Molecular production system and method

Abstract

A biomass reactor is disclosed having a closed fluid network including a plurality of vessels. In one embodiment, each vessel contains a volume of liquid medium for growing a microbial biomass in or on a liquid medium. The plurality of vessels are arranged in fluid communication to allow a recirculating flow of the liquid medium to flow in stages through the plurality of vessels. Methods for producing molecular products using the disclosed biomass reactor, and products resulting from operating the biomass reactor, are also disclosed.

Description

[Technical Field]

[0001] This international patent application claims priority from Australian Provisional Patent Application No. 2022903607, filed on 28 November 2022, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to biomass processing. In certain embodiments, the present disclosure relates to biomass processing to produce high-value molecules such as chemical compounds, nucleic acids, lipids, and proteins. The present disclosure also relates to biomass processing to detoxify or restructure waste products. [Background technology]

[0003] Technologies exist for processing biomass to produce molecules such as small molecules, nucleic acids, and proteins. These technologies are used in fermentation-based biotechnology and synthetic biology and involve "reactors" or "reactor systems." A "reactor" or "reactor system" involves growing microbial biomass as a cohesive mat or film on a liquid medium in a vessel or reservoir, and extracting molecular by-products from the liquid medium. Biomass can also grow as a film or clump along the submerged surface of the vessel.

[0004] Using existing technology, microbial biomass can be grown in a variety of ways. For example, they can be grown as fungi in mats called "syncytia," which allow for efficient distribution of nutrients and waste products by linking cellular components. In another example, organisms such as bacteria, yeast, and algae can form biofilms of individual cells on top of liquid media or cover submerged surfaces in reactors. In another example, non-biofilm-forming cells can be embedded in solid matrices or gels and float as biomass on top of liquid media in reactors.

[0005] Conventional reactors include a vessel or reservoir containing a liquid medium that is stirred and aerated. However, because such reactors contain large volumes of liquid medium, the aeration and aeration equipment is susceptible to mechanical failure due to the large volume of agitation and aeration. Filamentous organisms are also susceptible to mechanical failure. In addition to the risk of mechanical failure, agitation and aeration are also expensive and can be a source of contamination that can lead to biological breakdown of the biomass.

[0006] Furthermore, growing biomass is resource and time consuming. Existing reactors grow microbial biomass as a cohesive mat or film on a liquid medium, which then disturbs the biomass to extract molecules. In other systems, the liquid medium increases the concentration of hazardous waste, killing the biomass.

[0007] It would be desirable to provide a reactor system that overcomes at least some of the above-mentioned deficiencies of existing reactor systems. Summary of the Invention [Problem to be solved by the invention]

[0008] The following presents a simplified summary of one or more preferred embodiments of the present disclosure to provide a basic understanding of such embodiments. This summary is not an exhaustive overview of all contemplated embodiments of the present disclosure, and is not intended to identify key elements of all embodiments of the present disclosure or to delineate the scope of any or all embodiments of the present disclosure. Its sole purpose is to present some concepts of one or more embodiments of the present disclosure in a simplified form as a prelude to the more detailed description that is presented later. [Means for solving the problem]

[0009] One embodiment of the present disclosure provides a biomass reactor, the biomass reactor comprising a closed fluid network including a plurality of vessels, each vessel containing a volume of liquid medium for growing a microbial biomass in or on the liquid medium, the plurality of vessels being arranged in fluid communication to allow a recirculating flow of the liquid medium to flow in stages through the plurality of vessels.

[0010] In certain embodiments, each container includes a plurality of air inlets, each associated with an opening that is covered or blocked by a material that allows passive diffusion of air from the external environment to the interior space through the plurality of air inlets. In one embodiment, the material is a semi-permeable membrane. However, other suitable materials may be used.

[0011] In embodiments, the recirculating flow of liquid medium enters at least one first vessel of the plurality of vessels via at least one inlet and exits at least one final vessel of the plurality of vessels via at least one outlet, the first vessel and the final vessel being in direct fluid communication or indirect fluid communication in relation to one or more other vessels.

[0012] Each vessel of the plurality of vessels may have at least one respective inlet and at least one respective outlet. In embodiments, the at least one inlet of each of the at least one first vessel receives a flow of the liquid medium from a pump of the closed fluid network, and the at least one outlet of each of the at least one final vessel discharges the liquid medium to a sump of the closed fluid network. In embodiments, the sump is in fluid communication with the pump such that the pump can pump the liquid medium from the sump to the first vessel.

[0013] In certain embodiments, the at least one inlet and the at least one outlet of each vessel are juxtaposed at the base of each vessel.

[0014] In certain embodiments, the plurality of vessels are arranged in a multi-tiered network, with each tier comprising at least one vessel.

[0015] In embodiments, a biomass layer is disposed on the surface of the liquid medium contained in each vessel of the closed fluid network, wherein each vessel has a respective inlet and outlet configured to reduce disturbance of the biomass layer during the recirculating flow of the liquid medium.

[0016] In certain embodiments, the plurality of containers are arranged in a vertical stack of containers.

[0017] Yet another aspect of an embodiment of the present disclosure provides a biomass reactor, comprising: The biomass reactor is a closed fluid network including a plurality of vessels, each vessel containing a volume of liquid medium and microbial biomass growing in or on the liquid medium, each of the plurality of vessels arranged in fluid communication to allow the recirculation stream of liquid medium to flow in stages through the plurality of vessels, such that the recirculation stream of liquid medium enters at least one first vessel of the plurality of vessels through at least one inlet and exits at least one final vessel of the plurality of vessels through at least one outlet. a closed fluid network; a sump containing a constant addition amount of the liquid medium, the sump inlet in fluid communication with the at least one outlet of the closed fluid network; and a sump outlet. and a pump that generates a flow of the liquid medium between the sump outlet and the inlet of the closed fluid network to establish and / or maintain the recirculating flow of the liquid medium.

[0018] Yet another aspect of an embodiment of the present disclosure provides a method for producing a molecular product, comprising: The method includes providing a closed fluid network including a plurality of vertically offset vessels in fluid communication, each vessel containing a volume of liquid medium for growing a microbial biomass in or on the liquid medium; establishing fluid communication of the liquid medium between at least one final container of the closed fluid network and at least one first container of the closed fluid network to provide a recirculating flow of the liquid medium through the plurality of containers of the closed fluid network; The liquid medium obtained from the at least one final vessel is treated to extract one or more molecular products from the liquid medium.

[0019] Yet another aspect of an embodiment of the present disclosure provides a method of forming a biomass reactor for producing a molecular product, comprising: The method includes providing a closed fluid network including a plurality of vessels, each vessel containing a volume of liquid medium and a microbial biomass growing in or on the liquid medium, each of the plurality of vessels configured such that a recirculating flow of the liquid medium enters at least one first vessel of the plurality of vessels through at least one inlet and exits at least one final vessel of the plurality of vessels through at least one outlet; providing a sump containing a fixed additional amount of liquid medium, the sump being arranged in fluid communication to allow the recirculation flow of the liquid medium to flow in stages through the plurality of vessels, the sump having a sump inlet in fluid communication with at least one outlet of the closed fluid network, and a sump outlet; A pump is operated to control the flow of the liquid medium between the sump outlet and the inlet of the closed fluid network to establish and / or maintain the recirculating flow of the liquid medium.

[0020] These and other embodiments of the present disclosure will become apparent to those skilled in the art upon review of the following description of certain exemplary embodiments of the present disclosure in conjunction with the accompanying figures. Features of embodiments of the present disclosure are discussed in connection with the specific embodiments and figures below, and all embodiments of the present disclosure may provide one or more of the advantageous features discussed herein. In other words, although one or more embodiments may be discussed as having a particular advantageous feature, one or more such features may also be used in accordance with various embodiments of the present disclosure discussed herein.

[0021] Embodiments of the present disclosure will be described with reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0022] [Figure 1] FIG. 1 is a block diagram of a biomass reactor according to a first embodiment of the present disclosure. [Figure 2] FIG. 2 is an enlarged cross-sectional view of a portion of a vessel suitable for the biomass reactor shown in FIG. [Figure 3] FIG. 3 is an isometric view of an example vessel suitable for the biomass reactor shown in FIG. [Figure 4] FIG. 4 is a schematic diagram of a suitable vessel arrangement for a biomass reactor according to one embodiment of the present disclosure. [Figure 5] FIG. 5 is a schematic diagram of another vessel arrangement suitable for a biomass reactor according to embodiments of the present disclosure. [Figure 6] FIG. 6 is a perspective view of another vessel arrangement suitable for a biomass reactor according to embodiments of the present disclosure. [Figure 7] FIG. 7 is a cross-sectional view of the block diagram of the vessel arrangement of FIG. [Figure 8] FIG. 8 is a cross-sectional view of a sump suitable for a biomass reactor according to an embodiment of the present disclosure. [Figure 9] FIG. 9 is a block diagram of a biomass reactor according to another embodiment of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0023] 1, a biomass reactor 10 (hereinafter "reactor") is shown having a closed fluid network 12 including vessels 14a, 14b, and 14c. As shown, each vessel 14a, 14b, and 14c is configured to contain a volume of liquid medium 16 (hereinafter "medium") in which microbial biomass 18 (hereinafter "biomass") is grown to produce molecular products such as chemical compounds, nucleic acids, lipids, and proteins.

[0024] Before continuing, reference will be made throughout this specification to a "sealed fluid network." As used herein, reference to a "sealed fluid network" is understood to refer to a network that, during normal operation, supports direct or indirect fluid flow between elements of the network in a manner that prevents unfiltered fluid flow (e.g., unfiltered air) from the external environment from entering the elements of the network. In embodiments of the present disclosure, the sealed fluid network allows the contents of a vessel (e.g., vessels 14a, 14b, 14c) to remain sterile from the external environment, thus reducing the risk of contamination of the medium 16 or biomass 18 within the vessel.

[0025] Reference is also made throughout this specification to "liquid medium." As used throughout this specification, the term liquid medium is intended to refer to any aqueous solution or suspension of nutrients capable of maintaining the viability of biomass 18 suspended, suspended, or immobilized in the liquid medium 16. Non-limiting examples of liquid media include Czapeckdox, potato dextrose, yeast extract + sucrose (YeS), lysogeny broth (LB), terrific broth (TB), and soy-peptone sorbitol broth (SPSB). As one of ordinary skill in the art will appreciate, certain liquid media are suitable for use in combination with one or more types of microbial biomass.

[0026] In the context of this specification, reference to "biomass" is understood to refer to any biological cell mass that produces a molecular product as a by-product that is passively or actively exuded into the liquid medium during extraction of the molecular product from the reactor. In this regard, non-limiting examples of suitable biomass include fungi (e.g., Penicillium camenbergii, Aspergillus nidulans, or Parastagonospora nodrum) and bacteria (e.g., Escherichia coli, Vibrio natrigens, Bacillus subtilis, Streptomyces, or Anabaena azollae).

[0027] References are also made throughout this specification to a "vessel." As used herein, references to a "vessel" are understood to refer to any type of vessel suitable for containing a quantity of liquid medium capable of supporting the growth of a layer of biomass therein. Examples of suitable vessels include trays, tanks, cups, containers, cans, bottles, flasks, bowls, pans, buckets, and the like. The vessels may or may not have a separate or removable lid or cover. In some embodiments of the present disclosure, the vessels of the closed fluid network 12 are arranged or configured to provide an enclosed volume having a quantity of liquid medium 16 capable of supporting the growth of a layer of biomass 18 within each vessel of the closed fluid network 12.

[0028] In this example, vessels 14a, 14b, 14c are arranged in fluid communication such that a recycle stream of liquid medium 16 can flow stepwise through vessels 14a, 14b, 14c of closed fluid network 12 via sump 40, pump 50, and interconnecting tubing 42, 44, 46, 48, 52. In this regard, although the closed fluid network 12 of reactor 10 shown in FIG. 1 has three vessels 14a, 14b, 14c, of course, a different number of vessels may be arranged in fluid communication to form closed fluid network 12. Furthermore, in certain embodiments, the closed fluid network of reactor 10 may have at least one vessel in direct or indirect fluid communication with multiple downstream vessels and / or multiple upstream vessels.

[0029] In certain embodiments, the vessels 14a, 14b, 14c include a stimulating element (not shown) for stimulating the growth of the biomass 18. For example, each vessel 14a, 14b, 14c may have a stimulating element integrally formed with, disposed within, or disposed proximate to each vessel 14a, 14b, 14c, to generate a stimulating output for each vessel 14a, 14b, 14c. The stimulating element may be a suitably designed electronic module disposed in each vessel 14a, 14b, 14c.

[0030] The characteristics of the stimulus element, or stimulus output, can be selected, configured, or tailored depending on the biomass 18. Exemplary stimulus outputs include vibration, sound, electricity, magnetism, electromagnetic radiation (including infrared, ultraviolet, and gamma rays), and ionizing radiation. In one embodiment, the stimulus output is a light stimulus suitable for algal biomass growth. In another embodiment, the stimulus output is blue light having a wavelength suitable for inducing a gene cascade in a fungal biomass.

[0031] The sump 40 of the reactor 10 is a suitably sized vessel or container (such as a tank or jar) having a capacity to store the total volume of medium 16, which exceeds the total volume of medium 16 contained in the vessels 14a, 14b, and 14c, as described further below. In certain embodiments, the sump 40 is a vessel or container separate from the vessels. However, the sump 40 may be the lowest vessel of the biomass reactor 10, having a suitable capacity. In an alternative embodiment, the sump 40 is a tube or pipe containing a static mixer structure. In this regard, FIG. 8 illustrates an example of a sump 40 in the form of a pipe 70 incorporating a static mixer structure 72 (e.g., static impeller), a sensor device 78, a fluid dispenser 74, and a fluid supply 76. The fluid dispenser 74 is a container or port for supplying fluid into the pipe 70, and may be used, for example, for pH balancing.

[0032] As shown in FIG. 1, sensor devices 78, such as temperature sensors, pH sensors, oxygen sensors, flow sensors, etc., may be used to sense properties of the liquid medium 16 (or other parameters of the process) within, flowing into, or out of the sump 40 for monitoring and / or control purposes.

[0033] As will be explained in more detail below, when used to produce molecular products such as chemical compounds, nucleic acids, lipids, and proteins, a constant volume of liquid medium 16 is provided and maintained in each of vessels 14a, 14b, 14c and sump 40 by recirculating liquid medium 16 at a specific flow rate through closed fluid network 12 and, thus, through vessels 14a, 14b, 14c. The specific flow rate is controlled by pump 50, which in this embodiment is a low-power peristaltic pump, as described below. Tubing 42, 44, 46, 48, 52 may be rigid, semi-rigid, or flexible tubing having a diameter that allows medium 16 to pass through it at the specific flow rate.

[0034] A certain amount of liquid medium 16 in each vessel 14a, 14b, 14c forms a layer to support the layer of biomass 18 in the respective vessel 14a, 14b, 14c. Support of the layer of biomass 18 in each vessel 14a, 14b, 14c by the layer of liquid medium 16 may include the layer of biomass 18 floating on the surface of the liquid medium 16 or at least partially floating (i.e., partially submerged) in the liquid medium 16. For example, the biomass 18 may be supported on a suitable support structure (such as a support mesh or grid) in each vessel 14a, 14b, 14c to prevent the biomass 18 from being completely submerged in the medium 16. In such embodiments, the top surface of the layer of biomass 18 within each vessel 14a, 14b, 14c is exposed to and interfaces with the air 22 space contained within each vessel 14a, 14b, 14c of the closed fluid network 12, thereby allowing passive diffusion of air to oxygenate the layer of biomass 18 within each vessel 14a, 14b, 14c. In other embodiments, the biomass 18 is fully immersed or submerged in the medium 16. For example, in some embodiments, the biomass 18 may be located on or near the base of the vessel 14a, 14b, 14c.

[0035] With continued reference to FIG. 2, in this embodiment, the volume of air 22 within each vessel 14 a , 14 b , 14 c is in fluid communication with an external air mass 24 via one or more air inlets 26 .

[0036] In this embodiment, each air inlet 26 is blocked or covered by a semipermeable membrane 28 (see FIG. 2 ), forming a semipermeable barrier between the air 22 space and the external air mass 24. The semipermeable membranes 28 allow air 30 from the external air mass 24 to permeate through each air inlet 26 into the volume of air 22 contained within each vessel 14 a, 14 b, 14 c and passively diffuse across the layer of biomass 18. In this manner, contaminants present in the external air mass 24 that could contaminate the volume of air 22 and, therefore, the microbial biomass 18 and / or the liquid medium 16 are at least partially removed before the air enters the vessels 14 a, 14 b, 14 c via the air inlets 26. An advantage of providing air inlets 26 positioned to allow the aforementioned passive diffusion of air to oxygenate the layer of biomass 18 within the vessels 14 a, 14 b, 14 c is that mechanical failures and / or costs typically associated with stirring and aerating the liquid medium 16 may be avoided. In particular, by providing a layer of biomass 18 with an adequate surface area and configuring the reactor 10 for passive diffusion of air, the need for mechanical aeration may be avoided. Furthermore, the combination of the surface area of ​​the layer of biomass 18 and passive diffusion of air may also negate the need to use additional devices or means to dissipate heat. In this regard, conventional large reactors generate significant heat, and while cooling jackets can be used to remove the heat, this dramatically increases infrastructure and running costs.

[0037] While in this example, each of the one or more air inlets 26 is an inlet to a respective vessel, in another embodiment of a reactor (see FIG. 9 ), vessels 14 a, 14 b, 14 c are disposed within an enclosure 80, such as a shipping container, cabinet, box, compartment, or the like, and one or more air inlets 26 are formed within the enclosure 80. In such an embodiment, air within the enclosure 80 is blown in, and air from outside the enclosure 80 passes through a semipermeable membrane (not shown) of each air inlet 26 and passively diffuses through the layer of biomass 18. In this manner, contaminants that may contaminate the air within the enclosure 80, and thus the microbial biomass 1 and / or liquid medium 16, are at least partially removed before the air enters the enclosure 80.

[0038] 9 includes one vertical stack of vessels, the enclosure 80 can accommodate multiple vertical stacks of vessels, each with a separate respective pump 50 and appropriate interconnections to allow fluid communication between the vessels of the stack. An advantage of the embodiment shown in FIG. 9 is that it provides a self-contained reactor 10 that is suitable for use on an industrial scale and is easily scalable.

[0039] Returning now to FIG. 2, semipermeable membrane 28 may be any suitable material that allows air to pass through under normal atmospheric conditions while filtering out particles above a certain size. One example of a suitable semipermeable membrane material is surgical tape, such as Transpore® surgical tape manufactured by 3M®. Other suitable semipermeable membrane materials will be familiar to those skilled in the art. Non-limiting examples of other suitable semipermeable membrane materials include 2 micron polyester track etched PETE, polycarbonate track etched, polyethylene, polyethersulfone (PES), nitrocellulose, and the like.

[0040] As mentioned above, in this embodiment, the semipermeable membranes 28 are positioned to block the respective openings 32 of each air inlet 26 to form a semipermeable barrier between the air 22 space within each container 14a, 14b, 14c and the external air mass 24. Each opening 32 therefore defines the size of the air inlet 26 through which air can penetrate from the external air mass 24 through the semipermeable membranes 28 into the air 22 interior space of the respective container 14a, 14b, 14c and diffuse across and into the microbial biomass 18. Each air inlet 26 may have one opening 32 associated therewith or multiple openings 32.

[0041] 3, an embodiment of a container 14 suitable for use as the containers 14a, 14b, 14c of the closed fluid network 12 depicted in FIG. 1 is illustrated. In the illustrated embodiment, the container 14 has a generally square base 34 and upstanding side walls 36. Each side wall 36 includes a plurality of air inlets 26 covered with a semipermeable membrane 28 in the form of a length of semipermeable tape positioned over the plurality of inlets 26 in each side wall 36. A removable lid 56 sealably engages the upper edge of the container 14. The removable lid 56 can provide access to the interior of the container 14 for cleaning and / or sterilizing the container 14. In this example, each of the containers 14a, 14b, 14c of the closed fluid network 12 depicted in FIG. 1 has the same size and shape as the container shown in FIG. 2.

[0042] Before continuing further, it should be noted that while in this example, each of the vessels 14a, 14b, 14c of the closed fluid network 12 is the same size and shape, it is not necessary for the vessels 14a, 14b, 14c to have the same shape and size. In fact, it is contemplated that other embodiments of the reactor 10 may use vessels having different sizes and shapes. For example, in some embodiments, the size and shape of each vessel of the reactor 10 may be individually selected to provide a volume (V), depth (D), or sidewall perimeter configuration optimized for the growth of a particular microbial biomass. In other embodiments, the size and shape of each vessel may be selected to provide volumetric flow characteristics, such as a flow distribution of a given amount of liquid medium 16 within the vessel, that enhance the growth of the microbial biomass, for example, by providing a particular pressure head.

[0043] As will be appreciated, there is a relationship between flow rate, metabolism, and volume. For example, as the surface area of ​​vessel 14 increases, the metabolic and nutrient demands of the layer of biomass 18 also increase, necessitating a faster flow rate to ensure biomass 18 is maintained. Therefore, the appropriate flow rate depends on the size of reactor 10 in terms of vessel diameter and total system volume. A suitable flow rate for a 1-10 liter reactor consisting of a 30 cm x 30 cm tray is between 50 ml and 300 ml per minute.

[0044] It is also important, but not essential, to maintain the total volume of all vessels 14a, 14b, 14c less than the volume of sump 40. In this regard, if the total volume of all vessels 14a, 14b, 14c exceeds the volume of sump 40 and vessels 14a, 14b, 14c drain into sump 40 (and simultaneously pump sump 40 back into top vessel 14a), sump 40 may overflow or backpressure if pump 50 is turned off or fails.

[0045] 1 , and as outlined above, in this example, the closed fluid network 12 has three vessels 14a, 14b, 14c arranged and connected in fluid communication such that a recirculating flow of liquid medium 16 can flow stepwise through each vessel 14a, 14b, 14c of the network 12. In this example, cascade flow is achieved by arranging the vessels 14a, 14b, 14c in a vertically offset stacked arrangement to form a tower 38 in which the liquid medium 16 flows generally downward from vessel 14a through vessels 14b, 14c towards sump 40.

[0046] In the illustrated embodiment, tower 38 is formed by mounting container 14a on container 14b, which is mounted on container 14c. In this example, supports 54 are used to mount the containers to form tower 38. However, containers 14a and 14b could be directly mounted to or stacked on containers 14b and 14c, respectively. Suitable supports 54 include "standoffs" or spacers configured to space supports 54 apart.

[0047] The supports 54 may be formed, for example, integrally with the base 34 of the vessel 14 (see FIG. 3), or may be formed as part of separate posts, frames, or ribs interposed between the vessels 14a, 14b, and 14c. By providing gaps between the vessels 14a, 14b, and 14c, the air inlets 26 may be distributed throughout the top surface of each vessel 14 (see FIG. 3) rather than just the sidewalls 34. The advantage of distributing the air inlets 26 throughout the top surface of each vessel 14 is that it may allow for more evenly distributed airflow by reducing the average distance between a point on the surface of the liquid medium 16 and the nearest air inlet 26.

[0048] In operation, a steady state can be maintained by pumping a constant amount of liquid medium 16 into vessels 14a, 14b, and 14c at the same rate that the lowermost vessel 14c discharges into sump 40, maintaining a constant input during start-up of reactor 10. In this mode, liquid medium 16 contained in sump 40 is continuously pumped from sump 40 via tube 48 and into vessel 14a via tube 52 using pump 50, establishing a continuous recirculating flow of liquid medium 16 flowing stepwise through each vessel 14a, 14b, and 14c of closed fluid network 12. However, reactor 10 can also be operated in a "pulse" mode or cascade effect, whereby one vessel discharges into the next, thereby initiating flow. In this mode, the discharging vessel 14 stops discharging medium 16 until it is next "triggered." This continues down the stack, one vessel 14 triggering the next vessel, and then stops until the medium 16 is recirculated up to the top of the stack of stacked vessels.

[0049] 1, each of the vessels 14a, 14b, 14c includes at least one fluid inlet 58 and at least one fluid outlet 60. In this regard, although in this example each of the vessels 14a, 14b, 14c is illustrated with a single fluid inlet 58 and a single fluid outlet 60, in other embodiments each of the vessels 14a, 14b, 14c may include multiple fluid inlets 58 and / or multiple fluid outlets 60.

[0050] As shown in FIG. 1 , each fluid inlet 58 and fluid outlet 60 is positioned below the layer of biomass 18 in the medium 16. This arrangement allows the medium 16 to flow freely without disturbing the layer of biomass 18 floating or suspended above the medium 16. In this regard, during commissioning of the reactor 10, each vessel 14 a, 14 b, 14 c is filled with a certain amount of medium 16, so that, starting with the uppermost vessel 14 a, when the medium 16 reaches a certain height within each vessel 14, the outlet 60 of the vessel 14 is activated to discharge the medium 16 into the next vessel 14 or vessels 14 in the fluid network 12, filling and discharging in sequence. The final layer (in the illustrated example, the layer of medium 16 in vessel 14 c) is dispensed to an apparatus (not shown) for extracting high-value components secreted by the layer of biomass 18. For example, medium 16 from vessel 14c can flow through a resin to extract non-polar, medium molecular weight compounds, or a protein affinity column to collect proteins of interest from medium 16. Medium 16 then flows into a sterile vessel, shown here as sump 40, where it is replenished, including nutrient corrections as well as pH, osmolality, and oxygen. Once replenished, medium 16 flows back into upper vessel 14a, and the cycle repeats.

[0051] In this embodiment, the fluid inlets 58 and fluid outlets 60 of each vessel 14a, 14b, 14c are positioned on opposing sidewalls 36 and generally face toward the base 34 of each vessel 14a, 14b, 14c to form a vertical separation (S) between the height of the fluid inlets 58 and fluid outlets 60 and the height of the layer of biomass 18. Preferably, the fluid inlets 58 and fluid outlets 60 are positioned proximal to the base 34 to maximize the vertical separation (S).

[0052] Providing a vertical separation between the height of the fluid inlets 58 and fluid outlets 60 and the height of the layer of biomass 18 may reduce disturbance of the layer of biomass 18 that may result from the flow of liquid medium 16 into the vessels 14a, 14b, 14c via the fluid inlets 58 and / or from the vessels 14a, 14b, 14c via the fluid outlets 60. Such disturbance may have a detrimental effect on the growth of the biomass 18. Indeed, in embodiments, the layer of biomass 18 may be allowed to grow in a near-steady state, allowing the layer of biomass 18 to use incoming nutrients to produce desired molecules without the need to kill the biomass 18. Furthermore, orienting each fluid inlet 58 and fluid outlet 60 to generally face toward the base 34 of each vessel 14a, 14b, 14c potentially allows for a relatively shallow depth of the liquid medium 16 within each vessel 14a, 14b, 14c. Here, a shallow layer of medium 16 reduces wasted space within each container 14 and may therefore allow for a thicker layer of biomass 18 to grow.

[0053] During growth of the layer of biomass 18, the viscosity of the medium 16 may increase as the composition of the medium 16 changes, such as when the biomass 18 secretes molecules into the medium 16. As the viscosity of the medium 16 increases, the meniscus of the medium 16 may also change, altering the interaction of the medium 16 with the fluid inlet 58, fluid outlet 60, and tubing 42, 44, 46, 48, 52. For example, during operation, changes in the meniscus of the medium 16 may affect the flow of the medium 16 from the fluid outlet 60.

[0054] To maintain consistent flow, the diameters of the fluid inlet 58, fluid outlet 60, and tubing 42, 44, 46, 48, 52 should be selected to have the minimum diameter that supports fluid flow over the expected viscosity range of the medium 16. Here, the inner diameters of the fluid inlet 58, fluid outlet 60, and tubing 42, 44, 46, 48, 52 should be wide enough to avoid meniscuses that could create air bubbles within the tubing 42, 44, 46, 48, 52 and reduce the risk of the flow rate creating jets of medium 16 that could disturb the biomass 18. In embodiments, tubing with an inner diameter of 15 mm to 20 mm may be used. However, as the flow rate increases to accommodate increasing biomass 18, larger diameters may be required. It will be appreciated, therefore, that the minimum diameter will depend on the characteristics of the medium 16, particularly the viscosity of the medium 16 as its composition changes. It will also be appreciated that the minimum diameter of the fluid inlet 58 and outlet 60 will constrain the minimum depth of the vessel 14.

[0055] Referring now to FIG. 4, another example of a vessel arrangement 100 suitable for use with embodiments of the present disclosure is shown. For convenience, only two vessels 14a, 14b are shown, although it will be understood that additional vessels may be used. In the vessel arrangement 100 of FIG. 4, vessel 14a is positioned above and supported by vessel 14b. Vessels 14a, 14b have air inlets 26, as described above with respect to FIGS. 1-3. Tubes 102, 104, and 106 are functionally equivalent to tubes 52, 42, and 44 of FIG. 1. However, in the vessel arrangement 100 of FIG. 4, tubes 102, 104, and 106 are vertically oriented to provide downwardly depending fluid inlets 58 and fluid outlets 60 in this example. It will be understood, of course, that other arrangements of fluid inlets 58 and fluid outlets 60 may be used. For example, the arrangement of fluid inlets 58 and fluid outlets 60 may be configured or shaped to form a flow pattern that projects from a vertical axis.

[0056] 5, another example of a container arrangement 200 suitable for use with embodiments of the present disclosure is shown. For convenience, only two containers 14a, 14b are shown, although it will of course be understood that additional containers may be used.

[0057] In the vessel arrangement 200 of FIG. 5, vessel 14a is positioned above and supported by vessel 14b. Tubes 202, 204, and 206 have functional equivalents to tubes 52, 42, and 44 of FIG. 1. Vessel arrangement 200 has a central channel in the form of tube 208 extending vertically and centrally through vessels 14a and 14b. Tube 208 includes a portion 210 (shown here as an upper portion) having one or more air inlets 212 that are blocked or covered by a semipermeable membrane 28. Air can permeate from the external air mass 24 through the air inlets 212, which are covered by the semipermeable membrane 28, and through one or more air inlets 214 into the air 22 in the interior space of each vessel 14a and 14b. In this manner, tube 208 is configured to at least partially remove contaminants that could contaminate the air 22 in the interior space, and thus the biomass 18 and / or liquid medium 16, before the air enters vessels 14a and 14b. In the vessel arrangement 200 of FIG. 4, the tube 208 has a connection to the sump 40 (see FIG. 1). An advantage of the vessel arrangement of FIG. 4 is that the air inlet 214 also allows fluid flow from the vessels 14a, 14b to the sump 40 if the total amount of medium 16 and / or biomass 18 in the vessels 14a, 14b exceeds a threshold level. In other words, the air inlet 214 can be positioned to allow air to permeate the interior space air 22 from the external air mass 24, also reducing the risk of the vessel 14 overflowing. Although not shown in FIG. 5, the vessels 14a, 14b may additionally include an air inlet 26 of the type described in connection with FIGS. 1-4.

[0058] Continuing with reference to FIG. 6, another example of a container arrangement 300 suitable for use with embodiments of the present disclosure is shown. The container arrangement 300 includes eight containers 14a, 14b, 14c, 14d, 14e, 14f, 14g, and 14h. In this embodiment, each container 14 has multiple air inlets 26 of the type described above in connection with FIGS. 1-4. When assembled into a stack of vertically offset containers 14, a central channel 207, similar to the central tube 208 described in connection with FIG. 5, is formed by connecting tubular connectors 302 integrally formed with each of the containers 14a, 14b, 14c, 14d, 14e, 14f, 14g, and 14h.

[0059] 7, in container arrangement 300, containers 14a, 14b, 14c, 14d, 14e, 14f, 14g, and 14h have a serrated upper rim 304 that is contactable with a lower surface 306 above the serrated upper rim 304 to form a plurality of air inlets 26 (see FIG. 6). Depending on the location of the containers 14 in arrangement 300, surface 306 may be the base 34 (see FIG. 3) or lid of a container 14 that is above the serrated upper rim 304 of the lower container.

[0060] An advantage of embodiments of the present disclosure is that the disclosed reactor can be operated without aeration, eliminating the risk of contamination, due to the large surface area of ​​the biomass in contact with the air. As noted above, in embodiments, the vessel is separated from the outside air by a semipermeable membrane that allows gas to diffuse into the vessel in a manner that blocks contaminants. In this manner, oxygen can diffuse across the medium 16 and over the biomass 18. As a result, there is no need to supply oxygen to the medium 16, reducing costs, the risk of contamination, and the risk of degrading a high-value product.

[0061] It will be appreciated from the foregoing that various molecular products typically produced by biomass can be produced using the reactor and separated from the culture medium 16 for further use. Molecular products typically produced by bacterial, fungal, or algal biomass include small molecules, nucleic acids, amino acids, peptides, and proteins. The molecular products may be primary metabolic products of the bacteria, fungi, or algae present in the biomass, or may be secondary metabolic products (i.e., derivatives of primary metabolic products). Advantageously, the molecular products produced are of high or commercial value, such as pharmaceuticals, active ingredients, fermentation products, etc.

[0062] If desired, the molecular product can be separated or isolated from the liquid medium using known separation techniques such as precipitation, liquid-liquid extraction (SX), liquid-solid extraction, chromatography, and the like.

[0063] Other embodiments and applications of reactor 10 are also contemplated. One embodiment involves liquid-phase solvent extraction, in which medium 16 is pumped over a well, such as a chloroform well, before entering sump 40. In this example, the target molecule is more soluble in chloroform than in water, and therefore accumulates in the chloroform. The target molecule is then extracted from the chloroform and purified.

[0064] In another embodiment, it can be used for reversed-phase chromatography extraction. In such an embodiment, the medium 16 is pumped through a resin that captures the molecules of interest. The resin is packed as a fine powder, such as beads, into tubing placed between the final outlet of the tank and the sump. The tubing can be connected in a configuration that allows for quick removal and insertion of new tubing while the reactor is running. The reactor product can then be washed from the beads with a solvent, the beads washed, and the tubing returned to the system.

[0065] The operational aspects of reactor 10 will now be described in the following non-limiting examples.

[0066] Example 1: Trial run A reactor 10 according to one embodiment of the present disclosure, as shown in FIG. 1, can be commissioned for operation by a suitable commissioning process. In one example of a suitable commissioning process, vessels 14a, 14b, and 14c, as shown in FIG. 1, are sterilized with alcohol in a sterile chamber. The interconnecting tubing 42, 44, and 46 are sterilized with alcohol and connected as shown in FIG. 1. Vessels 14a, 14b, and 14c are filled with a microbially inoculated medium 16 to the minimum volume required to cover the inlet 58 and outlet 60. Vessels 14a, 14b, and 14c are assembled on top of each other, as shown, so that vessels 14a, 14b, and 14c form a vertically offset stacked arrangement. Tubing 46 and 48 to the filter (not shown), sump 40, and pump 50 are rinsed with alcohol and then connected to the stack of vessels 14a, 14b, and 14c.

[0067] The sump 40 is partially (e.g., half) filled with sterile medium 16. The reactor 10 is then removed from the sterile chamber and incubated at room temperature for several days until the biomass 18 covers the surface of the medium 16.

[0068] Pump 50 is then activated, pumping sterile medium 16 from sump 40 into the top vessel 14a of reactor 10. When the volume of medium 16 in top vessel 14a reaches a certain volume, outlet 60 of vessel 14a is activated and second vessel 14b begins to fill via its inlet 58. This process is repeated for vessels 14b and 14c.

[0069] Once reactor 10 is operational, biomass is established. Reactor 10 continues to operate until the biomass begins to degrade, at which point the filter (not shown) is replaced and molecular product is recovered as needed.

[0070] pH and dissolved oxygen are monitored and corrected as necessary (high oxygen indicates either more nutrients are needed or low metabolic activity, which could indicate the system is on the verge of failure). The entire medium 16 can also be replaced while the reactor 10 is in operation. For example, if the reactor 10 has been in operation for an extended period of time and salts have accumulated in the medium 16, the pump 50 can be connected to a new sump 40 containing fresh medium 16, and the previous sump 40 filled with old medium 16 can be discarded.

[0071] Example 2: Syncytium formation Biomass 18 was established in potato dextrose broth [Sigma] in a 1 litre vessel 14 and once reactor 10 was running, 100ml of 20x concentrated Czapek-Dox medium [Sigma] was pumped into sump 40 using a Grothen G328 12v peristaltic pump 50 at 100ml per minute.

[0072] Medium 16 was washed with chloroform and flowed into sump 40. In sump 40, medium 16 was stirred and the pH, ionic strength, and dissolved oxygen were measured. The pH was adjusted every 12 hours with a solution of sodium phosphate and sodium hydroxide.

[0073] The biomass 18 established after 48 hours, forming a 5 mm thick mass of white mycelia that covered the entire surface of the medium 16 in each vessel 14a, 14b, and 14c. The biomass 18 was not friable and maintained its integrity during operation at 22°C. In the absence of biomass 18, the bioreactor 10 was aerated to 8.7 mg / L, the maximum value at that temperature. In the presence of biomass 18, the medium 16 was depleted of oxygen (1.5 mg / L), and air directly supplied oxygen to the biomass 18.

[0074] Reactor 10 produced 71 mg of product, which was 10.5 times more efficient than a comparable process measured in volume per hour (i.e., liters per reactor day).

[0075] Example 3: Compound Production Sterile vermiculite (Brunnings®) and SFM medium (20 g / l sorbitol (Sigma® cat. 85529), 20 g / l soy flour (Lotus®)) were filled into four 30 ml containers 14 of a reactor 10 according to one embodiment. Each container contained 10 ml of liquid medium 16, and the remaining space was loosely packed with vermiculite.

[0076] Medium 16 was inoculated with Streptomyces spores and incubated at 25°C. After 7 days, the biofilm incorporated the vermiculite to form a cohesive mat. Reactor 10 was operated in pulse mode for 4 days, and the liquid medium 16 was drained from the vessel and passed through a fresh cellulose column (10 g Avicel® PH-101, [Sigma® cat. 11365]), after which 40 ml of fresh SFM medium was added to reactor 10. Reactor 10 was operated for 6 cycles, producing approximately 120 ml of spent liquid medium. Biomass 18 in reactor 10 was still viable and growing at the end of the experiment.

[0077] The cellulose column was washed with two volumes of ultrapure water, followed by two volumes of methanol at a rate of 1 ml per minute. The combined methanol fractions were stored overnight at -20°C, and the white, fluffy complex was precipitated and filtered. An internal standard of 0.2 mg / ml caffeic acid (Sigma® cat. C0625) was added to the filtered methanol fraction by dispensing 50 μl aliquots into tubes containing 50 μl of caffeic acid solution. The resulting 100 μl fraction was dried under vacuum at room temperature. This fraction was eluted with acetonitrile and water in a 50:50 ratio and compared with the niphimycin cocktail purified by high-performance liquid chromatography. The methanol extract, after removal of the white precipitate, contained the niphimycin cocktail of a purity comparable to that of the standard purified by column chromatography.

[0078] Example 4: Fungal production of multiple high- and low-value molecules Under sterile conditions, six 1-liter vessels 14 were assembled to form a reactor 10 having a configuration similar to that of the reactor 10 illustrated in FIG. 1 and having six vessels 14. The vessels 14 were stacked and connected with valved tubing to control flow between the vessels 14.

[0079] The valves were closed, and each vessel 14 in the stack was filled with 1 liter of liquid medium containing 100 g of sucrose (Sigma® cat. S0389), 100 g of yeast extract (Sigma® cat. 09182), and spores of Penicillium citrinum strain 5352. The vessels 14 were incubated at room temperature for 3 days to allow a mycelial mat biomass to form on the surface of the medium. Once the mycelial mat had formed, the last, or bottom, vessel 14 in the stack was connected to a sump 40 capable of containing the entire volume of the reactor 10.

[0080] Sump 40 was connected to tubing containing a static mixer, pH probe, and dissolved oxygen probe, and connected to nine parallel resin columns. Each column contained 50 g of Diaio® HP-20 resin (Sigma® cat. 13607), which was then connected to a peristaltic pump 50 that fed the fluid inlet of the first, or top, vessel 14 in the stack. The pump 50 was run at 100 ml per minute for five days, monitoring pH and dissolved oxygen. The pump 50 and monitoring device were powered by three 9-volt batteries, drawing 0.37125 kilojoules per liter per hour per day. After five days, the resin columns were removed and washed with one volume of distilled water, two volumes of methanol, and one volume of chloroform. The organic fractions were combined, and 50 μl of caffeic acid (Sigma® cat. C0625) was added to a final concentration of 0.2 mg / ml. The resulting mixture was analyzed by LCMS / MS. This analysis identified citrinin and dihydrolysergic acid at culture concentrations of 830 mg / l and 9 mg / l, respectively.

[0081] Example 5: Production of bacterial proteins Streptomyces bacteria were grown on minimal medium in reactor 10 according to embodiments of the present disclosure operated in pulsed mode as described above.

[0082] The minimal medium contained 10 g / L sorbitol [Sigma® cat. 85529], 10 g / L dextrin [Sigma® cat. 31400], 1 g / L sodium nitrate [Sigma® cat. S5506], trace elements, and was buffered to pH 7.0 with 10 mM phosphate. After 3 days, 37.5 ml of medium was harvested from the reactor 10, and a 50 μl aliquot was passed through Sephadex g-10 [Sigma® cat. G10120] to remove low molecular weight impurities.

[0083] Subsequent Nanodrop spectroscopic analysis showed that the protein content was 960 mg / l and reactor 10 produced 320 mg / l / day of total secreted protein.

[0084] Example 6: Fermented beverage production - continuous beer production Under sterile conditions, four 200 ml flasks were filled with 50 ml of liquid medium containing 20 g / l sucrose [Sigma® cat. S0389], 5 g / l yeast extract [Sigma® cat. 09182], and commercial ale yeast.

[0085] Cultivation was carried out for 48 hours at 30°C and 150 RPM. These cultures were then combined and evenly distributed into four 1 liter containers 14. The containers 14 were then stacked and connected via silicone tubing to assemble a reactor 10 of the general type described above with reference to Figure 1, where the four containers 14 were in the form of a tray.

[0086] Each vessel 14 was filled with 1 liter of liquid medium containing 1 g of dried brewer's hops and 100 g of dried dark malt extract. The starting specific gravity of the medium was 1.045. The vessels 14 were then sealed with plastic tape and incubated at room temperature for 3 days. The top vessel 14, i.e., vessel 14, was then connected to a peristaltic pump pumping malt-hop liquid medium from a 1-liter bottle at 0.5 ml / min, and the bottom outlet was connected to an empty 1-liter bottle. The liquid medium discharged from the reactor 10 was free of yeast sediment, had a hop, malt, and alcohol aroma, a specific gravity of 1.025, and an alcohol content of 2.63%.

[0087] Example 7: Production of fermented beverages - continuous production of kombucha The kombucha starter culture was homogenized at high speed in a kitchen blender and distributed evenly among four 1 liter containers 14 in a tray.

[0088] Next, the vessels 14 were stacked and connected via silicone to assemble a reactor 10 of the general-purpose type described above with reference to FIG. 1 , having four vessels. Each vessel 14 was filled with 1 liter of liquid medium containing 2 g of dried green tea (Lipto®), 80 g of sucrose (Sigma® cat. S0389), and 5 g of yeast extract (Sigma® cat. 09182). The vessels 14 were then cultured at room temperature for 14 days, drained, and refilled with the aforementioned tea-sucrose medium without yeast extract. The initial pH was 7.0. The top vessel 14, i.e., the first vessel 14, was then connected to a peristaltic pump that pumped the tea-sucrose liquid medium from a 1-liter bottle at 0.5 ml / min, and the bottom outlet was connected to an empty 1-liter bottle. The broth discharged from the reactor was free of bacterial pellets, smelled of acetic acid, and had a pH of 4.2, indicating that the tea-sucrose medium had fermented into an acidic kombucha beverage.

[0089] Example 8: Waste removal or valorization A liquid medium containing 10 g / L ethylene glycol (Sigma® cat. 102466), 1 g / L sodium nitrate (Sigma® cat. S5506), and trace elements was buffered to pH 6.5 with 10 mM phosphate buffer and sterilized. After sterilization, 500 ml of this medium was infused with Aspergillus niger spores and evenly distributed into five 100 ml containers (14) arranged in a tray.

[0090] The vessels 14 were then connected to assemble a reactor 10 of the general type described above with reference to FIG. 1, having five vessels 14, and incubated at room temperature. The vessels 14 were pulsed so that the medium was renewed every three days. After 14 days, a thick mycelial mat had formed over the entire surface of the tray, measuring 8 mm deep, indicating that the fungus was able to metabolize this toxic waste and assimilate it into fungal biomass.

[0091] The reference to prior art in this specification is not an acknowledgement or suggestion that such prior art forms part of the general public knowledge.

[0092] It will be understood that the terms "comprise" and "have," as well as their derivatives (e.g., include), as used in this specification and the claims that follow, are deemed to be inclusive of the features referred to by the terms and do not imply the exclusion of the presence of additional features, unless otherwise stated or implied.

[0093] In some cases, a single embodiment may combine multiple features for brevity and / or to aid in understanding the scope of the present disclosure. In such cases, these multiple features may be provided separately (in separate embodiments) or in any other suitable combination. Alternatively, if separate features are described in separate embodiments, these separate features may also be combined in a single embodiment unless otherwise stated or implied. This also applies to claims that can be recombined in any combination; that is, a claim can be amended to include features defined in any other claim. Furthermore, a phrase referring to "at least one" of a list of items refers to any combination of those items, including single members. As an example, "at least one of a, b, and c" refers to a, b, c, ab, ac, bc, and abc.

[0094] The present disclosure is not limited in its use to the particular applications or applications described, as will be understood by those skilled in the art. Nor is the present disclosure limited with respect to the particular elements and / or features described or depicted herein in its preferred embodiments. It will be understood that the present disclosure is not limited to the disclosed embodiment or embodiments, but is capable of numerous rearrangements, modifications, and substitutions without departing from the scope defined by the following claims.

Claims

1. a closed fluid network comprising a plurality of containers, each container containing a volume of liquid medium for growing a microbial biomass in or on said liquid medium; the plurality of vessels are arranged in fluid communication to allow a recirculation flow of the liquid medium to flow in stages through the plurality of vessels; Biomass reactor.

2. Each vessel includes a plurality of air inlets; each air inlet is associated with at least one opening that is covered or blocked by a semi-permeable membrane that allows passive diffusion of air from the external environment to the interior space through said plurality of air inlets; The biomass reactor of claim 1 .

3. The air inlet The top of each container, or one or more side walls of each container; or a top surface and one or more side walls of each container; uniformly distributed over the The biomass reactor of claim 2.

4. the recirculation stream of liquid medium enters at least one first vessel of the plurality of vessels through at least one inlet and exits at least one final vessel of the plurality of vessels through at least one outlet; the at least one first container and the at least one final container are in direct fluid communication or in indirect fluid communication with respect to one or more other containers; The biomass reactor according to any one of claims 1 to 3.

5. each vessel of the plurality of vessels having at least one respective inlet and at least one respective outlet; at least one inlet of each of the at least one first vessel receives a flow of the liquid medium from a pump of the closed fluid network, and at least one outlet of each of the at least one final vessel discharges the liquid medium to a sump of the closed fluid network; the sump is in fluid communication with the pump such that the pump can pump the liquid medium from the sump to the at least one first container; The biomass reactor of claim 4.

6. the at least one inlet and the at least one outlet of each container are juxtaposed to a base of the respective container; The biomass reactor according to claim 4 or 5.

7. the plurality of vessels being arranged in a multi-tiered network, each tier comprising at least one vessel; The biomass reactor according to any one of claims 1 to 6.

8. Further comprising a biomass layer disposed on the surface of the liquid medium contained in each vessel of the closed fluid network. The biomass reactor according to any one of claims 1 to 7.

9. In each vessel, the respective inlet and outlet are configured to reduce disturbance of the biomass layer during the recirculating flow of the liquid medium. A biomass reactor according to claim 8 which recites claim 4.

10. The plurality of containers are stacked and arranged with a vertical offset between the containers. The biomass reactor according to any one of claims 1 to 9.

11. a closed fluid network including a plurality of containers, each container containing a volume of liquid medium and a microbial biomass growing in or on said liquid medium, each of said plurality of containers arranged in fluid communication to allow a recirculation stream of said liquid medium to flow in stages through said plurality of containers, said recirculation stream entering at least one first container of said plurality of containers through at least one inlet and exiting at least one final container of said plurality of containers through at least one outlet; a sump containing a fixed addition volume of liquid medium, the sump having a sump inlet in fluid communication with the at least one outlet of the closed fluid network and a sump outlet; a pump generating a flow of the liquid medium between the sump outlet and the inlet of the closed fluid network to establish and / or maintain the recirculating flow of the liquid medium; Biomass reactor.

12. one or more sensors for sensing one or more parameters of the liquid medium exiting the at least one final container; and means for correcting the sensed one or more parameters in response to said sensing. The biomass reactor according to any one of claims 4 to 6 or 11.

13. providing a closed fluid network comprising a plurality of vertically offset containers arranged in fluid communication, each container containing a volume of liquid medium for growing a microbial biomass in or on said liquid medium; establishing fluid communication of the liquid medium between at least one final container of the closed fluid network and at least one first container of the closed fluid network to provide a recirculation flow of the liquid medium through the plurality of containers of the closed fluid network; treating the liquid medium obtained from the at least one final container to extract one or more molecular products from the liquid medium. A method for producing a molecular product.

14. a closed fluid network including a plurality of vertically offset vessels, each vessel containing a volume of liquid medium and a microbial biomass growing in or on said liquid medium, each of said plurality of vessels arranged in fluid communication to allow a recirculating stream of said liquid medium to flow in stages through said plurality of vessels, said recirculating stream of said liquid medium entering at least one first vessel of said plurality of vessels through at least one inlet and exiting at least one final vessel of said plurality of vessels through at least one outlet; providing a sump containing a fixed addition amount of liquid medium, the sump having a sump inlet in fluid communication with the at least one outlet of the closed fluid network and a sump outlet; activating a pump that controls the flow of the liquid medium between the sump outlet and the inlet of the closed fluid network to establish and / or maintain the recirculating flow of the liquid medium; A method for forming a biomass reactor for producing molecular products.

15. A product produced by operating the biomass reactor of any one of claims 1 to 10.

16. The product is a. syncytial products, b. bacterial protein products; c. a product containing citrinin; d. Products containing dihydrolysergic acid; e. a product containing mevastatin; f. fermentation products, and g. nucleic acid, including one or more of:

16. The product of claim 15.

17. 15. An installation for carrying out the method according to claim 13 or 14, comprising a biomass reactor according to any one of claims 1 to 12.

Images