Circulating Biofilm Bioreactor

Abstract

A system for producing hydrophobic chemicals includes a circulating biofilm bioreactor for synthesizing a hydrophobic product and continuous extraction into an organic solvent phase. Various configurations can be employed to assemble the bioreactors into production modules. The extraction operation can be performed in one or more extraction vessels. Optionally, the product and solvent are separated using membrane separation technology, and the solvent is rerouted to the extraction vessel. A method for bioconverting a feedstock into a chemical product is also described.

Description

[Technical Field]

[0001] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 396,634, filed August 10, 2022, which is incorporated herein by reference in its entirety. [Background technology]

[0002] REFERENCE TO ELECTRONIC SEQUENCE LISTING An electronic sequence listing conforming to the provisions of WIPO Standard ST.26 is hereby incorporated by reference in its entirety. The contents of the electronic sequence listing are encoded as XML in UTF-8 text. The electronic document, created on July 18, 2023, is titled "0412.0004 WO1_ST26.xml" and is 92,039 bytes in size.

[0003] In industrial-scale biomanufacturing, bioreactors or fermentors are used to grow cells to produce chemical products, typically in suspended cell reactors during periodic biomass doubling, known as logarithmic growth. To produce hydrophobic products using such traditional fermentation methods, the biomass is typically grown, the cells are harvested, and the product is then extracted from the cells.

[0004] Recently, a biofilm bioreactor has been developed, as described in U.S. Patent Application Publication No. 2021 / 0253990, the entire contents of which are incorporated herein by reference. This biofilm bioreactor allows for continuous product synthesis and extraction into an immiscible solvent phase. This biofilm bioreactor uses naturally occurring biofilms of hydrocarbon-clastic and / or oleaginous organisms grown on a solid support within a packed-bed reactor. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Application Publication No. 2021 / 0253990 [Non-patent literature]

[0006] [Non-Patent Document 1] "A Threshold-Based Bioluminescence Detector With a CMOS-Integrated Photodiode Array in 65 nm for a Multi-Diagnostic Ingestible Capsule", Liu, Q., Jimenez, M., Inda, ME, Riaz, A., Zirtiloglu, T., Chandrakasan, AP, Lu, TK, Traverso, G., Nadeau, P. and Yazicigil, RT, 2022. A Threshold-Based Bioluminescence Detector With a CMOS-Integrated Photodiode Array in 65 nm. IEEE Journal of Solid-State Circuits, 58(3), pp.838-851. [Non-patent document 2] Typically, the solvent layer 20 will contain: individual cells 104, cells in supported biofilm 106 (or in self-aggregated biofilm) as well as aqueous micelles 108, containing cells 104, cells in biofilm on supports 106 (or as self-aggregated unsupported biofilm). Summary of the Invention [Problem to be solved by the invention]

[0007] The main challenges to achieving high production yields from aerobic microorganisms in packed-bed biofilm reactors are oxygen supply, achieving substantial biomass, routing, and separation of immiscible fluids.

[0008] The present invention relates to an apparatus, system, and method for producing chemicals using a biofilm reactor in which supported or self-aggregated biofilm microorganisms are suspended in a reaction medium. While in conventional techniques, the biofilm and support material remain in a fixed location and a solvent is circulated over the biofilm, the present approach features a suspended solid support carrying a biofilm (or suspended biofilm self-aggregates) that is pumped from one type of reservoir (synthesis vessel) to a second type of reservoir (extraction vessel).

[0009] Generally, microorganisms are selected for inherent properties that provide reaction pathways for the conversion of carbon sources into desired products. In many embodiments, the organisms tolerate organic solvents, a property that allows for two-phase extraction of products while retaining functional biomass. [Means for solving the problem]

[0010] An embodiment of the invention features a system that includes one or more synthesis (growth) vessels and one or more extraction vessels.

[0011] In the synthesis vessel, a biofilm on a solid support (e.g., beads) or a self-aggregating biofilm is fluidized by air and / or liquid, e.g., hydraulic pressure. The cells in the biofilm convert the carbon source into products, such as hydrophobic chemicals. The carbon source is a compound or mixture of compounds that can be converted into desired (hydrophobic) chemicals by hydrocarbon-forming and / or oleaginous organisms. In examples, the carbon source is introduced into the synthesis vessel as a feedstock or as a component thereof. In many embodiments, an aqueous feedstock is used.

[0012] In the extraction vessel, the solid support bearing the biofilm, biofilm aggregates, and / or planktonic cells present in the medium extracted from the bioreactor is circulated through an organic solvent to extract the accumulated product.

[0013] Multiple synthesis vessels can be combined to form production modules or assemblies. In some embodiments, the vessels can be arranged in a parallel configuration for continuous operation, while in other embodiments, the vessels are arranged in a serial manner so that the feedstock and biomass are cycled sequentially from one column to the next until finally reaching the extraction vessel. In examples, aeration is provided to some or each column.

[0014] A series arrangement of vessels may also be employed. In some embodiments, aeration is provided to selected columns in series.

[0015] A single synthesis vessel or a set including multiple synthesis vessels can be used in combination with a single extraction vessel or multiple extraction vessels. In many embodiments, the extraction vessel is characterized by a medium, typically a water-containing medium, occupying the bottom region of the vessel, and a light solvent floating on top. An extraction module including one or more extraction vessels can further include a separator, which in one example utilizes a filtration membrane to separate the product from the solvent. The solvent can be returned to the extraction vessel, and the product can be recovered or further processed.

[0016] Some of the features, or sometimes characterizing aspects, of the present invention include one, and often two or more, of the following attributes: In some embodiments, for example, an oxygen-containing gas, often sterile air, is introduced into each column (or selected columns within a production module) and diffused to ensure adequate dissolved oxygen. Flow controllers that control the introduction of air can be provided for all or selected vessels. A source of biofilm microorganisms can be introduced as needed, and the amount introduced can be monitored.

[0017] In some embodiments, acid and / or base can be added to maintain a pH-controlled environment within the synthesis vessel. Waste materials can be continuously or periodically removed to ensure healthy bioreactor operation. Sensors for monitoring pH, dissolved oxygen (DO), temperature, pressure, carbon composition, etc. can be installed in one or more synthesis columns and / or one or more extraction vessels. Sensors can be hardwired or wireless. In some embodiments, one or more wireless sensors circulate through the system or its components (particular vessels, particular modules, etc.).

[0018] In some embodiments, the system is configured to simultaneously separate the extract, solvent, medium, and air. A condenser column can be included to recapture the solvent. In some cases, a carbon filter is utilized to prevent solvent leakage. A vent filter can be provided to allow the release of (sterile) air.

[0019] A specific embodiment features a filtration loop attached to the extraction vessel, allowing for continuous removal of solvent and product.

[0020] In some embodiments, the invention features a bioreactor design having two or more sets of vessels. One embodiment includes one set of vessels in which a biofilm on a solid support is fluidized by air and / or hydraulic pressure, and cells in the biofilm convert a carbon source into a product. In a second set of vessels, the biofilm-bearing solid support and the planktonic cells in the medium are circulated through an organic solvent to extract the accumulated product.

[0021] Thus, in a first embodiment, the bioreactor comprises two vessels: a first synthesis vessel, consisting of a solid support suitable for biofilm growth and providing a reservoir for a feed solution (typically an aqueous solution containing a carbon source, minerals, vitamins, and / or other nutrients) that contacts the biofilm; and a second extraction vessel, consisting of a vessel for an extraction solvent. One or more pumps can be used to keep the solid support in a fluid state and freely suspended in the feed solution. The pump also serves to transfer the suspended bead mixture from the first vessel to the second vessel (containing the extraction mixture) and back to the first vessel.

[0022] In a second embodiment, the bioreactor has multiple synthesis vessels, each consisting of a solid support suitable for biofilm growth and providing containment for a feed solution (e.g., an aqueous solution containing a carbon source, minerals, vitamins, and / or other nutrients) that contacts the biofilm. The multiple synthesis vessels interface with a single extraction vessel or multiple extraction vessels containing an extraction solvent. An extraction module containing one or more extraction vessels can further include associated equipment for phase separation. One or more pumps can be used to keep the solid support in a fluid state and freely suspended in the feed solution. The pump can also serve to transfer the suspended bead mixture from the synthesis vessel to the extraction vessel.

[0023] In some embodiments, the solid support is omitted and the microorganisms are allowed to form cohesive biofilms for product synthesis, which over time become "solid supports" and remain suspended in solution throughout the process.

[0024] As used herein, the term "support" encompasses biofilm aggregates (which become supportive of added microorganisms as the process progresses) and conventional solid supports (e.g., beads) made from materials different from the biofilm (e.g., plastic, glass, etc.). In the case of aggregates, the "support" can contain dead or live microorganisms. As used herein, the terms "biofilm particles" or "floating biofilm particles" generally refer to biofilms on solid supports (e.g., beads) and self-aggregated biofilms, the latter of which do not form on supports other than those formed from the microorganisms themselves.

[0025] Generally, floating biofilm particles, whether supported or in the form of self-aggregates, are encouraged to remain suspended and, in many instances, are generally not allowed to settle outside of designated settling tanks.

[0026] In some embodiments, inserts are placed at the bottom of the synthesis vessel to induce vortex and / or angular flow, which increases mixing, minimizes clogging of solid support particles, and reduces the required pumping force.

[0027] In other embodiments, a gas is pumped into one or more reservoirs. This gas can provide additional buoyancy to the biofilm material and introduce oxygen into the synthesis vessel(s). In a further embodiment, an oxygen-containing gas is supplied to the synthesis vessel and an inert gas is added to the extraction vessel to create an anoxic environment.

[0028] In yet another embodiment, the production module includes reservoirs, pumps, and valves that allow for the introduction of additional carbon-containing feedstock or various required nutrients. A sensor unit attachable to the bioreactor assesses the state of the bioreactor and communicates with a central processor, which controls the pumps and valves to introduce nutrients into the reactor.

[0029] In further embodiments, a membrane in a separation (e.g., filtration) loop interacts with the extraction vessel, concentrating the product and returning the raw solvent to the extraction vessel. In certain embodiments, a pump is used to remove the solvent-based feed from the extraction vessel and generate pressure across the membrane.

[0030] The present invention also relates to a method for synthesizing hydrophobic products using hydrocarbon- and / or oil-forming organisms.

[0031] Thus, as a result of the present invention and its embodiments, improved apparatus and methods are available that provide sufficient breathability with simplified extraction for the biological production of hydrophobic chemicals.

[0032] The above and other features and advantages of the present invention, including various novel details of construction and combination of parts, will now be more particularly described with reference to the accompanying drawings and pointed out in the claims. It will be understood that the particular method and device embodying the invention are shown by way of illustration and not as limitations of the invention.

[0033] The principles and features of this invention may be employed in many different embodiments without departing from the scope of the invention. [Brief explanation of the drawings]

[0034] In the accompanying drawings, reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention. In the drawings: [Figure 1] FIG. 1 is a schematic diagram of a system comprising multiple bioreactors in a parallel configuration, an extraction vessel, and a separator according to the present invention. [Figure 2] FIG. 1 is a longitudinal cross-sectional view of an exemplary bioreactor showing dimensions in centimeters (cm). [Figure 3A] FIG. 1 is a schematic diagram of a system comprising multiple bioreactors connected in series, an extraction vessel, and a separator according to the present invention. [Figure 3B] FIG. 1 is a schematic diagram of a system comprising multiple bioreactors connected in series, an extraction vessel, and a separator according to the present invention. [Figure 4] 1 is a schematic diagram of an extraction vessel, with the blow-up section showing components present in the interlayer during operation. [Figure 5] 1 is a schematic diagram of another embodiment of a system including multiple bioreactors, bubble traps, and two-phase extraction vessels according to the present invention; [Figure 6] FIG. 6 is a perspective view of the two-phase extraction vessel of FIG. 5 according to the present invention. [Figure 7] 6 is a perspective view of the two-phase extraction vessel of FIG. 5 further including a filtration loop attached to the solvent circulation loop according to the present invention. [Figure 8] FIG. 2 is a block diagram illustrating how various process parameters are controlled in a controller 200 based on sensor information. [Figure 9A] 1 illustrates several mixer designs that can be employed in practicing aspects of the present invention. [Figure 9B] 1 illustrates several mixer designs that can be employed in practicing aspects of the present invention. [Figure 9C] 1 illustrates several mixer designs that can be employed in practicing aspects of the present invention. [Figure 9D] 1 illustrates several mixer designs that can be employed in practicing aspects of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0035] The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which illustrative embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0036] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. Also, all conjunctions used should be understood in the most inclusive sense possible. Accordingly, the word "or" should be understood as having the definition of a logical "or" rather than a logical "exclusive or," unless the context clearly requires otherwise. Furthermore, the singular forms and articles "a," "an," and "the" are intended to include the plural forms as well, unless expressly stated otherwise.

[0037] Furthermore, as used herein, it will be understood that the terms "comprises," "includes," "including," and / or "comprises" specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. Furthermore, when an element, including a component or subsystem, is referred to and / or shown as being connected or coupled to other elements, it will be understood that the other elements may be directly connected or coupled or that intervening elements may be present.

[0038] Although terms such as "first" and "second" are used herein to describe various elements, it should be understood that these elements are not limited by these terms. These terms are used merely to distinguish one element from another. Thus, an element described below could be referred to as a second element, and similarly, a second element could be referred to as a first element without departing from the teachings.

[0039] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Furthermore, terms as defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the relevant technology, and will not be interpreted in an idealized or overly formal sense unless explicitly defined as such in this specification.

[0040] The devices, systems, and methods described herein preferably use hydrocarbon-destructive and / or oleaginous microorganisms to synthesize and isolate or extract desired products, such as hydrophobic chemicals. The synthesis approach relies on suspending microbial particles in a vessel or reactor.

[0041] In many of its aspects, the present invention employs a solid phase (or matrix) in the form of a particulate material to support a biofilm of, for example, hydrocarbon-destructive and / or oleaginous microorganisms. The solid phase can be provided in the form of granules or beads, such as, for example, silica or glass beads. Other possible carriers include, but are not limited to, magnetic beads, wood chips, plastic beads, etc.

[0042] For example, it has been found that the use of a carrier, i.e., a particulate solid phase such as beads, is not a hindrance and can actually increase efficiency. In the absence of a support, microorganisms self-assemble to form aggregates, referred to herein as "autoaggregates." Accordingly, some embodiments of the present invention utilize biofilm microorganisms that autoaggregate to form biofilm particles, e.g., 10 microns or larger. In illustrative examples, autoaggregates can have diameters or maximum dimensions ranging from about 1 millimeter (mm) to about 3 centimeters (cm). Autoaggregates can occur in microorganisms such as Escherichia coli, Pseudomonas aeruginosa, Pseudomonas stutzeri, and Clostridium thermocellus, to name a few. Furthermore, the formation of autoaggregates can occur not only in living biofilm microorganisms but also in dead biofilm microorganisms. Dead cells can also serve as a carbon source for living cells.

[0043] As with the supported type, the biofilm self-aggregates are maintained in suspension during the synthesis or manufacturing process. If necessary, sedimentation of the supported or self-aggregated biofilm can be carried out in a designated settling tank.

[0044] The growth or maintenance of biofilm microorganisms, whether supported or unsupported, is sustained by a carbon source (often supplied as an aqueous medium). The carbon source is converted into chemical products by biosynthetic pathways contained within the organism. Examples of usable carbon sources include, but are not limited to, acetate, succinate, lactate, glycerol, ethanol, plant by-products such as thin stillage, food waste by-products, anaerobically digested food waste, agricultural waste by-products, fermentation by-products, and combinations thereof.

[0045] The microbial communities that form biofilms can be used as microbial catalysts, with different microbial species able to metabolize different components of thin stillage or fermentation by-products or catalyze different steps in product formation.

[0046] The systems described herein include a synthesis vessel (also referred to herein as a "synthesis (or production) column," "bioreactor," or "reactor"), and an extraction vessel (or column). In many embodiments, the synthesis vessel receives fluid (liquid and / or gas) from the bottom of the column. The contents of the bioreactor are removed from the top of the synthesis column and fed to the top of the extraction vessel, where the product is removed from the cells.

[0047] The synthesis vessel or bioreactor is configured to create a dense suspension of particulate material suitable for the typical growth of oleaginous and / or hydrocarbon-clastic organisms, such as Marinobacter, and to continuously or periodically circulate the biofilm suspension through an organic solvent. This design differs from traditional bioreactor designs, such as those described in U.S. Patent Application Publication No. 2021 / 0253990, in which the biofilm and support material remain in a fixed location while the solvent is circulated over the biofilm. In this approach, the biofilm is allowed to self-aggregate, or a solid support bearing the biofilm is pumped from one reservoir (the synthesis vessel) to a second reservoir (the extraction vessel).

[0048] In many applications, synthetic vessels are designed to provide a well-mixed, carbon-, nutrient-, and oxygen-rich environment for biofilm-forming organisms to convert carbon sources (e.g., acetate, succinate, synstillage components, etc.) contained in aqueous feedstocks.

[0049] In a specific embodiment, the supports are kept in suspension by continuously pumping aqueous medium and support suspension (if supported biofilm is employed) from the extraction vessel into the bottom inlet of the synthesis vessel. If supports, e.g., beads, are not used, the flow returned to the bioreactor contains the self-aggregates of biofilm suspended in aqueous medium.

[0050] Air or other gases or gas mixtures can also be pumped into the synthesis vessel, typically through the bottom of the vessel. The gas can serve both as a supplemental lift source for biofilm support (or biofilm self-aggregation) and as an important oxygen source for the intensive metabolic processes occurring in the biofilm microorganisms. In some cases, the gas is oxygen-enriched air to further enhance aeration within the reaction vessel. Oxygen-containing gases, such as sterile air, can be introduced and diffused into the column to ensure sufficient dissolved oxygen (monitored by sensors circulating or fixed within the reactor). In other cases, gases, such as air, are enriched with CO2 to help buffer the growth medium.

[0051] While a single synthesis vessel may be employed, many embodiments feature multiple or multiple (i.e., two or more) vessels. As many as three, four, and as many as 10,000, e.g., at least 200, 500, 800, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, or 9000 synthesis or production modules or assemblies, can be combined. Configurations that can be employed include, for example, columns arranged in parallel or series.

[0052] The extraction vessel may be part of an extraction module or loop that may further include a separation device for separating the product from the solvent. Further operations may be performed to recover the product and / or recycle the solvent.

[0053] Once in the extraction vessel, the media, cells, biofilm, and in some cases, solid supports such as beads transferred from one or more bioreactors, fall by gravity through the solvent phase. As they pass through the solvent layer, hydrophobic products are extracted into the hydrophobic phase (solvent). Due to the inherent tolerance of biofilm-forming organisms to hydrophobic organic solvents, the productivity / viability of the biomass is maintained throughout the extraction process.

[0054] Shown in FIG. 1 is a system 10 including ten synthesis vessels 12 and extraction vessels 14 arranged in parallel.

[0055] The synthesis vessel 12, also referred to herein as a "synthesis column," "bioreactor," or "reactor," can be constructed from suitable materials, such as plastic materials (e.g., acrylic), glass, metals (e.g., aluminum, steel), fiberglass, or carbon fiber, and can have a volume ranging from about 1 liter (L) to about 100,000 L. While synthesis vessels often have a cylindrical shape, other shapes can also be employed. Depending on the process parameters, desired residence time, available footprint, ceiling height, or other factors, tall columns as well as square or short columns can be selected. In one example, the synthesis vessel 12 is cylindrical, has a height ranging from about 3 feet (ft) to about 20 feet (ft), and a diameter ranging from about 0.25 feet (ft) to about 3 feet (ft). Figure 2 shows a longitudinal cross-section of an exemplary synthesis vessel 12 with dimensions in centimeters (cm). Specifically, the vessel in this figure has a diameter of 156 cm and a height of 1022.15 cm.

[0056] In a typical approach, the synthesis (also referred to herein as "production") modules or "loops" (e.g., module 16 in Figure 1) contain the same type of column 12. In some cases, different column designs may be employed.

[0057] The extraction vessel 14 can be made of any suitable material, for example, materials similar to those employed in constructing the synthesis vessel. For continuous operation, it can have a capacity that depends on the volume of the synthesis module or other parameters such as flow rate, productivity (e.g., grams product / L / hour), product stability, etc. A suitable shape, for example, a cylindrical shape, can be selected, with a height ranging from about 1 foot to about 20 feet and a diameter ranging from about 1 inch (in) to about 2 feet.

[0058] Extraction vessel 14 may include a gas (e.g., air) gap 18, a solvent layer 20, and a media reservoir 22. In certain embodiments, solvent layer 20 is formed of a hydrophobic extraction solvent, such as hexane, heptane, dodecane, oleic acid, soybean oil, castor oil, corn oil, avocado oil, another suitable water-immiscible solvent, or a combination thereof. Typically, media reservoir layer 22 contains an aqueous composition below the solvent layer, from which the solvent spontaneously phase separates. Due to the lower density of the solvent phase, the solvent layer remains above the aqueous phase.

[0059] The system can also include a separation arrangement or loop 39 using, for example, filtration through a membrane system 29. Specific examples include the use of chemically or size-selective membranes, gravity or centrifugation, or column-based separations similar to chromatographic separations. The separation loop or module can be interfaced with a solvent reservoir 20 to, for example, continuously remove product from the system and recycle the solvent to the extraction vessel.

[0060] Some product molecules, e.g., retinoids, are highly sensitive to oxygen. The reducing environment within cells maintains these molecules in a desirable state prior to extraction. However, once extracted from the cells, these substances begin to degrade. To reduce oxidation, the extraction vessel can be purged with an inert gas, such as nitrogen, reducing or preventing the product's exposure to oxygen as the molecules are extracted into the solvent phase.

[0061] The media exits a media reservoir layer 22 at the bottom of the extraction vessel 14 and is fed using a pump 24 to a manifold 26 that connects to the bottom of each reactor 12 .

[0062] Optionally, supplemental fresh medium can be added from a reservoir, such as reservoir 28, using pump 30. Materials such as acid or base, pumped by pumps 32 and 34, respectively, can be added as needed from reservoirs 36 and 38 under the control of controller 200. Flow from the reservoirs can be controlled by motorized ball valves 31, 33, and 35, which are operated by controller 200.

[0063] In some embodiments, air, nitrogen, oxygen mixtures, or another suitable gas is added to a reservoir, such as reservoir 28, to provide the desired environment for the feed medium and to add buoyancy to the supported or self-aggregating biofilm. Certain gases or gas mixtures, such as CO, CO, methane, ethane, etc., can also serve as a carbon source for the organisms.

[0064] Gas is supplied in the direction of the arrows from a gas source 40, for example a sterile air source, to each column 12. Oxygen gas or oxygen-enriched air can be used in addition to or as a substitute for air.

[0065] 1, the gas is supplied through manifold 42. Other approaches can also be implemented. For example, the gas can be supplied through the same manifold employed for the media supply. The flow rate can be monitored or controlled by device 44, for example, a mass flow controller under the control of controller 200.

[0066] Generally, gas can be introduced into any one of the columns or vessels described herein using a device such as, for example, a diffuser stone, a sparger, drilled (or perforated) piping, or other device capable of generating gas bubbles, particularly small (fine) gas bubbles that promote dissolution of the gas into the liquid medium. In many embodiments, the gas is supplied from the bottom of the synthesis vessel, and a bubble-generating device, e.g., a diffuser stone, is located at the bottom of the vessel. However, other arrangements are also contemplated.

[0067] In some embodiments, different gas compositions or flow rates can be applied to different columns, thereby creating different synthesis zones, where a more oxidizing or a more reducing environment can be created.

[0068] As an alternative or addition to a bubble generator (e.g., a diffuser stone), the synthesis vessel can include oxygen-permeable tubing made, for example, from a fluoropolymer (e.g., fluoroethylene propylene), low-density polyethylene, or silicone. The tubing can increase gas (e.g., air) transport within the bioreactor without generating gas bubbles, and in a specific example, receives air or another suitable gas from an inlet port at the bottom of the synthesis vessel and extends through the vessel's interior to an outlet port. In certain embodiments, the inlet and outlet ports are staggered relative to one another, resulting in a configuration in which the permeable tubing is not parallel to the vertical axis or the bioreactor. Other configurations, such as helical tubing, coiled tubing, or multiple tubes connecting pairs of inlet and outlet ports, can also be employed.

[0069] The contents from the reactor are removed from the top of column 12 via manifold 46 and directed to the overhead or extraction vessel 14. A condensation column 48 can be installed at the top of vessel 14 to capture solvent vapors and return them to the extraction vessel. Gases, e.g., air, exiting the condensation column can be passed through a carbon filter 50 (to control solvent leakage) before being released at a vent 52 equipped with a vent filter 54 to ensure the release of sterile air.

[0070] The product-containing stream from solvent layer 20 is conducted (via conduit 56) to separation arrangement 39, which may include one or more chemoselective and / or size-selective membranes. The membranes separate the feed stream (product-containing solvent) into its components. In one embodiment, the solvent passes through the membrane material (as the permeate fraction), while the product, e.g., hydrophobic chemicals, is retained (as the retentate or concentrate fraction).

[0071] In another embodiment, two membranes are included: one to concentrate the stream (purified solvent passes through as permeate) and a second to remove the product as permeate.

[0072] In yet another multi-membrane embodiment, a first membrane has a size smaller than the target molecule, and a second membrane has a size larger than the target molecule. In the first stage, the permeate is returned to the reactor, and the retentate is sent to the second stage. This concentrates the product in the solvent. In the second stage, the protein passes through the membrane, removing cellular debris and larger proteins, while the retentate is returned to the reactor. The product can be recovered for further purification, such as affinity tag purification or other chromatographic separation methods.

[0073] In another embodiment, the product and solvent pass through a first membrane to remove impurities, a second immiscible solvent is mixed in a second membrane, and the product and second solvent are removed through a second membrane.

[0074] A high-pressure (e.g., 0-800 psi) pump 58, controlled by controller 200, can be added to generate the driving force necessary to push the solvent through the membrane. This solvent is then returned (via conduit 60) to the solvent layer 20 of the extraction vessel. The retained (separated) product is recovered as the membrane fraction through outlet 62, from which it can be subjected to further downstream operations such as further purification, blending, or other processes.

[0075] The waste material 64 may be continuously or intermittently drawn from the media bed 22 using a pump 66.

[0076] Valves such as valves 70, 72, 74, or 76 can be used to open, close, and / or adjust flow rate under the control of controller 200. Conduit 98 and valve 99 can be used to bypass the extraction vessel and recirculate the media to the production loop and circulate through the biofilm reactor without passing through the extraction vessel.

[0077] Sensors 164 can be provided on one column, selected columns, or all columns in production module 16, which can form a sensor array. In some embodiments, the sensors are hardwired to controller 200. Additionally, or alternatively, one or more or all columns can be equipped with wireless sensors (e.g., sensors 169) that communicate with a wireless interface board that is typically external to the bioreactor and is typically part of controller 200. These wireless sensors can include individual elements for pH, oxygen, fluorescence-based sensing, or electrochemical measurements via cyclic voltammetry, electrochemical impedance spectroscopy, or other techniques. Data generated by these sensors is received and analyzed by controller 200.

[0078] Non-limiting examples of wireless sensors include, for example, "A Threshold-Based Bioluminescence Detector With a CMOS-Integrated Photodiode Array in 65 nm for a Multi-Diagnostic Ingestible Capsule," Liu, Q., Jimenez, M., Inda, ME, Riaz, A., Zirtiloglu, T., Chandrakasan, AP, Lu, TK, Traverso, G., Nadeau, P. and Yazicigil, RT, 2022. A Threshold-Based Bioluminescence Detector With a CMOS-Integrated Photodiode Array in 65 nm. IEEE Journal of Solid-State Circuits, 58(3), pp. 838-851. Other types of wireless sensors known in the art or developed in the future can also be employed.

[0079] The sensors 164 can be located at the bottom of each column or selected columns 12, attached to the interior surface of the column, or in other suitable locations. Sensors that circulate with the flow in a circulating reactor can also be employed. Some embodiments utilize wireless sensors that actively circulate in the flow path with the cells through the reactor system. Sensors can also be placed in the conduits to assess the condition and / or composition of the passing fluid. The type of sensor used and / or how the sensor is positioned within the vessel can be the same or different for each column.

[0080] For example, sensors such as those described above may also be provided in the extraction vessel to enable monitoring by the controller 200. For example, in the embodiment of Figure 1, the extraction vessel 14 is monitored by a sensor (or sensor array) 165 in the solvent layer 20 and from a sensor (or sensor array) 167 in the media layer 22.

[0081] Controller 200 is configured to receive inputs from various sensors in one, more than one, or all of the columns in production module 16 and / or from sensors in at least one extraction vessel 14. In one example, controller 200 receives input 202 from sensor 164, input 204 from sensor 165 (in solvent layer 20), and input from sensor 167 (in media reservoir layer 22).

[0082] Based on the data received from the various sensors (oxygen level, pH, chemical content, etc.), the controller 200 issues various responses, such as controlling the air supply via the flow control device 44 (output signal 208), controlling the valve 70 (output signal 210), and / or controlling one or more valves 31, 33, 35 (output signal 212), allowing feedback control of the process.

[0083] Communication to and / or from the controller 200 can be wired or wireless.

[0084] It is possible for all columns to be operated in the same way and for their contents to be transferred to the extraction module simultaneously. However, in many embodiments, each column (or group of columns) in the production module 16 is operated independently from the rest of the columns in the module. Biofilm-containing medium is transferred from some columns but not from others, depending on the stage it has reached in the synthesis vessel.

[0085] In another approach, shown in Figures 3A, 3B, and 5, the synthesis vessels of production module 16 are arranged in series for continuous operation, with aeration being fed to selected columns. More specifically, media from media reservoir layer 22 (of extraction vessel 14), supplemental media from reservoir 28, recycled media via loop 98 and valve 99 in the open position, and components such as acid from reservoir 36 and / or base from reservoir 38 are fed to the bottom of the first column, column 12a.

[0086] The extracted material from the top of column 12a is transferred to the top of column 12b via conduit 80. The material from column 12b is transferred from the bottom of column 12b to the next column, column 12c, via conduit 82. This pattern continues from one column to the next via conduits 80 and 82 until the last column, column 12L, is reached.

[0087] From the bottom of this final column, the contents of the bioreactor are directed to an extraction module via conduit (line) 92. In the embodiment of Figure 3A, the destination is gas layer 18, while in the embodiment of Figure 3B, line 92 connects to condensation column 48 at the top of vessel 14.

[0088] Gas from source 40, typically sterile air, pure oxygen, or a precise mixture of oxygen and nitrogen, is delivered to the bottom of selected columns, e.g., columns 12a, 12c, 12e, 12g, and 12j, using manifold 94. In some cases, the columns that do not receive air (e.g., columns 12b, 12e, etc.) can be made narrower than the columns with aeration (e.g., columns 12a, 12c, etc.), thereby minimizing the time that the cells do not receive active aeration.

[0089] Each column of the production module 16 is provided with a sensor 164 (hardwired or wireless) attached to the bottom of the vessel and a sensor 169 that can circulate with the circulating fluid in the vessel (or through the system or part thereof) and is typically wireless.

[0090] Controller 200 receives data from sensors, such as sensor 164 and (input 202) in the column of production module 16, and / or from sensors (or sensor arrays) 165 and 167 in extraction vessel 14. As a result, (mass) flow controller 44 can be activated or adjusted to allow more or less gas into the vessel. Valves, such as valve 70 or motorized ball valves 31, 33, 35, can be controlled via output signals 214 or 212. In one embodiment, sensor 183 (in extraction vessel 14) is a level height sensor, such as an optical line break sensor, to detect the occurrence of foam.

[0091] Sensors (or sensor arrays) such as sensor 171 (monitoring the air in container 14), sensor 173 (monitoring waste), sensor 175 (monitoring product), and 177 (monitoring supply from supply reservoir 28) provide additional information to controller 200 to take further action as necessary.

[0092] For product extraction, biofilm suspension, removed from a synthesis vessel such as column 12 in Figure 1 or column 12L in Figures 3A or 3B, is transported through an organic solvent phase as it circulates through an extraction vessel under the control of controller 200. The suspension and medium cascade out of the top inlet of the extraction vessel into a gas phase 18, a hydrophobic extraction solvent such as hexane, heptane, dodecane, or oleic acid. Below solvent layer 20 is an aqueous layer from which the solvent undergoes spontaneous phase separation. Due to the solvent's low density, the solvent phase remains above the aqueous phase, forming medium layer 22. The mixture of medium, cells, biofilm, and, if used, a solid support such as beads, falls by gravity through the solvent phase. As it passes through the solvent, the hydrophobic product is extracted into the hydrophobic phase. Due to the biofilm former's inherent resistance to hydrophobic organic solvents, biomass productivity / viability is maintained throughout the extraction process. After natural separation of the aqueous and solvent phases, the aqueous phase collects at the bottom of the extraction vessel (media layer 22) and can serve as a reservoir from which the aqueous medium can be pumped (e.g., using pump 24) to the synthesis vessel.

[0093] The process performed in extraction vessel 14 is illustrated in more detail in Figure 4. When cell suspension 100 is transferred from a synthesis vessel (e.g., reactor 12 in Figure 1 or 12L in Figure 3) to extraction vessel 14, the aqueous mixture of biofilm (on a support or as self-aggregates) cascades from extraction vessel inlet 102 through gap 18, then through solvent layer 20 and settles into media reservoir 22. Typically, the solvent layer 20 will contain: individual cells 104, cells in supported biofilm 106 (or in self-aggregated biofilm), as well as aqueous micelles 108 containing cells 104, cells in biofilm on supports 106 (or as self-aggregated unsupported biofilm). Contact between the cells and the solvent results in the transfer (or extraction) of product 110 into the solvent. The aqueous medium from media layer 22 is removed at outlet 112 and can be directed to one or more synthesis columns, as described above.

[0094] Illustrated in FIG. 5 is an arrangement employing a production module 16 having columns in a series configuration (as described with reference to FIGS. 3A and 3B) and an extraction module including a set of vessels. In the embodiment of FIG. 5, the contents from column 12L are directed to the bottom of bubble trap 120, which is used to separate gas, e.g., air, shown in upper region 122 of the bubble trap, from the media occupying lower region 124 of bubble trap 120. The top of bubble trap 120 connects to condensation column 48 (to recapture solvent) and carbon filter 50 (to prevent solvent leakage). Gas, e.g., air, collected in upper region 122 of the bubble trap is released at vent 52, which may include vent filter 54 (to allow for the evacuation of sterile air). The media (lower region 124) can be directed from bubble trap 120 to the bottom of column 12A (using pump 24 and open valve 70), to waste by open valve 72, or to the upper region of extraction vessel 14 (in this case, a two-phase extraction column) by closing open valves 70 and 72.

[0095] In the embodiment of Figure 5, extraction vessel 14 no longer includes gas layer 18 as shown in Figures 1 and 3. Rather, as also shown in Figure 6, the solvent forms an upper clarified solvent layer 130, and the clarified medium settles as a bottom layer 132, while the solvent and medium mixture forms an intermediate layer 134. The product-containing solvent is removed from vessel 14 (using pump 58) and can be recovered or further processed, as generally indicated by arrow 138. Solvent stream 140 can be returned to extraction vessel 14, for example, using distribution plate 142.

[0096] Another distributor plate, distributor plate 160, can be employed to introduce the media into extraction vessel 14. The media exiting the bottom of extraction vessel 14 can be directed to the bottom of column 12A using pump 24 and open valve 70.

[0097] In a specific embodiment, distributor plates 142 and / or 160 are positioned at or near the fluid injection point (inlet) to reduce disruption of the layer arrangement within the extraction vessel.

[0098] The extraction vessel 14 can be fitted with a sensor 167, which can be located at the bottom or wall of the vessel, or another suitable location. A sensor 181 can be added to monitor the solvent-medium mixture in the intermediate layer 134. In the bubble trap, sensors 183 and 168 monitor the air and medium, respectively.

[0099] Backpressure valves 144 and 146 control the flow through the extraction vessel 14 back to the reactor system and the flow through the solvent loop, respectively.

[0100] Figure 7 shows an arrangement in which a separation loop 39, e.g., a filtration loop (allowing for continuous product removal from the system), is attached to the solvent circulation described with reference to Figure 6. In this approach, a pump 58, controlled by a controller 200, pushes the product-containing solvent drawn from the top of the extraction vessel 14 through a membrane system 29, where the two are separated. Outlet 62 allows the product to be recovered or directed to further processing equipment. The solvent can be returned to the vessel 14, for example, through a distribution plate 142. Valves such as valves 190, 192, and 194 control the flow of the various streams.

[0101] The flow control device 44 is controlled based on input 202 received by the controller 200. Input 202 provides information to the controller 200 from a sensor in the extraction vessel 14, and input 216 provides data from the sensor 168 in the bubble trap. In response (output signals 210, 212, 214), the controller 200 controls the pump 24, opening valve 70, and motorized ball valves 31, 33, 35 essentially as described above.

[0102] Sensors such as sensors 164 and 169 (synthesis vessel), sensors 167 and 181 (extraction vessel), sensors 168 and 183 (bubble trap), and / or other sensors (e.g., sensors 171, 173, 175, 177) can be employed to enable controller 200 to monitor process parameters such as surface temperature, pressure, internal temperature, pH, DO, CO2, etc. In some embodiments, electrochemical or optical sensors are provided to track and quantify pH, CO2, O2, or carbon sources (e.g., acetate, lactate, succinate, glycerol, etc.) found within the bioreactor. More specifically, the sensors used can monitor biofilm health, system oxygenation, pH, and / or chemicals present in the feedstock or extraction solution. In some examples, controller 200 dynamically responds to sensor readings to alter flow rates, adjust nutrient or gas concentrations, or notify an operator of system failures or contamination events.

[0103] In most cases, the sensor is in contact with the media at the bottom of the reactor and can be embedded directly into the bottom or wall of the reactor. Sensors that float within the vessel (reactor or extraction vessel) or that circulate through the entire system or part of it are also available, and removable sensors are also available. Media can also be sampled by aspirating it into a secondary detection chamber. Split extraction and offline analysis can also be employed.

[0104] Information from sensors, pumps, valves, and reservoirs can be used to change or adjust the media composition, for example, by introducing more carbon or by treating with acid / base to adjust the pH. The controller 200 often operates autonomously, executing closed-loop system control algorithms.

[0105] In one embodiment of the invention, controller 200 is a computer (such as a single-board computer) or other digital control system for controlling process parameters and / or system components (e.g., pumps, valves, thermal systems, etc.) and monitoring sensors. The computer can process sensor inputs, send signals to thermal jackets to regulate temperature, open or close valves, or adjust pump speeds to control the introduction of raw materials and / or extraction solution, and alert the operator to poor performance, contamination, or blockages.

[0106] 8 is a block diagram illustrating a process 300 in which the controller 200 acts on several sensor data. In a loop 310, for example, a sensor (e.g., one of the sensors 164) detects the DO level in a vessel and sends the data to the controller 200 for evaluation or comparison 314 against a DO setpoint (set value) 316. A logic controller component 318 controls the flow control device 44 (e.g., adds air or reduces the air supply), thus adjusting the parameters of the entire continuously running process 400.

[0107] In some examples, the logical control component may be a thread executing on a processor, or may be a separate hardware system or subsystem.

[0108] In pH loop 330, a pH sensor (e.g., one of sensors 164) transmits data that is compared (step 334) to a setpoint 336. Acid or base can be added from reservoir 36 or 38 (by opening motorized ball valve 35 or 33 and activating pump 32 or 34) in response to logic controller component 338, thus optimizing process 400.

[0109] Flow loop 350 receives process flow readings from one or more sensors, such as, for example, sensor 169 (in one of the synthesis columns of FIG. 3A, FIG. 3B, or FIG. 5) or sensors circulating through various system components. As a possible result of comparison 354 to flow setpoint 256, logic controller component 358 can adjust a pump (e.g., pump 24) and thus the flow rate throughout process 400.

[0110] In a chemical analysis (optical density and / or ultraviolet (UV) wavelength) loop 370, a sensor, such as one of the wireless sensors 169 (or a spectrometer), provides chemical composition information for comparison (step 374) with a setpoint value 376. Based on input from a logic controller component 378, the pump 30 is turned on or off, thus optimizing the overall process 400. Additionally, the logic controller can set the circulation rate, vent rate, waste rate, solvent circulation rate, and solvent removal rate, each of which can be adjusted by this control loop.

[0111] Loop 390 depicts an optical density loop in which information from one of the sensors 169 (or spectrometers) provides data that is compared (step 394) to an optical density setpoint 396. Based on input from a logic controller component 398, the pump 30 can be opened or closed, and the media supply from the reservoir 28 can be opened or closed, thus optimizing the overall process 400. In some embodiments, the logic controller sets the circulation rate, aeration rate, waste rate, solvent circulation rate, and solvent removal rate, each of which can be regulated by this control loop.

[0112] As seen in the figures above, many embodiments of the present invention include reservoirs, conduits, pumps, valves, flow controllers, sensors, and computer control systems. Solutions are flowed between these components using fluid flow paths. Mixers, arrangements for gas introduction, output analysis, phase separators, and / or product purification modules may also be present. One or more vents for releasing pressure buildup may be provided in the synthesis module, the extraction module, or both.

[0113] For example, devices for controlling the introduction and removal of fluids include suitable containers for the liquids, fluid flow paths (tubes, pipes, joints, etc.), valves, pumping systems, flow sensors, computer control of fluid flow, etc. In some cases, a gravity-based configuration may eliminate the need for one or more pumps.

[0114] As discussed above, various devices (such as diffusers, spargers, distributor plates, etc.) can be employed to provide the desired conditions for introducing fluids (whether in liquid or gas form) into a vessel, e.g., a bioreactor and / or extraction vessel. In some embodiments, the system incorporates devices that promote mixing. Specific examples utilize such devices to promote fluidization and contact between materials within the synthesis vessel. The mixing device can be located where desired, often at the bottom of the reactor, e.g., at the inlet for supplying medium to the bioreactor.

[0115] Possible mixer or vortex generator designs are shown in Figures 9A-9D. Shown in these figures is a mixer or vortex generator 180 including a mixer body 182 made from a suitable material, such as, for example, plastic, metal, fiberglass, ceramic, or the like. The vortex generator 180 defines a plurality of flow channels 184. The flow channels may be formed by drilling, such as laser drilling, or other suitable techniques, as known in the art. In one example, the vortex generator 180 including the channels 184 is fabricated by 3D printing using PETG (polyethylene terephthalate glycol modified).

[0116] 9A and 9B, the channels are curved, while the designs of FIGS. 9C and 9D include straight channels 184. In all cases, the channels are not parallel to an axis perpendicular to the base 186 of the mixer body 182. Rather, they are inclined or angled, often pointing outward. Angled flow can increase mixing efficiency, increase cleaning rate, reduce or minimize clogging, and / or lower pumping requirements.

[0117] The vortex generator 180 can be sized according to the vessel or process requirements. In one example, the mixer body 182 has a height in the range of about 6 inches to about 12 inches and a maximum diameter in the range of about 6 inches to about 24 inches. The diameter of the mixer body 182 matches the inner diameter of the bioreactor vessel. Often, all of the channels 184 have the same shape, diameter, and / or slope angle. In one example, the channels 184 have a diameter in the range of about 0.25 inches to about 6 inches and a length in the range of about 6 inches to about 24 inches. The height of the mixer body 182 is determined by the distance required to form an appropriate fluid vortex for a particular pipe diameter.

[0118] Heating or cooling can be achieved by heat exchangers, heating tapes, thermoelectric elements, Peltier cooling elements, or other elements known in the art. In one example, the vessel is enclosed in a heating jacket. A temperature sensor can indicate the need for temperature adjustment, automatically turning the heating element on and off.

[0119] The synthesis / extraction cycle can be run continuously for weeks or months until the reactor becomes less productive or fouled. Semi-continuous or even batch modes can also be used in some cases.

[0120] (Genetically engineered microorganisms suitable for use in recirculating biofilm reactors) Generally, the microorganisms employed to form biofilms, either on slide supports or as self-aggregates, are selected based on their innate mechanisms for converting carbon sources into desired products (often hydrophobic chemicals). Examples of products that can be biologically produced using the techniques described herein include, but are not limited to, isoprenoids, carotenoids, retinoids, or other substances. In many embodiments, the organisms tolerate organic solvents, a property that allows for two-phase extraction of products while retaining functional biomass.

[0121] The recirculating biofilm reactors for producing isoprenoids, carotenoids, and retinoids described herein are comprised of suitable biofilm-forming, solvent-tolerant, hydrocarbon-forming organisms. Specifically, engineering hydrocarbon-destructive organisms (also known as hydrocarbon-degrading bacteria) with pathways to produce isoprenoids and retinoids can enable more efficient methods of biological isoprenoid and retinoid synthesis, for example, by direct extraction using organic solvents, using biofilms or biofilm reactors according to the invention.

[0122] These genetically engineered hydrocarbon-forming organisms are selected from prokaryotic or archaeal species capable of degrading and utilizing hydrocarbon compounds as carbon and energy sources. As described herein, these hydrocarbon-degrading organisms are used in methods for the biosynthesis of compounds called isoprenoids or terpenoids (terpenoids / isoprenoids are organic compounds derived from the 5-carbon compounds isoprene and isoprene polymers, terpenes). Hydrocarbon degradation and utilization are characteristic of hydrocarbon-degrading bacteria such as Marinobacter (Gauthier, 1992; Handley, 2013) and Pseudomonas (Isken, 1998).

[0123] Specifically included are hydrocarbon-forming microorganisms that have been genetically engineered to have increased biological activity compared to their wild-type organisms, for synthesizing / producing isoprenoids, carotenoids, or retinoids (e.g., where the product molecule / compound is, for example, retinal or retinol) at high yields in biofilms or biofilm bioreactors. Such genetically engineered microorganisms and their nucleic acid sequences are described in detail in U.S. Patent Application Publication No. 7 / 722,182 (hereinafter the '182 application), the teachings of which are incorporated herein by reference.

[0124] Hydrocarbon-destructive microorganisms suitable for use in the present invention possess two important characteristics: the ability to form stable biofilms (e.g., in a biofilm reactor) and tolerance to hydrophobic organic solvents. Examples of such microorganisms include Marinobacter species and Pseudomonas species. More specifically, the present invention encompasses biofilm-forming, hydrocarbon-forming microorganisms, such as the genus Marinobacter, particularly Marinobacter atlanticus, that are capable of forming biofilms and are tolerant to hydrophobic organic solvents. Such hydrocarbon-forming organisms are genetically engineered to contain one or more nucleic acid or amino acid sequence mutations / mutations in one or more (e.g., multiple) genes constituting the mevalonate and / or carotene synthesis pathways. Nucleic acids (DNA) encoding engineered operons and / or genes in the mevalonate pathway, β-carotene pathway, and retinol pathway are engineered, for example, via codon harmonization, for expression in Marinobacter. Exemplary genetically engineered microorganism DNA sequences are, for example, SEQ ID NOS:1-30, shown in Figures 1-30 of the '182 application. These sequences are also incorporated into the present application as set forth above.

[0125] Operons are responsible for gene expression and protein synthesis in prokaryotes. As described herein, an operon is a group (or region) of one or more related genes / gene sequences that are expressed to produce one or more biologically active enzymes / proteins. An operon consists of one or more gene sequences encoding a desired protein, a promoter sequence, and an operator sequence (the operator sequence can be located within the promoter sequence or as a separate sequence). The operon is responsible for transcription of DNA into messenger RNA (mRNA), which is then translated into the desired protein or enzyme product in prokaryotes.

[0126] As described herein, the present invention encompasses mutant operons / genes encoding enzymes / proteins with different biological activities than their corresponding wild-type (non-mutated) operons / genes. One example of a mutant operon / gene having increased biological activity over its wild-type counterpart is an increased yield of a desired product, such as a retinoid compound. Another biological activity described herein is the ability / ability to form more stable biofilms, for example, in a bioreactor. Yet another biological activity described herein is improved stability to organic solvents. One example of an increased biological activity of a mutant operon / gene over its wild-type counterpart is enabling the expression of genes and subsequent enzymes required in hydrocarbon-forming / oogenic biofilm-forming organisms.

[0127] For example, in some embodiments of the invention, the variant gene encodes a promoter sequence that has been engineered to increase expression of a desired enzyme, resulting in increased synthesis, yield, or stability of the desired retinoid compound. In another example, certain genes that make up an operon can be rearranged to result in a mutant operon that has a different biological activity than the wild-type operon, again resulting in increased synthesis, yield, or stability of the desired retinoid product.

[0128] More specifically, the present invention encompasses genetically engineered (also referred to herein as genetically modified or variant) hydrocarbon-forming microorganisms in which the operon genes encoding the enzymes required for the mevalonate production pathway have been codon-matched (also referred to herein as synthetic genes), the pathway being designed to direct the native acetyl-CoA pool of Marinobacter to the production of isoprenoids such as retinal or retinol, or to produce isoprenoids such as retinal or retinol from similar hydrocarbon-forming organisms. Such microorganisms can be further modified to contain mutated genes / operons in the β-carotene synthesis pathway designed to direct β-carotene to the production of retinal, retinoate, retinol, or retinyl esters.

[0129] As described herein, these enzymes (also referred to herein as mutant proteins or synthetases) from these pathways are engineered to improve performance, i.e., the nucleotide sequences encoding these modified enzyme sequences are optimized (e.g., by codon matching; the mutant enzyme differs in sequence and biological activity from its wild-type / naturally occurring counterpart by codon matching, mutation, insertion, or modification) to alter / modify (typically increase) the biological / catalytic activity of the enzyme compared to (i.e., relative to) the enzymatic activity encoded by the operon genes of a wild-type (unmodified) microorganism. Methods for genetically engineering microorganisms are described herein, and methods for assessing the enzymatic / biological activity of proteins are also described herein and are known to those of skill in the art.

[0130] The genetically engineered microorganism contains multiple genes for retinal or retinol synthesis specifically arranged in an operon, where the genes are arranged such that enzyme expression is optimized for the highest yield of product synthesis.

[0131] Specifically, described herein are organisms containing mutant genes in the mevalonate synthesis pathway (also referred to herein as the MVA pathway) that encode enzymes that convert acetyl-CoA to isopentenyl pyrophosphate (IPP), the building block of all isoprenoids (U.S. Patent No. 7,172,866, the teachings of which are incorporated herein by reference in their entirety.) Such mutant genes include nucleic acid S5, 6, 7, 8, 9, 10, or 15 (SEQ ID NOS: 5, 6, 7, 8, 9, 10, or 15), or sequences comprising about 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 5, 6, 7, 8, 9, 10, or 15 of the '182 patent application.

[0132] In some embodiments, the organism is also genetically engineered with one or more additional mutant genes introduced into the organism, such genes comprising the β-carotene pathway encoding enzymes that convert IPP to β-carotene. (See Figure 32.) Such mutant genes include nucleic acid sequences 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 27, 28, or 29 (SEQ ID NOS: 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 27, 28, or 29), or sequences comprising about 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to Sequences 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 27, 28, or 29 of the '182 patent application.

[0133] In one embodiment, the genetically modified organism comprises the introduction of a mutant blh gene encoding a 15,15'-dioxygenase (SEQ ID NO: 16 of the '182 patent application), wherein the introduction of the mutant blh gene results in the production of retinal and / or retinol.

[0134] Also included in the present invention is a mutant human retinol dehydrogenase 12 (RDH12) gene encoding a retinol dehydrogenase consisting of SEQ ID NO: 30, and its encoded protein consisting of SEQ ID NO: 30. The retinol dehydrogenase gene (RDH12) can consist of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18; or SEQ ID NO: 20 (SEQ ID NOS: 17, 18, or 30), or a sequence comprising about 80, 85, 90, 95, 96, 98, or 99% sequence identity to SEQ ID NO: 18, 19, or 20. The encoded RDH12 protein can also include a sequence comprising about 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 30, wherein the protein has aldehyde dehydrogenase biological activity comparable to mutant RDH12 activity. Further encompassed by the present invention is a genetically engineered organism, wherein the organism comprises the introduction of a mutant retinol dehydrogenase 12 (RDH12) gene (SEQ ID NO: 17, 18, or 20) that expresses a mutant RDH12 gene (SEQ ID NO: 30), where the introduction of the mutant RDH12 gene results in the conversion of retinal to retinol. These sequences are described in patent application '182.

[0135] Also included are genetically modified organisms, which contain the introduction of a mutant ybbO gene (SEQ ID NOS: 19 or 21), which introduction of the mutant ybbO gene results in the conversion of retinal to retinol.

[0136] Also included herein are genetically modified organisms comprising the variant operon sequences described herein. For example, an organism of the invention can comprise a variant upper mevalonate pathway operon consisting of SEQ ID NO:1 (SEQ ID NO:1) and / or a variant lower mevalonate pathway operon consisting of SEQ ID NO:2 (SEQ ID NO:2).

[0137] The genetically modified organisms of the present invention may further comprise a mutant operon sequence of the beta-carotene pathway, wherein the mutant operon sequence is selected from the group consisting of: SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:22; SEQ ID NO:23; SEQ ID NO:24 or SEQ ID NO:25 (SEQ ID NOS:3, 4, 22, 23, 24 or 25).

[0138] One specific embodiment comprises a genetically engineered organism, wherein the mutant mevalonate pathway gene(s) comprise a mutant operon of SEQ ID NO:1 and SEQ ID NO:2, and the mutant carotene pathway gene(s) comprise a mutant operon of SEQ ID NO:3 and SEQ ID NO:4. Another specific embodiment includes a genetically modified organism, wherein the mutant mevalonate pathway gene(s) comprise a mutant operon of SEQ ID NO:1 and SEQ ID NO:2, and the mutant carotene pathway gene(s) comprise a mutant operon of SEQ ID NO:22 and SEQ ID NO:26.

[0139] All nucleic acid and amino acid sequences described herein include sequences having about 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to the described sequences. Such sequences will have equivalent biological activity (essentially the same within several activity scales) as the described sequences when assessed using standard techniques.

[0140] In some embodiments of the present invention, these genes / operons are introduced into an expression vector, such as a plasmid, suitable / adapted for expression of the genes in a competent host cell. Specifically, the host described herein is a hydrocarbon-forming microorganism, specifically a Marinobacter species organism, more specifically a Marinobacter atlanticus microorganism. After introduction of the expression vector comprising the mutant gene of the present invention, under suitable conditions well known to those skilled in the art, the mutant gene is integrated / inserted into the genome of the host organism for expression. Techniques for introducing genes into cells are known to those skilled in the art. The present invention also encompasses host cells containing the vectors or plasmids described herein.

[0141] In some embodiments, the hydrocarbon-forming organisms produce isoprenoids, carotenoids, or retinoids from aromatic or aliphatic molecules. In still other embodiments, the hydrocarbon-forming organisms produce isoprenoids, carotenoids, or retinoids from short-chain fatty acids. In some of these embodiments, the short-chain fatty acids are lactates from dairy waste.

[0142] The present invention further encompasses biofilms comprising the genetically engineered hydrocarbon-destructive microorganisms described herein, and their use in the biofilm reactors described herein.

[0143] Also included in the present invention are methods for producing / synthesizing isoprenoids, carotenoids, and retinoids in biofilms or biofilm bioreactors using genetically engineered hydrocarbon-forming organisms, such as the Marinobacter species described herein or Pseudomonas species. In particular, the methods described herein include the production of the isoprenoids β-carotene, retinal, retinol, or squalane. Importantly, these methods involve the use of organic solvents (e.g., non-polar solvents) to extract isoprenoids without significant or substantial degradation of the isoprenoid product, resulting in higher yield, synthesis, and / or stability of the desired product. For example, the synthetic biofilms and biofilm bioreactors described herein and methods for synthesizing isoprenoids and retinoids can include using hexane, dodecane, or oleic acid as the extraction solvent for the desired product, which can be determined by techniques known to those skilled in the art.

[0144] In some embodiments, the extraction solvent specifically contains an antioxidant or encapsulating agent to prevent oxidation or degradation of the product. For example, the extraction solvent can contain molecules such as cyclodextrin to stabilize the product molecules. In other embodiments, the product is simultaneously extracted and encapsulated in liposomes using lipid molecules dispersed in solvent microdroplets.

[0145] In a specific embodiment of the present invention, the method involves the production / synthesis of an isoprenoid for use as an ingredient or component in a cosmetic formulation, wherein the cosmetic ingredient is substantially free of contaminants that may be found in isoprenoids produced by conventional methods. For example, retinol products are often used in cosmetic creams and ointments manufactured for human use, so the purity of retinol is extremely important. The purity of the final product, i.e., the degree of contamination or lack of contamination, can be assessed using methods known to those skilled in the art. Specifically, the product of the method described herein is a cosmetic ingredient suitable for veterinary or human use (e.g., retinol in a cosmetic facial cream), and the extraction solvent of the method is a component or suitable additional ingredient of a cosmetic preparation / formulation. For example, the cosmetic ingredient can be an emollient, and in one embodiment, the emollient is squalane.

[0146] Embodiments of the present invention are illustrated in the following non-limiting examples.

[0147] Example 1: Synthesis of retinol in a bioreactor The bioreactors are stacked with 2 x 6 inch (in) diameter x 20 inch high sight glasses and connected to each other using tri-clamp fittings to form a compound vessel. The synthesis vessel is connected to a third 6" x 20" sight glass, which serves as the extraction vessel, using 0.5" stainless steel tubing from the outlet port at the top of the sight glass. An air-driven pump connects the bottom of the extraction vessel to the inlet on the perimeter of the bottom of the synthesis vessel. The bottom of the synthesis vessel has a central inlet for adding carbonate stones. The reactor contains ~30 L of medium and is loaded with 1-20 lbs of glass beads, which serve as a solid support for the biofilm. These beads can be pre-seeded with a liquid culture of retinol-producing cells or planktonic retinol-producing cells and introduced into the bioreactor to seed biofilm growth. The reactor is circulated for 12-24 hours to allow biofilm formation, at which point 100 mL to 10 L of hexane, heptane, or dodecane is added to the extraction vessel. The biofilm suspension is circulated through the bioreactor at a rate of 0.1-10 L / min. The product is continuously extracted into the solvent phase and collected from the separation module.

[0148] Example 2: Use of a Vortex Generator Inducing angular flow in a bioreactor can promote better mixing at lower pumping speeds. To achieve this angular flow, vortex generators are 3D printed out of polyethylene terephthalate glycol (PETG) and placed at the bottom of each column of the bioreactor. Vortex generators can also be printed out of PP or other materials and can be steam sterilized in situ. Initial experiments have confirmed that these inserts improve reactor flushing rates by more than an order of magnitude.

[0149] (Example 3: Wireless sensor for controlling bioreactor operation) Small, pill-sized wireless sensors (e.g., "A Threshold-Based Bioluminescence Detector With a CMOS-Integrated Photodiode Array in 65 nm for a Multi-Diagnostic Ingestible Capsule," Liu, Q..., Jimenez, M., Inda, ME, Riaz, A., Zirtiloglu, T., Chandrakasan, AP, Lu, TK, Traverso, G., Nadeau, P. and Yazicigil, RT, 2022. A 65 nm Threshold-Based Bioluminescence Detector with a CMOS-Integrated Photodiode Array for a Multi-Diagnostic Ingestible Capsule. IEEE Journal of Solid-State Circuits, 58(3), pp. 838-851.) are distributed throughout the bioreactor system. The sensors are either fixed in place within the reactor system or actively circulate along the flow path with the cells within the reactor system. These sensors measure the state of the bioreactor at a specific location and moment using optical or electrochemical signals, as described by Liu et al. Specific optical or electrochemical signals correspond to measurements of the levels of feedstock components such as pH, carbon dioxide, oxygen, glycerol, lactate, acetate, other organic acids, glucose, and other sugars. The electrochemical fingerprints (cyclic voltammetry and / or electrochemical impedance) from these sensors provide a snapshot of the bioreactor state (e.g., cell density, cell health, available feedstock).

[0150] At the start of the process, the reactor is filled with sterile medium and inoculated. Air flow to the columns is initiated, and oxygen saturation is monitored by different sensors. Wireless sensors within the bioreactor transmit instantaneous oxygen saturation to a wireless receiver connected to a microprocessor. This microprocessor uses a PID algorithm to determine the appropriate airflow setting for each column and sends a signal to a mass flow controller or proportional valve (I / P or E / P converter) that converts a variable current or voltage signal into a proportional compressed air output. Dissolved oxygen typically ranges from 1 to 9 mg / L, with specific setpoints related to the desired growth rate and redox environment. Cell density is continuously measured at different points within the reactor using either electrochemical impedance measurements, which change the dielectric properties of the reactor environment as biomass increases, or optical density measurements, which change due to increased scattering associated with increased cell density. These data are wirelessly transmitted to a microprocessor-based controller, which processes the information, along with information about oxygen consumption, and controls the introduction of fresh feedstock into the reactor. To introduce fresh ingredients, the microprocessor sends a digital signal to open an ingredient control valve, such as a ball valve or solenoid valve. The microprocessor then sends a digital signal to operate a pump at a flow rate specified by the algorithm to introduce the additional ingredient.

[0151] Once the desired biomass density is achieved, the microprocessor switches to continuous operation mode. In this mode, the feed is continuously introduced at a specified rate. Sensors are constantly monitored along the reactor. Electrochemical measurements taken at each column are sent to the microprocessor, which uses these measurements to assess cell health and productivity. Depending on the electrochemical signature, the reactor performs controlled dilution within the reactor by (1) increasing or decreasing the feed rate, (2) adjusting the pH, (3) introducing nutrient feeds, (4) modifying the airflow, (5) changing the circulation flow rate, or (6) sending some medium to waste and introducing fresh feedstock. If measurements indicate contamination or are outside the normal range of measurements, the microprocessor will notify the operator by email or text message, or by illuminating a warning light or sounding an alarm to indicate the problem.

[0152] Sensors installed in the extractor optically or electrochemically measure the product concentration and quality. Signals are sent wirelessly to a microprocessor, which analyzes the data. For example, if retinol is the target product, an optical absorption spectroscopy sensor can measure absorbance at light wavelengths of 325 nanometers (nm), 350 nm, and 368 nm. The microprocessor controller uses the ratio of these signals to determine the relative concentrations of retinol, retinal, and retinoic acid. If the retinoic acid concentration is too high, the microprocessor can adjust the oxygen concentration in the final reactor stage to create a more reducing environment or increase the overall flow rate to ensure product removal is more frequent. If the total retinoid concentration is too high, the sensor can change the flow rate from the extractor to the nanofiltration system.

[0153] Example 4: Parallel operation of multi-column bioreactors for producing hydrophobic proteins For example, the bioreactor system depicted in Figure 1 is configured with 10 columns operating in parallel and feeding a single extractor. Culture medium is introduced into the reactor and inoculated with a Marinobacter strain engineered to overexpress and excrete hydrophobic proteins. Cells are grown to a cell density of between 5 and 30 OD600 and maintained at this density using a microprocessor that monitors sensors to control feeding, aeration, the introduction of nutrients and acid-base solutions for pH balancing, and media waste. Sensors in each column are continuously monitored to ensure balanced flow throughout the system, consistent cell density and feed rate, and to identify any declines in cell vitality or potential contamination events.

[0154] Cells, biofilm, and media (feedstock) are all continuously pumped upward through each column and circulated through an extractor containing an organic solvent. The media falls through the organic solvent by gravity and is pumped back into the reactor. Maintaining media balance within the extractor is important to ensure the solvent remains within the reactor. This is achieved by continuously monitoring the solvent level within the reactor using sensors and balancing the media level. During the initial biomass accumulation and protein maturation phases at the start of operation, a bypass valve is used to allow the extractor to skip a portion of the extract or media through the extractor. A level height sensor, such as an optical line break sensor in the partial air extractor, monitors the occurrence of foam. If the media / solvent / foam level becomes too high, the line break sensor signals a microprocessor controller to stop or terminate the pump and / or aeration, allowing foaming to decrease. As the cells produce protein, the protein is excreted from the cells and accumulates in the solvent layer as the cells and media are washed through the extractor. The solvent is continuously circulated by the controller through a filtration membrane, allowing size-selective extraction of the protein of interest. In one embodiment, a multi-stage membrane process is used, with the first membrane being smaller in size than the target molecule and the second membrane being larger in size than the target molecule. In the first stage, the permeate is returned to the reactor by the controller 200, and the retentate is sent to the second stage, which concentrates the protein in a solvent. In the second stage, the protein passes through the membrane, removing cellular debris and larger proteins, while the retentate is returned to the reactor. The product can be recovered for further purification, such as affinity tag purification or other chromatographic separation methods.

[0155] The process is designed to run continuously until either enough protein is produced or the productivity of the cells declines.

[0156] Example 5: Serial operation of multi-column bioreactors for retinoid production A bioreactor system such as that depicted in Figure 3A or 3B is configured with 10 columns operating in series and feeding a single extractor. This system can alternatively be configured with an air trap before the extractor, as shown in Figure 5, to relieve pressure from the system before extraction and reduce emulsification in the extractor.

[0157] Figures 3A, 3B, and 5 show bioreactor configurations with paired columns (one column in each pair is aerated and the other is not). In some embodiments, the non-aerated column is narrower than the aerated column and acts like a pipe connecting one reactor to the next.

[0158] During process startup, the reactor is filled with sterile medium and inoculated with a retinoid-producing strain of Marinobacter. Cells can be optionally circulated through the reactor without entering the extractor during startup to achieve the desired biomass. A biomass sensor monitors the biomass, and once the target biomass (typically at least 1-5 OD600) determined by the controller 200 is achieved, the cells are circulated through the extractor. During each cycle through the columns, the feedstock is introduced by the controller 200, and the cells convert the feedstock into products as they pass through each column, ultimately reaching the extractor. In the extractor, the aqueous solution is dispersed in an immiscible solvent and falls by gravity through the solvent, extracting the products. With each cycle, a portion of the aqueous phase is removed from the reactor by the controller 200 to allow for the addition of more feedstock and maintain a constant liquid level.

[0159] To achieve optimal operation, the residence times and feed rates of the cells in the series of columns can be balanced to achieve a target feed consumption during the cycle through the reactor—for example, 50% of the feed rate at a flow rate of 1 L / min, or 90% of the feed rate at a flow rate of 0.1 L / min. The microcontroller can monitor sensor signals in real time to assess product production via optical signals in the extractor and quantify the feed consumption of each column based on wireless sensors in each column. The microcontroller can then adjust the feed rates and flow rates to achieve the target feed consumption during each cycle.

[0160] The biosynthetic pathway for retinol production involves 14 steps driven by 13 genes, the final step of which requires reducing conditions. The multi-column design of the bioreactor system allows us to tailor reactor circulation to the kinetics of retinoid production and operate the reactor in distinct zones: an oxygen-rich zone for the initial production of acetyl-CoA, key coenzymes such as NADH / NADPH, and early pathway precursors, and a more reductive zone where final product conversion occurs. Sensors in each column measure oxygen levels and electrochemically characterize the local redox environment. This signal is analyzed by a microprocessor, which can increase or decrease the air pressure in individual columns to create the desired oxygenation profile.

[0161] Once the retinoid product is extracted into the solvent, it is circulated through a nanofiltration system to concentrate and size-selectively purify retinol from other cellular components. With the appropriate choice of nanofiltration membrane, it is possible to selectively purify retinol, retinoic acid, or retinal.

[0162] While the present invention has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as encompassed by the appended claims. [Explanation of symbols]

[0163] 10 system, 12 synthesis vessel (column), 12a, 12b, 12c, 12e, 12g, 12j, 12L column, 14 extraction vessel, 16 production module, 18 gap (gas layer), 20 solvent layer, 22 medium reservoir (media layer), 24 pump, 26 manifold, 28 reservoir, 29 membrane system, 30 pump, 31 motorized ball valve, 32 pump, 33 motorized ball valve, 34 pump, 35 motorized ball valve, 36 reservoir, 38 reservoir, 39 loop (separate arrangement), 40 gas source, 42 manifold, 44 flow control device, 46 manifold, 48 condensation column, 50 carbon filter, 52 vent, 54 vent filter, 56 conduit, 58 pump, 60 conduit, 62 outlet, 64 waste, 66 Pump, 70, 72, 74, 76 Open valve, 80 Conduit, 82 Conduit, 92 Conduit (line), 94 Manifold, 98 Conduit (loop), 99 Valve, 100 Cell suspension, 102 Extraction vessel inlet, 110 Product, 112 Outlet, 120 Bubble trap, 122 Upper region, 124 Lower region, 130 Upper clarified solvent layer, 132 Bottom layer, 134 Middle layer, 138 Arrow, 140 Solvent flow, 142, 160 Distribution plate, 144, 146 Back pressure valve, 164 Sensor, 165 Sensor (sensor array), 167, 168 Sensor (sensor array), 169, 171, 173, 175, 177, 181 Sensor, 180 Vortex generator (mixer), 182 Mixer body, 183 Sensor, 184 Multiple flow paths (channels), 186 Base, 190, 192, 194 Valve, 200 Controller, 202, 204, 216 Input, 208, 210, 212, 214 Output signal, 256 Flow setpoint, 300 Process, 310 Loop, 314 Comparison, 316 DO setpoint, 318 Logic controller components, 330 pH loop, 334 Step, 336 Setpoint, 338 Logic controller components, 350 Flow loop, 354 Comparison, 358 Logic controller components, 370 Loop, 374 Step, 376 Setpoint value, 378 Logic controller components, 390 Loop, 394 Step, 396 Optical density setpoint, 398Logical controller component, 400 processes.

Claims

1. A bioreactor system comprising: a first vessel containing a substrate coated with a biofilm comprising hydrocarbon-clastic and / or oleaginous organisms, said substrate being maintained in suspension; a second vessel containing an organic solvent for extracting products from the biofilm; and a circulation system for transferring liquid from the first container to the second container and maintaining the liquid in suspension in the first container; A bioreactor system comprising:

2. 10. The bioreactor system of claim 1, wherein a separation module comprising a chemically selective or size-selective membrane is used to separate product from the extraction vessel and recycle solvent to the extraction vessel.

3. 10. The bioreactor system of claim 1, wherein sensors are used to monitor process conditions and adjust media components by introduction of reagents from the secondary reservoir.

4. 10. The bioreactor system of claim 1, further comprising sensors for monitoring temperature, pressure, dissolved oxygen, pH, or chemical constituents.

5. 10. The bioreactor system of claim 1, further comprising a reservoir, a conduit, a pump, a valve, and / or a flow controller.

6. 10. The bioreactor system of claim 1, further comprising a computer control system.

7. 10. The bioreactor system of claim 1, wherein the first vessel is a synthesis vessel within a production module comprising a plurality of synthesis vessels.

8. 8. The bioreactor system of claim 7, wherein the plurality of vessels are arranged in series or parallel.

9. 8. The bioreactor system of claim 7, wherein the plurality of synthesis vessels feed one or more extraction vessels and phase separation devices.

10. 10. The bioreactor system of claim 1, wherein the second vessel is an extraction vessel that is part of an extraction module, the extraction module comprising one or more extraction vessels.

11. 10. The bioreactor system of claim 1, wherein the first vessel comprises a device for generating vortices and / or angular flows.

12. 10. The bioreactor system of claim 1, wherein the first vessel comprises a diffuser for introducing a gas into the first vessel.

13. 10. The bioreactor system of claim 1, wherein the second vessel receives fluid through a distribution plate.

14. 10. The bioreactor system of claim 1, wherein the substrate is a solid support or a biofilm mass.

15. 15. The bioreactor system of claim 14, wherein the microorganisms in the biofilm mass are live or dead microorganisms.

16. 10. The bioreactor system of claim 1, wherein the carbon source provided to the first vessel comprises an ethanol plant by-product, a food waste by-product, an agricultural waste by-product, or any combination thereof.

17. 10. The bioreactor system of claim 1, further comprising at least one wireless sensor.

18. 20. The bioreactor system of claim 17, wherein the at least one wireless sensor circulates within the bioreactor system.

19. 1. A method for synthesizing a hydrophobic product, comprising: providing a bioreactor containing a microorganism capable of producing a hydrophobic product; introducing a water-soluble raw material into the bioreactor and allowing the microorganism to convert the water-soluble raw material into the hydrophobic product; contacting the microorganism with a solvent effective to extract the hydrophobic product; and recovering the hydrophobic product from the solvent; A method comprising:

20. 1. A process for synthesizing a hydrophobic chemical, the process comprising: suspending biofilm particles in a recirculating bioreactor, said biofilm comprising microorganisms capable of producing said hydrophobic chemical; introducing an aqueous feedstock into said bioreactor; the microorganism converts a carbon source provided in the aqueous feedstock to the hydrophobic chemical; and extracting said hydrophobic chemical into a hydrophobic solvent; A process involving:

21. 21. The process of claim 20, further comprising separating the hydrophobic chemical from the hydrophobic solvent.

22. 22. The process of claim 21, wherein the hydrophobic chemical is separated from the hydrophobic solvent by a chemically selective or size selective membrane.

23. 22. The process of claim 21, wherein the solvent is recycled.

24. 21. The process of claim 20, wherein the process is carried out continuously.

25. 21. The process of claim 20, further comprising introducing a gas into the recirculating bioreactor.

26. 21. The process of claim 20, wherein the aqueous feedstock and biofilm particles are fed from a bottom layer within an extraction vessel.

27. 21. The process of claim 20, wherein the process is computer controlled.

28. 21. The process of claim 20, further comprising monitoring process parameters.

29. 21. The process of claim 20, further comprising adjusting process parameters in response to input from one or more sensors.

30. 21. The process of claim 20, further comprising wireless communication between at least one sensor and a controller.

31. 21. The process of claim 20, wherein the process further comprises circulating at least one wireless sensor through the bioreactor system.

32. 21. The process of claim 20, further comprising generating vortices and / or angular flows within the recirculating bioreactor.

33. 21. The process of claim 20, wherein the fluid is introduced through a diffuser or distribution plate.

34. 21. The process of claim 20, wherein the feedstock comprises an ethanol plant by-product, a food waste by-product, an agricultural waste by-product, or any combination thereof.

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