Biofilm Bioreactor

Abstract

Described herein are biofilm bioreactors for synthesis at the interface between two liquids and methods of using such bioreactors for the bioconversion of feedstocks into chemical products. Extraction of such products is also contemplated.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 978,428, filed February 19, 2020, which is incorporated herein by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT The U.S. Government has certain proprietary rights in this invention. For a license, contact the Technology Transfer Office, U.S. Naval Research Laboratory, Code 1004, Washington, DC 20375, USA; +1.202.767.7230; techtran@nrl.navy.mil, Reference NC112,479. [Background technology]

[0003] Fermentation and algae bioreactors are used to produce bioethanol, biodiesel, and other products. In industrial-scale biomanufacturing, products are typically produced in suspended cell reactors during so-called logarithmic-scale growth, where the biomass undergoes periodic doubling. Downstream processing, such as distillation or solvent-phase extraction followed by dehydration, may be required to extract the final product.

[0004] Biofilm bioreactors are a type of bioreactor that use cells immobilized on a solid scaffold for product synthesis. Various biofilm bioreactors have been developed for different applications, particularly wastewater treatment. These include packed-bed reactors (Holliday et al., 1978), which use a granular matrix packed into a column as a scaffold; two-phase partitioning bioreactors (Daugulis et al., 1997), in which a non-aqueous phase is added to a microorganism-containing aqueous phase to promote the continuous and gradual migration of hydrophobic substrates into the aqueous phase; and extractive membrane bioreactors (Livingston, 1998), in which a membrane separates the aerated biomedia from the feedstock (typically wastewater). Volatile organic compounds diffuse across the membrane and are degraded by the biofilm growing on the biomedia side of the reactor.

[0005] Biofilm bioreactors utilize naturally occurring consortia of microorganisms and have been applied in applications such as the removal of toxic substances from wastewater or the production of products from gaseous sources such as carbon monoxide.

[0006] Hydrocarbon-degrading organisms, such as Marinobacter spp., can form biofilms and ingest alkanes and aromatic compounds (Gauthier et al., 1992; Lattuati et al., 2002; Ennouri et al., 2017; Mournier et al., 2018; Arroyo et al., 2013), converting them into products such as wax esters, which have commercial value as lubricants. Organisms capable of accumulating lipids, such as wax esters, are commonly referred to as oleaginous organisms. Evidence of extracellular neutral lipids produced by Marinobacter spp. was reported by Nakano et al. in 2012. These pathways can also be repurposed to produce other products, such as phloroglucinol (Meyer et al., 2019).

[0007] Additionally, organic solvents have been utilized as a means of routinely extracting products from planktonic algae such as B. brunaii or D. salina in a process known as milking (Hejazi et al., 2004; Jackson et al., 2018; Hejazi et al., 2005; Sayre, 2009). Milking techniques allow for the non-destructive removal of hydrocarbons from the bioreactor into the solvent phase, which can dramatically reduce the amount of downstream processing required. Several systems have been developed for product extraction from milking. One mixer-settler system operates in a single, continuous stage. In the mixing segment, the solvent and medium are agitated to provide contact between the solvent and the cells. The solvent-culture mixture is then transferred to the settling segment, where an upper solvent phase containing the product and a lower aqueous phase containing the cell fraction are separated. A second system, a column extractor, passes droplets of organic solution through a medium solution to extract the product. In both cases, the cells are planktonic, cultured in separate containers, and periodically transferred to an extraction system for removal.

[0008] Key challenges in biofilm-based processes include mass transfer limitations, control of biofilm formation, and challenges in product extraction. Summary of the Invention

[0009] In a first embodiment, the bioreactor includes a vessel containing a solid support suitable for biofilm growth, a vessel for containing a feedstock solution in contact with the biofilm, and an extraction solution configured to be delivered to the vessel containing the biofilm. The bioreactor is configured to host biofilm-forming microorganisms that are tolerant of both solutions and capable of carrying out biotransformation. The feedstock provides a chemical source that can be biologically converted into a desired product. The extraction solution, upon contact with the biofilm, allows for removal of the desired product. The biofilm-forming microorganisms should be tolerant of both solutions and capable of carrying out biotransformation of the feedstock into the product. The microorganisms preferably undergo stable growth in the biofilm. The product can be extracted in a continuous or periodic (batch) manner through exposure to the extraction solution.

[0010] For example, a bioreactor system may include a column comprising a substrate coated with a biofilm comprising hydrocarbon-degrading organisms and / or oleaginous organisms, a first reservoir operably connected to deliver a first liquid comprising a feedstock to the column, a second reservoir operably connected to deliver a second liquid to the column, and a phase separator operably connected to receive both liquids from the column, wherein the biofilm is effective to convert the feedstock to a desired product and the phase separator is effective to separate the product from one or both of the liquids.

[0011] In a further embodiment, the bioreactor resembles a packed-bed reactor, in which a biofilm of hydrocarbon-degrading and / or oleaginous organisms is grown on beads packed into a column. During operation, an aqueous feedstock flows through the column, providing both nutrients for the biofilm and chemicals that are converted by the organisms into desired products. Periodically, a bolus of organic solvent is introduced into the reactor to collect the products.

[0012] In another embodiment, the bioreactor resembles an extractive membrane bioreactor. In this embodiment, a porous scaffold is used to grow a high-surface-area biofilm of hydrocarbon-degrading and / or oleaginous organisms. The porosity can be designed so that membrane growth completely occupies the pores, forming a barrier layer between the two solutions. Optionally, the membrane can be chemoselective to a particular product, and biofilm grows only on the feed side of the bioreactor.

[0013] In some embodiments, the membrane may be in a tubular geometry and contained in a column. Such a coaxial geometry would provide the ability to continuously introduce feedstock on one side of the biofilm and perform extraction on the other side of the biofilm.

[0014] In yet another embodiment, a method of bioconversion includes providing a bioreactor of any of the preceding embodiments (or combinations thereof), supplying the bioreactor with a feedstock and an extraction solution, allowing the viable biofilm-forming microorganisms and the bioreactor to convert the feedstock into a product, and extracting the product.

[0015] For example, a method for converting a hydrophobic feedstock to an aqueous phase product may include providing a bioreactor containing live microorganisms capable of producing a water-soluble product, circulating a nutrient medium through the bioreactor to establish a biofilm of the microorganisms, introducing both an aqueous solution and the hydrophobic feedstock into the bioreactor, where the aqueous solution provides supplemental nutrients to the microorganisms, allowing the microorganisms to convert the hydrophobic feedstock to the product, and recovering the product from the aqueous solution.

[0016] As another example, a method for converting an aqueous feedstock to a hydrophobic product may include providing a bioreactor containing a microorganism capable of producing the hydrophobic product, introducing a water-soluble feedstock into the bioreactor and allowing the microorganism to convert the feedstock to the hydrophobic product, contacting the microorganism with a solvent effective to extract the product, and then recovering the product from the solvent. [Brief explanation of the drawings]

[0017] [Figure 1] 1 shows an end-to-end schematic of an exemplary bioreactor system for converting crude feedstock into purified products. The components shown include various flow controllers, each labeled 101, vent 102, waste 103, feedstock-containing vessel 104, supplemental nutrients 105, product extraction solvent (aqueous or non-aqueous) 106, pump 107, mixer 108, aerator 109, heat-jacketed bioreactor 110, acid / base 111, sensors 112, output analysis 113, phase separator 114, product purification 115, product storage 116, recirculation reservoir tank 117, gas source 118, and computer 119. [Figure 2] Figure 2 shows a cross-sectional view of an embodiment of a biofilm bioreactor with a packed-bed design in which solvent droplets circulate through the reactor to extract products. In this diagram, 201 represents the feedstock (e.g., fatty acids, oils, waste products, and combinations thereof), 202 represents the product (which is initially obtained in the solvent layer and may include, for example, lubricants, fuels, fatty acids, vitamins, etc.), 203 represents the organic solvent droplets that function to extract the product, 204 represents support beads (e.g., incorporating Marinobacter sp.) with biofilm, and 205 represents water (which may be recycled). A close-up view shows individual support beads with biofilms carrying out the bioconversion. [Figure 3] 3 shows a cross-sectional view of an embodiment of a biofilm bioreactor in which a biofilm intercalates or coats a membrane between the feedstock solution and the extraction solution. In this diagram, 301 represents the feedstock, 302 represents the product (in the solvent phase), 303 represents the organic solvent that functions to extract the product, and 304 represents the microbial (e.g., Marinobacter spp.) biofilm intercalating in membrane 305. A close-up view shows the biofilm within the membrane. [Figure 4]Figure 4 shows a cross-sectional view of an embodiment of a biofilm bioreactor in which a biofilm is growing on one side of a membrane that may be optionally chemoselective. In some embodiments, the membrane may be molecularly selective. Here, 401 represents the feedstock, 402 represents the product (in the solvent phase), 403 represents an organic solvent that functions to extract the product, and 404 represents a microbial (e.g., Marinobacter) biofilm on the surface of membrane 405. The enlarged view details the biofilm on the membrane. [Figure 5] Figure 5 shows an example of the conversion of a hydrophobic feedstock (e.g., petroleum) to water-soluble products. In this diagram, 501 represents the hydrophobic feedstock (e.g., petroleum, alkanes, aromatics, etc., including combinations thereof), 502 represents the water-soluble products such as acetate, lactate, etc., 503 represents the membrane, and 504 represents the aqueous phase collection. [Figure 6] Figure 6 shows an example of the bioconversion of an aqueous mixed feedstock into solvent-soluble (hydrophobic) products, where 601 represents the aqueous feedstock (e.g., short chain fatty acids, sugars, liginol, etc., including combinations thereof), 602 represents hydrophobic products such as wax esters, lubricants, styrene, etc., 603 represents the membrane, and 604 represents the organic solvent for extraction. [Figure 7] Figure 7 shows an example of aqueous-to-aqueous bioconversion where a biofilm was grown on a selective membrane, where 701 represents aqueous feedstock (e.g., short chain fatty acids, sugars, liginol, etc., including combinations thereof), 702 represents water-soluble products such as acetate, lactate, etc., 703 represents the membrane, and 704 represents the aqueous extract. [Figure 8] FIG. 8 is a diagram of the wax ester biosynthetic pathway and potential conversion to alternative products. [Figure 9A] Figures 9A and 9B show exemplary aromatic / alkane pathways (adapted from Evans et al., 2018). [Figure 9B] Figures 9A and 9B show exemplary aromatic / alkane pathways (adapted from Evans et al., 2018). [Figure 10]Figure 10 shows transmission electron micrographs of planktonic and biofilm cells of M. hydrocarbonclasticus SP17 grown on lactate, hexadecane, or paraffin (adapted from Branchu et al., 2017). Bright inclusions are associated with lipid accumulation. Bars represent 1 μm. DETAILED DESCRIPTION OF THE INVENTION

[0018] definition Before describing the present invention in detail, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not necessarily intended to be limiting. Although many methods, structures, and materials similar, modified, or equivalent to those described herein can be used in the practice of the present invention without undue experimentation, the preferred methods, structures, and materials are described herein. In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set forth below.

[0019] As used herein, the singular forms "a," "an," and "the" do not exclude plural referents unless the content clearly dictates otherwise.

[0020] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0021] As used herein, the term "about," when used in conjunction with a stated numerical value or range, means a numerical value that is slightly more or slightly less than the stated value or range, within ±10% of the stated numerical value.

[0022] As used herein, the term "oleaginous organism" refers to an organism capable of metabolizing carbon substrates into lipids.

[0023] As used herein, the term "hydrocarbonoclastic organism" refers to an organism capable of metabolizing aliphatic and / or aromatic hydrocarbons.

[0024] Overview The bioreactor is configured to provide a high-surface area solid scaffold suitable for the growth of oleaginous and / or hydrocarbon-degrading organisms, such as Marinobacter spp., optimize mass transfer, and allow for continuous extraction of products between two liquid phases. This is expected to enable more efficient bioconversion of hydrophobic (e.g., petroleum) feedstocks to aqueous products, or aqueous feedstocks to hydrophobic products (e.g., lubricants).

[0025] Bioreactors, as described herein, can take on numerous configurations, including packed-bed and extractive membrane types. For some embodiments of bioreactor design, many columns can be paralleled to increase or decrease product production while maintaining optimal mass transfer and fluid flow within the reactor. This modular design facilitates the removal and replacement of individual columns as they become necessary (e.g., in the event of fouling) with minimal impact on overall bioreactor productivity.

[0026] For example, a bioreactor system may include a combination of some or all of the following features: vessels for holding feedstock, supplemental nutrients, and extraction solutions; pumps and flow controllers for controlling the introduction of fluids; fluid channels that allow solutions to be delivered to the bioreactor; valves that allow control of individual fluid streams; a fluid mixer; an aeration device for the introduction of dissolved gases; a bubble trap for the removal of gas bubbles; a thermally jacketed bioreactor chamber(s) used for product synthesis; a system for introducing acid or base to adjust pH; and controls for controlling pH, dissolved gases, planktonic cell density, biofilm cell density, temperature, and flow rate. sensors for monitoring reaction conditions including conductivity, pressure, bioavailable nitrogen species, dissolved organic carbon, organic acids, genetic material, and / or minerals; connection of the output to a system for chemical analysis (HPLC, GC-MS, ICP, etc.); a phase separator for separating the solvent and aqueous phases; a module for product purification; a product storage vessel; fluid paths and associated pumps and valves that allow recirculation of the product extract solution into the bioreactor; fluid paths and associated pumps, valves, and reservoirs that allow recirculation of the feed solution into the bioreactor; and a computer control system operably connected to monitor the sensors and operate the valves.

[0027] The biofilm-forming microorganism can be one or more of a number of different organisms. Preferably, it can be hydrocarbon-degrading and / or oleaginous. For example, naturally occurring Marinobacter sp. can be used to convert aromatic compounds, alkanes, short-chain fatty acids, or other feedstocks into wax esters. In an alternative embodiment, engineered Marinobacter sp. can be used to redirect carbon flux from fatty acid ester production to the production of alternative products.

[0028] Organisms suitable for use in this technology include Aestuariibacter aggregatus WH169, Aestuariibacter OTU3, Acinetobacter baylyi ADP 1, Acinetobacter sp. H01-N, Alcanivorax borkumensis SK2, Alcanivorax jadensis T9, Alkanidiges, Alteromonas sp. TK-46 strain, Alteromonas macleodii, Alteromonas macleodii NBRC 102226, Alteromonas macleodii 107, Arcobacter UTICA-S4D1, Arcobacter MARC-MIP3H16, Bacillus subtilis, Colwellia, Cycloclasticus, Escherichia coli, Escherichia fergusonii, Escherichia fergusonii ATCC 35469, Halomonas, Halomonas sp. TG39 strain, Marinobacter adhaerens, Marinobacter algicola, Marinobacter alkaliphilus, Marinobacter antarcticus, Marinobacter aquaeolei, Marinobacter aquaeolei VT8, Marinobacter atlanticus, Marinobacter gudaonesis, Marinobacter hydrocarbonoclasticus, Marinobacter hydrocarbonoclasticus SP17, Marinobacter maritimus, Marinobacter sahuginis, Marinobacter santoriniesis, Marinobacter squalenivorans, Marinobacter sp.Examples of isolates include, but are not limited to, UTICA-S1B6, Mortierella isabellina, Oceanospirillales, Oleispira, Pseudoalteromonas, Pseudoalteromonas sp. strain TG12, Pseudomonas aeruginosa, Pseudomonas aeruginosa LST-03, Pseudomonas aeruginosa PST-01, Pseudomonas putida, Pseudomonas putida IH-2000, Pseudomonas putida DOT-TIE, Pseudomonas putida S12, Rhodococcus opacus PD630, Rhodocuccus ruber, Saccharomyces cerevisiae, Thalassospira sp. TK-13, Thalassospira xanhenis P-4, Thalassolitus, Yarrowia lipolytica, and isolates from Bacosa et al., 2018. Additional organisms suitable for use in the bioreactor include Streptomyces atlanticus, Streptomyces griseus, Streptomyces lividans, Streptomyces coelicolor, Labrenzia aggregata, Labrenzia alexandrii, and members of the Biocathode MCL Consortium from Wang, 2015. Combinations of any of the above organisms are further contemplated herein.

[0029] Systems for controlling the introduction and removal of fluids may be included. Suitable containers for the liquid, fluid channels (such as tubing, pipes, and fittings), valves, pumping systems, flow sensors, and computer control of fluid flow are contemplated. In various cases, a gravity-based configuration may eliminate the need for one or more pumps. Sensors may also be included to monitor biofilm health, system oxygenation, pH, and / or chemicals present in the feed or extraction solutions. The computer control system may dynamically respond to sensor readings to change flow rates, adjust nutrient or gas concentrations, or notify the operator of system failures.

[0030] Further contemplated are methods of using bioreactors to convert petroleum products into aqueous products, methods for converting aqueous feedstocks into hydrophobic products, methods for converting complex aqueous feedstocks into a single aqueous product, and methods for the production of lubricants.

[0031] Various embodiments may include electrodes that can act as a final electron sink or source effective to drive the enzymatic process.

[0032] Detailed Description of the Preferred Embodiments Biofilms for biosynthesis or biotransformation Biofilm-forming organisms form layers on surfaces, entrapping cells in a self-assembled extracellular matrix. Biofilms can consist of individual types of organisms or a consortium of multiple different species / strains. Cells often exhibit altered gene expression upon growth in biofilms, which can result in more efficient nutrient utilization or increased production and / or accumulation of specific biosynthetic products. Additionally, biofilms can support significantly higher biomass densities than suspension cell cultures, which can enable higher kinetics of biosynthesis and higher concentrations of end products. Biofilms are particularly well suited for syntheses requiring multistep enzymatic pathways (Muffler et al., 2014), because the system readily facilitates spatial segregation of individual reaction steps, eliminating the need for product purification between each step. Organisms in biofilms exhibit enhanced robustness, exhibiting higher durability and improved chemical resistance compared to planktonic cells. These properties make biofilms a favorable medium for biosynthesis or bioconversion.

[0033] Continuous Manufacturing Because the biofilm remains supported on a solid scaffold, it can facilitate continuous production of products in stationary phase. Unlike many cell suspension fermentation reactions, where significant resources are used to produce cellular biomass along with product synthesis, in stationary phase synthesis, the total biomass remains virtually constant throughout the entire course of product synthesis.

[0034] Oleaginous and hydrocarbon-degrading organisms Species from the genera Marinobacter, Thalassolituus, Oleispira, Acinetobacter, and Alkanidiges, among others, can metabolize alkanes and other hydrocarbon compounds. Many of these organisms can survive using hydrocarbons as their sole carbon source, and n-alkane oxidation produces acyl-CoA metabolites. Alkanes are first converted to alkanols by alkane hydroxylase, which are then converted to alkanals by alcohol dehydrogenase. Alkanals are converted to acyl-CoA by acyl-CoA synthase (Manila-Perez, 2010). Excess carbon is often channeled into the production of wax esters (Knutson, 2017), which can accumulate intracellularly as inclusion bodies or potentially be excreted by the cell (Manilla-Perez, 2010). Wax esters are enzymatically produced in cells through the activation of fatty acids to acyl-CoA or acyl-ACP substrates. Some of these substrates are then converted to fatty alcohols. Fatty alcohols and fatty aldehyde CoA (fatty aldehyde ACP) combine to form the final wax ester product. The wax ester pathway can be modified to produce other compounds, as demonstrated by the production of phloroglucinol by Meyer et al. (2019). Often, these organisms form biofilms at the interface between a hydrophobic solution, such as petroleum, and an aqueous medium, allowing them to survive on compounds from the hydrophobic solution as the organism's sole carbon source. In association with biofilms, Marinobacter spp. and other organisms can exhibit increased productivity compared to suspension cell cultures (Klein et al., 2007).

[0035] Type II secretion systems (T2SSs) are important for biofilm formation and are involved in the mass transfer of alkanes into cells (Ennouri et al., 2017). In some embodiments, upregulation of T2SSs and related proteins that support alkane uptake is used to increase the rate of alkane metabolism. Furthermore, AupA and AupB membrane proteins are involved in alkane uptake into cells (Mounier et al., 2018). Upregulation of the expression of these proteins can be used to increase the efficiency of alkane uptake.

[0036] In some hydrocarbon-degrading organisms, lipids can be transported from the cell and used to facilitate better product extraction (Manilla-Perez et al., 2010; Nakano et al., 2012). In some embodiments, organisms that produce biosurfactants, such as glycolipids, can be used to increase product extraction.

[0037] Certain species of the genera Marinobacter, Oleispira, Alcanivorax, and Halomonas, among others, are halotolerant (can be classified as mildly to severely halophilic) and grow in salinities ranging from 1.7 to 30%. The use of these organisms can facilitate biosynthesis in seawater or even more extreme salinity conditions.

[0038] One advantage of the Marinobacter genus is that experimental data show that Marinobacter atlanticus, in particular, can produce stable biofilms that can be only 10 μm thick. Thin, stable biofilms are advantageous in biofilm bioreactors because they improve mass transport and reduce the risk of adhesion / clogging within the bioreactor. To optimize the transport of reagents and products into and out of the biofilm, the total thickness should be less than 150 μm.

[0039] Controlling biofilm spread Natural biofilms undergo cycles of growth and spreading driven by environmental factors. To promote efficient continuous synthesis, it is desirable to control cell planktonicity and prevent spreading during synthesis. Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a signaling molecule responsible for regulating cell surface properties, secretion, cell adhesion, motility, and biofilm formation (Hengge, 2009). c-di-GMP is synthesized from guanosine triphosphate by diguanylate cyclase enzymes and degraded by phosphodiesterase enzymes. Diguanylate cyclase enzymes contain a GGDEF (SEQ ID NO: 1) domain responsible for c-di-GMP synthesis, while phosphodiesterase enzymes contain an EAL domain responsible for c-di-GMP degradation. Overproduction of GGDEF (SEQ ID NO: 1) domain proteins has been demonstrated to increase the synthesis of molecules required for biofilm formation, such as adhesins and matrix components, while disrupting motility. As a corollary, overproduction of EAL domain proteins resulted in the opposite behavior. In one embodiment of the present invention, biofilm-forming organisms are engineered to control the expression of GGDEF (SEQ ID NO: 1) and EAL domain proteins. The use of an inducible promoter allows microorganisms to establish an initial culture by inducing EAL domain proteins and repressing GGDEF (SEQ ID NO: 1) domain proteins, and then grow in planktonic phase with minimal spreading during biofilm formation due to the expression of GGDEF (SEQ ID NO: 1) domain proteins and the repression of EAL domain proteins (Hengge, 2009). In an alternative embodiment, expression of CsrA, a carbon storage regulator, can be regulated to control biofilm formation. A suitable activator of the inducible promoter can be provided, for example, when introducing feedstocks and / or nutrients.

[0040] Packed Bed Bioreactor In one embodiment of the present invention, product synthesis is carried out in a packed-bed bioreactor. Packed-bed reactors, also known as fixed-bed bioreactors, contain a granular solid support for biofilm growth. These particles are tightly packed within the bioreactor to maximize the surface area to volume ratio. Particle sizes can range from 5 μm to 20 mm. Particle size can be used to determine pore size and control feedstock permeation through the packed-bed reactor. In some embodiments, multiple particle sizes can be used to control packing density and biofilm formation. In further embodiments, particle sizes range from 5 to 100 μm. Small particle sizes with thin biofilms can maximize the total biomass in the reactor while minimizing the potential for clogging.

[0041] The vessel containing the support beads has a cylindrical geometry with a fluid inlet at one end and an outlet at the opposite end. The column diameter can be proportional to the particle size and ranges from 5 to 1000 times the particle size. In certain embodiments, the column diameter can be greater than 1000 times the particle size; in these configurations, specialized fittings can be used to match the impedance between the flow from the input source and the flow within the column. Specifically, a distribution plate can be used at the column inlet to distribute the flow evenly across the column cross-section. The column length is selected based on the contact time with the biofilm required to process the majority of the feedstock. In some embodiments, shorter columns can be used, with the feedstock recycled back into the column. The column can be made of any suitable material, such as polyvinyl chloride or glass.

[0042] Product recovery from the packed bed To overcome the product extraction limitations experienced in packed reactors, the solvent phase is periodically or continuously circulated through the reactor to extract hydrophobic products soluble in the solvent phase. In some embodiments, droplets of the water-immiscible liquid phase form plugs that completely displace the aqueous phase when introduced into the bioreactor. The aqueous phase solution is introduced following the plugs, and water pressure pushes the plugs through the system. An inlet valve allows switching between the two liquid phases. The time the cells are exposed to the solvent in the reactor must balance the required extraction time and cytotoxicity, and the duration of exposure is set by the flow rate and the height / volume of the immiscible plug. The immiscible phase can be recirculated through the reactor to fully extract the desired product. In another embodiment, the solvent phase is dispersed into small droplets that are introduced into the reactor as an aqueous suspension, so that at the reactor outlet, the solvent phase droplets can coalesce and separate from the aqueous phase.

[0043] At the outlet of the bioreactor, a separator is used to separate the aqueous and organic streams. In some embodiments, a hydrophobic membrane is used to reject the aqueous phase while allowing the hydrophobic phase to pass. In other embodiments, an active valve is used to send the hydrophobic phase to a separate channel. In yet other embodiments, the mixed solution is sent to a chamber that separates the two liquid phases using gravity separation.

[0044] Magnetic carrier beads In one embodiment, the carrier beads may be made from a ferromagnetic material. To extract a product from a biofilm immobilized on the carrier beads, a magnetic field can be applied to move the carrier beads from the aqueous feed phase into the product extraction solvent phase.

[0045] Scaffolding materials for packed-bed bioreactors A variety of different scaffold materials may be used to support biofilm growth. In some cases, these scaffolds may be roughly spherical particles. In other cases, the scaffolds may be small ring-like structures. Suitable materials include, but are not limited to, polymers, carbonized agricultural by-products, expanded clay, silica, alumina, iron oxide, metals, graphite, or carbon nanostructures. In some embodiments, the support material is electrically conductive, allowing electron transfer into and out of the biofilm.

[0046] Potential degradation of the scaffold material by the organism itself or components of the feedstock or extraction solution should be considered as a selection criterion. In some cases, particles may be metabolized by the biofilm, serving as a primary feedstock or providing supplemental nutrients to the biofilm. In other cases, the support material may be resistant to temperature and / or chemical treatments, which allow for the elimination of the biofilm and subsequent regrowth of a new biofilm to alter the product produced in the reactor or restore reactor productivity.

[0047] Membrane Bioreactor In an embodiment of the present invention, a bioreactor with a membrane supporting a biofilm between two liquid phases is used for product biosynthesis and extraction. Extractive membrane bioreactors, a type of membrane bioreactor, are currently used for wastewater treatment. Extractive membrane bioreactors consist of a membrane between two aqueous liquids, a wastewater zone that may contain high levels of organic compounds, and a biological zone where waste is metabolized / degraded. Both membranes between the two zones support biofilms, facilitating the selective transport of organic matter to the biological zone.

[0048] Product recovery from membrane bioreactors. In one embodiment, a solvent is introduced to one side of the membrane and an aqueous feedstock is introduced to the other side of the membrane. Hydrophobic products are produced in the biofilm on the aqueous side of the membrane, and the solvent flows continuously across the other side of the membrane to extract the products from the biofilm. The solvent may be recirculated within the system to concentrate the extracted products. In an alternative embodiment, a hydrophobic feedstock is introduced to one side of the membrane and a hydrophilic product is produced by the biofilm on the other side of the membrane and extracted into the aqueous phase. In yet a further embodiment, an aqueous feedstock is introduced to the biofilm-containing side of the chemoselective membrane and the purified product is extracted on the opposite side of the membrane.

[0049] In some embodiments, the membrane is formed within a tubular structure, with one liquid phase on the outside of the tube and the other liquid phase on the inside of the tube. Liquids from two different sources are introduced into the tube through a concentric manifold. Valves upstream of the manifold allow aqueous media to be delivered to both sides of the membrane, which can be used to help promote biofilm growth, rinse the system, or for other purposes.

[0050] Membrane structure and materials In some embodiments, the membrane is a highly porous scaffold intercalated with biofilm, creating a barrier layer between the two liquid phases. This highly porous scaffold can be a polymer, graphene, porous glass, or metal. In some implementations, the scaffold material is a cryogel, a macroporous structure created by rapid freezing in a cryobath and subsequent thawing (Berillo et al., 2019). In alternative embodiments, the membrane is chemoselective. These membranes can be made from polymers, ceramics, or graphene oxide. The membrane material is selected based on the target chemical and can function through size exclusion, electrostatic repulsion, or other hydrophobic / hydrophilic interactions.

[0051] Bioreactor System In a bioreactor system, the bioreactor chamber contains the biofilm used to synthesize and extract the desired product. In addition to the bioreactor chamber, the bioreactor system typically includes reservoirs, pumps, valves, flow controllers, sensors, and a computer control system. Solutions are routed between these components using fluidic channels. Some embodiments of the invention may also include a mixer, a system for introducing gases, output analysis, a phase separator, and a product purification module.

[0052] A pumping system can be used to propel fluids through the bioreactor system. Separate pumps can be used for each liquid stream, or valves can be used to allow a single pump to be used for multiple streams. In one embodiment, a pump that does not require direct contact with the fluid, such as a peristaltic pump or a pressure-driven pump, is used. Non-contact pumping is advantageous because the system is less susceptible to potential biofouling and clogging. In various cases, a gravity-based configuration can eliminate the need for one or more pumps.

[0053] Some embodiments of the invention will have additional inlets for the introduction of gases. In some cases, the gases may be used as feedstocks (e.g., ethylene, methane, carbon dioxide) and / or may be necessary for respiration (oxygen).

[0054] To prevent gases resulting from bacterial respiration from clogging the reactor, some embodiments employ bubble traps, a type of fluidic structure that uses a porous polytetrafluoroethylene membrane to remove gases from the reactor. In other embodiments, organisms capable of fixing carbon dioxide are introduced into the biofilm community to facilitate gas removal, thereby lowering the overall carbon footprint of the reactor system. Organisms such as Candidatus Tenderia electrophaga are known to fix carbon dioxide and live naturally in biofilm consortia that include organisms such as the Marinobacter genus (Eddie et al., 2016). To promote efficient carbon fixation, some embodiments use electrically conductive biofilm scaffolds with an applied electrical potential. Controlling surfactant production by the biofilm or introducing additional surfactants into the bioreactor provides yet another means of removing gas bubbles, as the use of soluble surfactants has been shown to facilitate the dissolution of gas bubbles (Hanwright et al., 2005).

[0055] In some embodiments, multiple biofilm-containing vessels are connected in parallel and a fluid manifold is used to distribute the solution to the individual vessels. Paralleling vessels in this manner can allow for scaling production up and down without altering shear forces and mass transfer within the entire reactor, thus mitigating some of the challenges traditionally associated with scalability of biofilm reactors.

[0056] Some embodiments include a thermal jacket to heat or cool the reactor. The thermal jacket may surround the entire reactor or an individual vessel. The thermal jacket is used to maintain an optimal temperature for the synthesis. Temperature sensors (thermocouples, resistance temperature detectors, etc.) can monitor the temperature, which can be regulated under closed-loop control.

[0057] One or many sensors can be used to monitor and regulate fluid flow and reaction conditions. Sensors can include, but are not limited to, pH, temperature, oxygen (and other dissolved gases), optical properties of chemical components (absorbance, Raman, fluorescence), cell density (of biofilm or planktonic cells), pressure, and flow rate. Pressure and flow rate sensors can provide information about conditions that may cause reactor clogging and / or shear forces to disrupt biofilms. In embodiments where the bioreactor contains multiple vessels in parallel, sensors can monitor both the performance of the entire system or the performance of individual vessels.

[0058] A computer (such as a single-board computer) or other digital control system is used to control the pumps, valves, thermal systems, and monitor sensors. The computer can process sensor inputs and send signals to the thermal jacket to regulate temperature, open or close valves or adjust pumping rates to control the introduction of raw materials and / or extraction solution, and can alert the operator to poor performance, contamination, or clogging.

[0059] In one embodiment, three reservoirs for the feedstock solution, supplemental nutrients, and extraction solution are connected to the inlet of a heat-jacketed bioreactor via a series of fluid channels. Each reservoir outlet fluid channel may have an individual pump and flow controller (FC). These fluid channels are directed to a series of multi-way valves that unify the three fluid channels, allowing controlled routing of individual source solutions or mixtures thereof into a single channel entering the bioreactor.

[0060] In alternative embodiments, for example, for a membrane bioreactor, two or more fluid channels enter the bioreactor, and a series of channels and valves allow for optional mixing of three source solutions introduced into each inlet of the bioreactor. A mixer may be included in-line before the bioreactor inlet to thoroughly mix or emulsify the solutions before entering the bioreactor. Some embodiments may contain an aerator before the bioreactor inlet to facilitate the introduction of oxygen or other gases that can serve as feedstocks for breathing as needed. To allow for adjustment of pH in the bioreactor, acid and base reservoirs may be connected to the bioreactor, along with pumps and multi-way valves that allow for the controlled introduction of acid or base.

[0061] The reactor outlet can be directed to recycle, purified to a product reservoir, and / or to a waste stream. A series of fluid channels and valves control the path of the output solutions in response to process conditions. Immediately after the outlet, a phase separator can be used to route immiscible solutions to separate process paths. Downstream of the bioreactor outlet and the phase separator can be a module for product purification and extraction. The outlet of this module can be directed to a product storage tank.

[0062] In some embodiments, spent feedstock can be recycled with the addition of additional feedstock molecules, nutrients, or other components introduced as needed. Additionally, product-containing solutions can be recycled to extract more product and increase the total product concentration in the solution. Valves control the routing of the outlet solution, allowing for controlled recirculation. Storage reservoirs can be included in the recirculation fluid path(s), along with pumps and valves that allow controlled introduction of recirculated solutions in response to process conditions. Valves can be connected to allow for recirculating flow or flow to further downstream processing.

[0063] A sensor module may be included at the outlet of the bioreactor to monitor process conditions. Each reservoir and system module may also contain additional sensors to monitor quality and allow adjustment of process conditions. In some embodiments, valved fluid paths may allow fluid sampling of the output of the bioreactor, phase separator, and / or purification module for chemical analysis. The fluid may be routed to a GC-MS, LC-MS, ICP, or other analytical instrument for this analysis.

[0064] Multi-step enzymatic process To facilitate multi-step enzymatic processes, in some embodiments, the bioreactor system contains multiple biofilm reactor modules, each performing a separate enzymatic step necessary to produce the desired product. The bioreactor system contains fluid channels that route intermediates or products between each module. Some embodiments may utilize membrane bioreactors and packed-bed bioreactors in individual modules. For example, products extracted with solvent droplets in a packed-bed reactor can be routed to a membrane bioreactor for subsequent process steps. In some embodiments, purification or separation modules are utilized between the biofilm reactor modules.

[0065] Biofilm freeze-drying Biofilms can be resistant to desiccation, allowing them to be completely dehydrated and still remain viable upon rehydration. To enable production of specific products on demand, biofilms containing one or more organisms can be established on a solid support (such as a membrane or carrier beads) and dried by techniques such as freeze-drying for storage. In some embodiments, the carrier beads will be packed into a bioreactor column, the biofilm will be established, and then dried, while in other embodiments, the biofilm will be established on the carrier beads, the biofilm will be dried, and the beads will be packed into a bioreactor column and rehydrated prior to synthesis. [Example]

[0066] Example 1: Conversion of a hydrophobic feedstock into an aqueous phase product Hydrophobic feedstocks refer to liquid solutions that are immiscible with water. Examples of hydrophobic feedstocks include crude oil, refined petroleum, and waste solvents. Typically, these feedstocks consist of non-polar organic chemicals, such as aromatic or aliphatic compounds. The compounds in the feedstock can be metabolized by the organisms in the biofilm or otherwise biotransformed by enzymes in the organisms themselves or the extracellular matrix of the biofilm.

[0067] To produce water-soluble products such as acetate or caprolactone from hydrophobic feedstocks: - The Marinobacter genus is modified to produce the desired water-soluble product. o Grow Marinobacter species in suspension culture to an OD of 0.1-2. The suspension culture is circulated through the bioreactor to inoculate the membrane or support particles. The bioreactor circulates the nutrient medium for 1 to 400 hours until a sufficient biofilm is formed. The feedstock is continuously introduced into the bioreactor along with an aqueous solution from which the product is extracted. The aqueous solution also contains fixed nitrogen species (ammonium, nitrate, nitrite), sodium ions, and trace minerals that support the biofilm. ■ In a packed bed bioreactor, the raw material is introduced as an emulsion containing an aqueous phase. Droplets of raw material are trapped in the biofilm and metabolized / converted into water-soluble products which are then excreted by the organisms and collected in the aqueous phase. In a membrane bioreactor, hydrophobic feedstock flows on one side of the membrane and aqueous solution flows on the other side. The hydrophobic feedstock diffuses through the membrane into the biofilm and is metabolized / converted into products that are excreted by the organisms and collected in the aqueous phase on the other side of the membrane. The aqueous phase can be recycled into the reactor to concentrate the product if necessary. The product is collected and purified from the aqueous solution. Purification steps may include desalting and evaporation.

[0068] Example 2: Conversion of aqueous feedstocks to hydrophobic products such as wax esters To produce wax esters from aqueous feedstocks: Marinobacter species were grown in suspension culture to an OD of 0.1–2. The suspension culture was circulated through the bioreactor to inoculate the membrane or support particles. The bioreactor was run for 1-400 hours with nutrient medium circulating until a sufficient biofilm was formed. In a packed bed reactor, the feedstock was circulated through the bioreactor for 1-24 hours, and then once optimal product formation was achieved, a valve was activated to introduce a solvent bolus into the bioreactor to extract the product. In a membrane reactor, the feed can be circulated on the outside of a tubular membrane and the solvent is circulated through the center of the tubular membrane. o Wax esters are miscible with the solvent phase and can therefore be extracted into the solvent. o The solvent phase can be recycled until it is saturated with product. The product was extracted from the solvent and purified.

[0069] Example 3: Conversion of mixed aqueous feedstocks to aqueous products To produce acetate from mixed organic waste feedstock: o Biofilm-forming organisms are grown in planktonic culture to an OD of 0.1-2. The suspension culture is circulated through the bioreactor to inoculate the membrane or support particles. The bioreactor is a packed bed reactor where the nutrient medium is circulated and the feedstock is circulated through the bioreactor until a biofilm is fully formed, for 1-400 hours. The packed bed is contained within a chemoselective membrane that allows the product to pass through into a harvest stream. In a membrane reactor, a biofilm grows inside a chemoselective membrane. As products are synthesized, they pass through the membrane and are extracted. o Pressure across the membrane can concentrate the product in the collection stream. Collect the product and remove the water.

[0070] conclusion While the present invention has been described in terms of its preferred embodiments, it will be understood by those skilled in the art that additions, deletions, modifications, and substitutions not specifically described may be made without departing from the spirit and scope of the invention. The terms used herein should not be construed as "means-function" terms unless the term "means" is expressly used in connection therewith.

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Claims

1. 1. A bioreactor system comprising: a column comprising a substrate coated with a biofilm comprising hydrocarbon-degrading organisms and / or oleaginous organisms; a first reservoir operably connected to deliver a first liquid containing the raw material to the column; a second reservoir operably connected to deliver a second liquid comprising an extraction solvent to the column; a phase separator operably connected to receive both liquids from said columns; Equipped with the biofilm has a thickness of 150 μm or less and is effective in converting the feedstock into a desired product; The bioreactor system, wherein the phase separator is effective to separate the product from one or both of the liquids.

2. a mixer operable to (1) introduce a dispersion of either of the two liquids into the column, or (2) introduce a plug of the second liquid into a stream of the first liquid; 10. The bioreactor system of claim 1, further comprising:

3. 10. The bioreactor system of claim 1, wherein the column is a packed bed containing particles having a particle size of 5 μm to 100 μm.

4. 10. The bioreactor system of claim 1, wherein the substrate is a membrane effective to separate the two liquids.

5. 5. The bioreactor system of claim 4, wherein the membrane is chemoselective.

6. A bioreactor system as described in claim 1, wherein the substrate is conductive and promotes electron transfer into and out of the biofilm.

7. 10. The bioreactor system of claim 1, further comprising a plurality of columns connected in parallel.

8. 10. The bioreactor system of claim 1, wherein the substrate serves as additional feedstock for the biofilm.

9. 10. The bioreactor system of claim 1, further comprising a pump and valves configured to recirculate liquid through the column or to direct liquid to the phase separator.

10. a mixer operable to (1) introduce a dispersion of either of the two liquids into the column, or (2) introduce a plug of the second liquid into the stream of the first liquid; a thermal jacket effective to regulate the temperature within said column; one or more sensors for monitoring reaction conditions; a computer control system operatively connected to the one or more sensors, the pump, and the valve; 10. The bioreactor system of claim 9, further comprising:

11. A method for converting a hydrophobic feedstock into an aqueous phase product using the bioreactor system of claim 1, comprising: providing a bioreactor containing live microorganisms capable of producing a water-soluble product; circulating a nutrient medium through the bioreactor to establish a biofilm of the microorganisms; introducing both an aqueous solution and a hydrophobic feedstock into the bioreactor, the aqueous solution providing supplemental nutrients to the microorganisms; allowing the microorganism to convert the hydrophobic feedstock to the product; and recovering the product from the aqueous solution; A method comprising:

12. The method of claim 11 , wherein the feedstock comprises a compound that is metabolized by the microorganism.

13. 12. The method of claim 11, wherein the step of recovering the product involves the use of a phase separator.

14. 12. The method of claim 11, wherein the microorganism is provided in the form of a solid support coated with a freeze-dried biofilm of the microorganism.

15. 12. The method of claim 11, wherein electron transfer into and out of the conductive solid support allows for increased productivity of the biofilm.

16. A method for converting an aqueous feedstock into a hydrophobic product using the bioreactor system of claim 1, comprising: providing a bioreactor containing a microorganism capable of producing a hydrophobic product; introducing a water-soluble feedstock into the bioreactor and allowing the microorganisms to convert the feedstock into the hydrophobic product; contacting the microorganism with a solvent effective to extract the product; and then recovering the product from the solvent; A method comprising:

17. 17. The method of claim 16, wherein the water-soluble raw material is provided in the form of particles that act as a substrate for the growth of the microorganisms.

18. 17. The method of claim 16, wherein the step of recovering the product involves the use of a phase separator.

19. 17. The method of claim 16, wherein the microorganism is provided in the form of a solid support coated with a freeze-dried biofilm of the microorganism.

20. 17. The method of claim 16, wherein electron transfer into and out of the conductive solid support allows for increased productivity of the biofilm.

21. A bioreactor system as described in claim 1, wherein the microorganisms include the genus Marinobacter.

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