal conduit (9) having an inlet (10). A gas outlet (8) is provided in the separation device and a gas outlet (13) is provided in the bioreaction compartment, to allow removal of gas introduced in the separation device. This can be injected gas and/or gas produced by the biocatalyst. The gas outlet (8) as shown in figure 1 mounds into the bioreaction compartment. However, it is also possible to have such outlet mounding outside the bioreaction compartment. Advantageously the gas outlet (8) is a vent hole. Advantageously the gas outlet (13) is a vent hole.

The bioreactor system can further have one or more further items (not shown), as known in the art, such as one or more selected from outlets for bleed, sampling reaction compartment contents, sensors, etc.

Legend to the drawings: 1. Bioreactor system 2. Bioreaction compartment 3. Agitator 4. Inlet for reactor contents a. Inlet for aqueous phase (substrate, micro-organism suspension) b. Inlet for non-aqueous phase (product recovery phase)

5. Liquid-liquid separation device 6. liquid phase inlet opening into the separation device 5 7. fluid holding space inside the separation device 5 a. lower part of the fluid inside the holding space 7 bh. upper part of the fluid inside the holding space 7, enriched in product 8. gas outlet of separation device 5 9. withdrawal conduit for withdrawing a liquid enriched in the product phase from separation device 5 10. inlet of liquid phase withdrawal conduit 9 11. outlet of liquid phase withdrawal conduit 9 12. gas inlet into bioreaction compartment 2 13. gas outlet from bioreaction compartment 2 14. top (head plate) of bioreaction compartment 2 15. lower extremity (bottom) of the separation device 5 16. outlet for liquid from bioreactor compartment (e.g. for bleeding or sampling)

Ai: inner cross sectional area of liquid phase inlet opening 6

As: inner cross sectional area of holding space 7 (adjacent to the liquid phase inlet opening (6) of the elongated separation device)

Ab: inner cross sectional area of the bioreactor compartment

Hs: height of the bioreaction compartment

Le: distance between inlet 10 into conduit 9 and liquid/gas interface inside the separation device 5

Ls: length of the elongated separation device 5 inserted in the bioreaction compartment

La: length of the liquid level in the elongated separation device 5

Claims

Conclusions

1. A multiphase bioreactor system (1) for biocatalytically producing a substance, comprising a bioreaction compartment (2) for holding a liquid reaction mixture comprising a biocatalyst and a substrate for the biocatalyst, the bioreaction compartment (2) comprising - a mixing device (3) configured for turbulently mixing an aqueous liquid, for example a stirrer or a gas injector; - an inlet or inlets for feeding liquid reactor components, such as at least one of: an aqueous phase containing substrate (4a) and a non-aqueous product recovery phase (4b); and - a gravitational liquid-liquid separation apparatus (5), said separation apparatus (5) comprising a liquid phase inlet opening (6) to an internal space (7) for holding liquid in said separation apparatus (5), said internal space having an inner cross-sectional area (A), said liquid phase inlet opening (6) having an area (A;) smaller than the inner cross-sectional area (A;) of said internal space (7) of said separation apparatus (5), said separation apparatus further comprising a gas outlet (8) (e.g., a hole or gas exchange opening) and a liquid phase withdrawal channel (9) extending into said internal space (7) of said separation apparatus (5), said withdrawal channel (9) having an inlet (10) for withdrawing the liquid phase into said internal space (7) at a position between said liquid phase inlet opening (6) and said gas outlet (8) of said separation apparatus (5).

2. The multiphase bioreactor system according to claim 1, wherein the area (A;) of the liquid inlet opening of the elongated separation device is 1-50%, preferably 2-25%, more preferably 4-16% of the cross-sectional area (As) of the internal space (7) for holding liquid of the elongated separation device (at the position of the liquid inlet opening).

3. The multiphase bioreactor system according to claim 1 or 2, wherein the multiphase bioreactor system comprises one or more separation devices (5), wherein the total (average) internal cross-sectional area of the internal space (7) of said separation device(s) is 0.1-25 %, preferably 0.1-15 %, more preferably 0.3-2.5 % of the (average) internal cross-sectional area of the bioreaction compartment (2).

4. The multiphase bioreactor system according to claim 1, 2 or 3, wherein the elongated separation device inserted into the bioreaction compartment (2) has a length (Ls) of 50-99%, preferably 70-99%, more preferably 90-99% of the height (Hp) of the bioreaction compartment.

5. The multiphase bioreactor system according to at least one of claims 1 to 4, wherein the elongated separation device (5) has a spiral or otherwise wound structure defining the internal space (7).

6. The multiphase bioreactor system according to at least one of claims 1 to 5, wherein the internal space (7) of the elongated separation device (5) has an at least substantially constant cross-section.

7. The multiphase bioreactor system of claim 6, wherein the elongated separation device (5) is at least substantially cylindrical.

8. The multiphase bioreactor system according to at least one of claims 1 to 5, wherein the cross-section (A) changes over the length of the separation device, for example has a conical shape.

9. The multiphase bioreactor system according to at least one of claims 1 to 8, wherein the elongated separation device is configured obliquely, preferably at an angle of 10-60° to the vertical axis (when the bioreactor system is upright), more preferably at an angle of 40-50° to the vertical axis, in particular at an angle of 45° to the vertical axis.

10. The multiphase bioreactor system according to at least one of claims 1 to 9, wherein the elongated separation device (5) has at least one liquid phase opening (6) at a lower end of the elongated separation device (5).

11. The multiphase bioreactor system according to at least one of claims 1 to 10, wherein the bioreaction compartment (2) comprises a gas inlet (12) for aerating the liquid reaction mixture in the bioreaction compartment (2).

12. A gravitational liquid-liquid separation apparatus (5), said separation apparatus (5) comprising a liquid phase inlet opening (6) to an internal space (7) for holding liquid in said separation apparatus (5), said internal space having an inner cross-sectional area (A; s), said liquid phase inlet opening (6) having an area (A; s) smaller than the inner cross-sectional area (A; s) of said internal space (7), said separation apparatus further comprising a gas outlet (8) (e.g., a hole or gas exchange opening) and a liquid phase withdrawal channel (9) extending into said internal space (7) of said separation apparatus (5), said withdrawal channel (9) having an inlet (10) for withdrawing the liquid phase into said internal space (7) at a position between said liquid phase inlet opening (6) and said gas outlet (8) of said separation apparatus (5).

13. The gravitational liquid-liquid separation apparatus (5) according to claim 12, wherein the apparatus is as defined in at least one of claims 2, 5, 6, 7, 8 or 10.

14. A method for obtaining a substance, comprising the use of the multiphase bioreactor system according to at least one of claims 1 to 11 or the liquid-liquid separation apparatus according to claim 12 or 13, the method comprising - providing a reaction mixture comprising a liquid carrier, a biocatalyst and a substrate in a bioreaction compartment (2) of the bioreaction system; - biocatalytically converting the substrate, while the reaction mixture is mixed in the bioreaction compartment (2), to form the substance; - withdrawing a liquid comprising the produced substance from the bioreaction compartment (2) using the elongated separation apparatus (5), wherein the liquid comprising the produced substance enters the internal space (7) through the liquid phase inlet opening (6); - subjecting the liquid comprising the produced substance to a liquid-liquid phase separation under the influence of gravity, wherein in an upper part of the liquid in the internal space (7) a liquid phase (7b) enriched in the produced substance is formed; and - withdrawing liquid phase (7) enriched in the produced substance through the withdrawal channel (9) of the elongated separation device (5).

15. The method of claim 14, wherein a product recovery phase is fed into the bioreaction compartment, which is dispersed in the liquid carrier, the product recovery phase having a lower density than the liquid carrier, and wherein the liquid phase (7b) enriched in the produced substance is a product recovery phase containing the produced substance.

16. The method of claim 15, wherein the liquid carrier is an aqueous liquid and the product recovery phase comprises a liquid selected from liquid hydrocarbons, liquid organic acids, liquid alcohols, liquid ketones, liquid aldehydes, liquid cyclic carboxylic esters, liquid non-cyclic esters, liquid lipids, liquid amines, including mixtures thereof.

17. The method of claim 14, 15 or 16, wherein the substance produced has a lower density than the liquid carrier.

18. The method according to at least one of claims 14 to 17, wherein the organic substance produced is selected from the group consisting of hydrocarbons, in particular monoterpenes, sesquiterpenes, aromatic hydrocarbons; 1-soprenoids (terpenoids); organic acids, in particular C5-C24 fatty acids, more in particular C12-C20 fatty acids; alcohols, in particular alcohols with at least 4 carbon atoms; ketones, in particular ketones with at least 5 carbon atoms; aldehydes, in particular aldehydes with at least 5 carbon atoms; cyclic carboxylic esters, in particular lactones; non-cyclic esters, in particular non-cyclic esters with at least 5 carbon atoms; lipids, in particular glycerides; amines; amino acids; and peptides.

19. The method according to at least one of claims 14 to 18, wherein the biocatalyst comprises a living organism and the substance produced is secreted into the reaction mixture or wherein the biocatalyst comprises an isolated enzyme or combination of isolated enzymes dispersed or dissolved in the reaction mixture or wherein the biocatalyst is an isolated enzyme or combination of isolated enzymes immobilized on one or more carrier materials dispersed in the reaction mixture.

20. The use of an elongated gravitational liquid-liquid separation apparatus according to claim 12 or 13 for recovering a biocatalytically produced substance from a liquid reaction mixture comprising the produced substance and a biocatalyst.

21. Method for renewing a bioreactor, in particular a continuously stirred bioreactor, comprising mounting a gravitational liquid-liquid separation device according to claim 12 or 13 in the bioreactor.