Bioreactor Systems Used for Valorization of Corn Ethanol and Brewery Co-Products

Abstract

The present invention provides a method and apparatus for upgrading thin stillage, a by-product of corn ethanol production, and by-products from distilleries and breweries, into value-added hydrophobic chemicals, such as retinoids and lubricants, using biofilm-type bioreactors and hydrocarbon degrading organisms. [Solution] The bioreactor system comprises at least one biofilm-type bioreactor containing an organism capable of metabolizing at least one component of a by-product stream, and a means of connection to the by-product stream for introducing the by-product stream into the bioreactor.

Description

Related Applications

[0001] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 326,428, filed April 1, 2022, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Most commercially available biosynthetic chemicals today are produced using fermentation in a batch process in which suspension cells are grown in large vessels containing refined sugar-containing growth medium and allowed to simultaneously produce a product that is eventually isolated from the aqueous medium or from the cells themselves.

[0003] In contrast, biofilm bioreactors can use more diverse and complex carbon sources as well as produce products in a continuous flow manner, which can be more efficient than batch synthesis-extraction cycles. Hydrocarbonoclastic organisms are able to metabolize hydrocarbons as a food source, and because such organisms have high carbon flux pathways that can be exploited to build value-added chemicals, they are solvent tolerant, making continuous-flow extraction techniques feasible.

[0004] The continuous-flow biofilm bioreactor (Glaven et al., US 2021 / 0253990 A1; hereafter referred to as Glaven, the entire contents of which are incorporated herein by reference) architecture combines the advantages of biosynthesis with water-, energy-, and time-saving solvent extraction steps to economically produce complex hydrophobic chemicals from low-value feedstocks.

[0005] In a dry-grind corn ethanol plant, corn is taken in and mixed with yeast, water, and enzymes to convert the starches present in the corn into sugars. The ethanol plant then ferments the sugars into the desired molecule, ethanol. The fermentation liquid is passed through a series of distillation columns to extract the ethanol. The remaining by-product is usually called "whole stillage." Whole stillage contains the liquid and suspended solids left over from the fermentation and is separated in a centrifuge. The extracted solids, called wet cake, are sent to a dryer for dehydration and turned into dried distillers grains (DDG), an animal feed additive used primarily in the cattle, hog, and poultry industries. The liquid portion that leaves the centrifuge is turned into thin stillage, a nutrient-rich mixture consisting mainly of water.

[0006] Ethanol plants also profitably use thin stillage as an oil source and animal feed additive. Typically, the thin stillage is first dehydrated to efficiently extract the oil and finally dried for use as a feed additive. In ethanol plants, the water is removed by passing the thin stillage through a series of evaporators to form a concentrated thin stillage called condensed distillation solubles (CDS; also known as syrup).

[0007] The ethanol plant then centrifuges the CDS (or "syrup") in large centrifuges to extract another by-product, currently called "distillers corn oil", which is a very profitable corn oil that is sold to customers such as biodiesel plants. The "de-oiled" syrup is then sent back for additional evaporation before being added to the wet cake (the solid portion of the total stillage centrifuge) to enhance the nutritional content of animal feed. The wet cake that is dried without the addition of syrup is called dried distillers grains (DDG). The wet cake with syrup added is called dried distillers grains with solubles (DDGS). Occasionally, due to maintenance issues or other plant imbalances, ethanol plants may sell the syrup by tanker truck to local farmers at a lower profit.

[0008] Thus, of the three corn ethanol co-products (DDGS, crude corn oil, and distillers condensed syrup), the syrup has the lowest value. Even in the most favorable circumstances, the syrup is either added to DDGS at market value (about 40%) or sold to farmers for animal feed as concentrated distillers syrup at about one-tenth its dry weight value.

[0009] Microbiological approaches to valorize by-products of bioethanol plants. Various approaches have been proposed, including the production of protein-rich fungal biomass (Non-Patent Document 1), the production of organic acids by granular fermentation (Non-Patent Document 2), and even the more classical anaerobic digestion. In all of these approaches, the separation of product chemicals from complex feedstock mixtures is a key challenge.

[0010] Thin stillage is also used as a feedstock for oil-producing organisms such as the pink yeast Rhodotorula glutinis, which produces biodiesel (see Non-Patent Document 3). Microorganisms have also used thin stillage as a feedstock to produce medium-chain fatty acids, such as succinic acid, lactic acid, and caproic acid. Granular fermentation has Caproic acid can be produced at high rates from thin distillation residues that contain no solids (see Non-Patent Document 2 and Non-Patent Document 4). [Prior art documents] [Patent documents]

[0011] [Patent Document 1] US Patent Application Publication No. 2021 / 253990 [Non-patent literature]

[0012] [Non-Patent Document 1] Bulkan, G., Ferreira, JA, Rajendran, K. and Taherzadeh, MJ, 2020. Techno-economic analysis of bioethanol plant by-product valorization: exploring market opportunities with protein-rich fungal biomass production. Fermentation, 6(4), p.99. [Non-Patent Document 2] Carvajal-Arroyo, JM, Candry, P., Andersen, SJ, Props, R., Seviour, T., Ganigue, R. and Rabaey, K., 2019. Granular fermentation enables high rate caproic acid production from solid-free thin stillage. Green Chemistry, 21(6), pp.1330-1339. [Non-Patent Document 3] Yen, HW, Yang, YC and Yu, YH, 2012. Using crude glycerol and thin stillage for the production of microbial lipids through the cultivation of Rhodotorula glutinis. Journal of bioscience and bioengineering, 114(4), pp.453-456 [Non-Patent Document 4] Fortney, NW, Hanson, NJ, Rosa, PR, Donohue, TJ and Noguera, DR, 2021. Diverse profile of fermentation byproducts from thin stillage. Frontiers in Bioengineering and Biotechnology, 9, p.695306 Summary of the Invention [Problem to be solved by the invention]

[0013] Petrochemical replacements produced from bio-based resources often rely on feedstocks that are also important food crops, such as oil from soybeans and sugar from corn, beets, and sugarcane. Technologies that can efficiently produce chemicals biologically from non-edible carbon sources could be key to building sustainable chemical supply chains away from petroleum. [Means for solving the problem]

[0014] Connected to existing ethanol, distillery, brewery or similar plant infrastructure and continuously converting their wastewater streams into hydrophobic chemicals in pure organic solvents, this approach could enable sustainable chemical production with fewer downstream treatment requirements.

[0015] In this approach, biofilm bioreactors (such as those described in US 2021 / 0253990 A1, incorporated herein by reference) are used to convert renewable carbon sources that are not currently consumed by humans as food and are not particularly valuable into valuable organic chemicals such as retinol (vitamin A) and lubricants. Specifically, such biofilm bioreactors may employ a two-phase approach in which aqueous feedstocks are converted into hydrophobic products that can be extracted with an immiscible organic solvent, thereby isolating the products from the aqueous phase foreign matter.

[0016] The present invention encompasses methods and apparatus for converting low value streams such as thin stillage in corn ethanol plants and distilleries and breweries into hydrophobic chemical products using a biofilm bioreactor system that can be integrated into fermentation plants (such as corn oil, distilleries, etc.) to take the by-products of the fermentation process (e.g., ultra-thin waste, concentrated stillage, etc.) and convert them into high value chemicals.

[0017] More specifically, the present specification discloses a bioreactor system with at least one biofilm-type bioreactor that converts an aqueous raw material into a hydrophobic product that can be extracted with an organic solvent. The bioreactor is equipped with a biofilm of microorganisms that form a stable biofilm, metabolize the components of the substrate (raw material), and are resistant to organic solvents. Examples of microorganisms that can be used include the genera Marinobacter, Pseudomonas, Chromatiacea, and Labrenzia.

[0018] The biofilm bioreactor comprises a solid phase, such as a packed bed, solid support or solid matrix. The solid phase is made of particles or beads suitable for supporting a biofilm of hydrocarbon degrading microorganisms. Such a bioreactor has an inlet for introducing a medium that supports the growth of the biofilm organisms (e.g., a medium containing a carbon source such as thin stillage). The medium is converted into chemical products by biosynthetic pathways contained within the organisms.

[0019] In some embodiments, the invention features a biofilm-type bioreactor with beads that hold the biofilm and oxygen-permeable tubes that provide oxygen content during medium recirculation. This design improves oxygenation to the reactor while reducing, suppressing, or eliminating bubble formation.

[0020] In some embodiments, biofilm-forming microbial communities are used as microbial catalysts, where different microbial species may metabolize different components in the thin stillage or fermentation by-products or catalyze different steps in product formation.

[0021] In some embodiments, the organisms are tolerant to organic solvents, a property that allows them to remain functional biomass while also allowing for two-phase extraction of products from the bioreactor.

[0022] Some embodiments of the invention feature a system designed to be integrated in the plant before the evaporation means (used to concentrate the thin stillage). The medium used is compatible with the organisms in the bioreactor, which converts the medium into a product. Prior to the inlet to the bioreactor, important components can be introduced into the medium by a mixing system.

[0023] Some embodiments of the invention feature a system configured to use concentrated thin stillage (or distillation solubles (CDS; also referred to as "syrup")). The reactor is integrated into the plant after one or more evaporation cycles. In this process, a pump may recirculate the medium through the bioreactor. As the carbon is consumed, the concentrated thin stillage is gradually introduced into the bioreactor.

[0024] Some embodiments of the invention feature a system configured to use distilled corn oil (or similar stream) as a feedstock. The reactor is integrated into the plant after the centrifugation step. The process may use an inlet emulsifier module, e.g., a mixer, that disperses small droplets of corn oil into the medium. Cross-contamination of the product stream extracted from the feedstock is minimized by flushing the column with a side medium stream that does not contain corn oil prior to product extraction with an organic solvent. The configuration of the system after the centrifugation step (and before the final drying step) also allows for the utilization of the residue by-product after the extraction of crude corn oil.

[0025] The invention also encompasses a method for converting thin stillage, crude corn oil, or other suitable by-products into hydrophobic chemical products using hydrocarbon-degrading platform organisms such as Marinobacter spp. or Pseudomonas spp. in a biofilm bioreactor. In some embodiments, the method uses hexanes, heptanes, dodecane, corn oil, or the like as the extraction solvent. The hydrophobic phase is sent to downstream processing. Any aqueous by-products that are generated during this process that can no longer support a biofilm culture are discharged from the system. In some embodiments, the aqueous by-products are sent back to the evaporation stream, and in other embodiments, the aqueous by-products are evaporated and composted.

[0026] In one aspect, the invention features a method of modulating a hydrophobic chemical, the method including directing a by-product of a biorefinery to a bioreactor system including a plurality of biofilm bioreactors, each of which contains an organism capable of metabolizing the by-product to produce a hydrophobic chemical, circulating the by-product to at least one of the plurality of biofilm bioreactors, and operating the at least one of the biofilm bioreactors in response to an assessment from a sensor associated with the at least one biofilm bioreactor.

[0027] In another aspect, the invention features a bioreactor system including a plurality of biofilm bioreactors each housing an organism capable of metabolizing a by-product in a biorefinery (e.g., a corn ethanol plant, a brewery, a distillery, etc.) to produce a hydrophobic chemical, each configured to recycle the by-product and / or introduce at least one additional material and / or recover the hydrophobic chemical. The biofilm bioreactor system further includes a sensor for evaluating a parameter of each biofilm bioreactor and a controller for controlling each biofilm bioreactor in response to the evaluation of the sensor.

[0028] The described systems and methods allow for the conversion of complex, low-value carbon sources that are fermentation by-products into useful hydrophobic chemicals that require minimal costly downstream processing. In some applications, the present invention advantageously upgrades even the lowest value by-products, such as thin stillage / syrup, into high value cosmetic ingredients and lubricants.

[0029] The configurations described herein can be retrofitted into existing plants (without the need for new configurations), and aspects of the present invention provide numerous retrofit options and versatility to best suit the plant and process goals and / or constraints.

[0030] It is fundamentally difficult to isolate a desired product from a complex crude medium. Implementation of a continuous flow design as described herein allows the product to be extracted with a secondary phase (e.g., a hydrophobic solvent) and separated from the feedstock without cross contamination, reducing the burden on downstream processing. This is often important for the economical production of chemicals, especially high-end chemicals.

[0031] The design of the film bioreactor using oxygen permeable tubes that deliver oxygen-containing gas to the reactor interior can reduce, limit and generally eliminate bubble formation.

[0032] The above and other features of the invention, including various novel details of the combination and construction of parts, as well as other advantages, will now be particularly described with reference to the accompanying drawings and pointed out in the claims, and it will be understood that specific methods and apparatus embodying the invention are shown by way of illustration only and not as limitations of the invention.

[0033] In the accompanying drawings, reference characters refer to the same structures / components throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention. [Brief description of the drawings]

[0034] [Figure 1] A schematic showing the end-to-end production of ethanol at an ethanol plant, with the primary goal being animal feed (DDGS), crude corn oil, and distillers condensed syrup (CDS) as co-products. [Diagram 2] FIG. 1 is a schematic diagram of a biofilm-type bioreactor consisting of a column packed with biofilm-coated glass beads (the inset shows the beads covered with a bacterial biofilm). [Diagram 3] Schematic showing an end-to-end overview of an ethanol plant, with a continuous-flow biofilm bioreactor containing hydrocarbon degrading organisms inserted between the corn oil extraction and final evaporation steps and final drying means. [Figure 4] FIG. 1 is a schematic diagram showing an end-to-end overview of an ethanol plant, with continuous-flow biofilm bioreactors containing hydrocarbon degrading organisms inserted between any of the steps in the multi-stage evaporation procedure after centrifugation of thin stillage from the whole stillage. [Diagram 5] FIG. 1 is a schematic showing an end-to-end overview of an ethanol plant, with a continuous-flow biofilm bioreactor containing hydrocarbon degrading organisms inserted between the centrifuge of thin stillage from the whole stillage and a multi-stage evaporation cascade. [Figure 6] FIG. 1 is a schematic diagram showing an overview of a biofilm-based bioreactor system that includes a bioreactor equipped with a valve to periodically introduce thin stillage and a sensor to monitor the amount of carbon source, and which recirculates culture medium within the reactor. [Figure 6A] FIG. 7 is a flow diagram of a process implemented in the structure shown in FIG. [Figure 7] FIG. 1 is a schematic diagram showing an overview of a biofilm-based bioreactor system in which salts and minerals are mixed with thin stillage by a mixing means and introduced into the bioreactor. [Figure 7A] FIG. 8 is a flow diagram of a process that can be implemented in the structure shown in FIG. [Figure 8] FIG. 1 is a schematic diagram of a biofilm-type bioreactor equipped with an oxygen permeable tube that increases gas transport while preventing air bubble formation through the center of the reactor. [Figure 9] This is a table of the chemical composition of thin stillage (adapted from Kim, Y., Mosier, NS, Hendrickson, R., Ezeji, T., Blaschek, H., Dien, B., Cotta, M., Dale, B. and Ladisch, MR, 2008, Composition of corn dry-grind ethanol by-products: DDGS, wet cake, and thin stillage. Bioresource technology, 99(12), pp.5165-5176.). [Figure 10] 1 is a plot of relative fluorescence units (RFU) versus time (hours). [Figure 11] UV-vis absorbance spectra of solvent overlays collected from retinoid-producing M. atlanticus cultures grown on different concentrations of corn mash with and without glycerol. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which exemplary embodiments of the invention are shown. However, the present invention may 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. Any conjunctions used should be understood in their most inclusive sense. That is, the terms "or", "or", and "alternative" should be understood as having a logical OR definition and not an exclusive OR, unless otherwise clearly indicated from the context. Additionally, the singular forms "a", "an", and "the" are intended to include the plural unless otherwise specifically indicated. Additionally, the terms "having" and / or "comprising" and / or "comprising" and / or "including" and / or "having" as used herein should be understood to specify the presence of stated structures and / or elements and / or steps and / or processes and / or components and / or ingredients, but should not exclude the presence or additional of one or more other structures and / or elements and / or steps and / or processes and / or components and / or ingredients and / or collections thereof. Additionally, when an element (e.g., a component or subsystem) is described and / or illustrated as being connected or coupled to another element, it should be understood that the element may be directly connected or coupled to the other element, or that there may be intervening elements present.

[0037] In this specification, the terms "first", "second", etc. are used to describe each component, but it should be understood that the components should not be limited by such terms. These terms are used only to distinguish one component from another. That is, the following component can be named the second component, and similarly, the second component can be named the first component, without departing from the teachings of the present invention.

[0038] Unless otherwise specified, all terms (such as technical and scientific terms) used in this specification 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 this technical field, and should not be interpreted as an idealized meaning or an overly formal meaning unless clearly defined in this specification.

[0039] As used herein, the singular forms "a", "an" and "the" do not exclude a plural reference unless the content is clear.

[0040] As used herein, the term "about," when used in conjunction with a numerical value or range description, is intended to refer to a numerical value or range that is slightly above or below the stated numerical value or range, within +10% of the stated numerical value or range.

[0041] In some embodiments, the invention features a bioreactor system that includes at least one biofilm-type bioreactor containing an organism capable of metabolizing at least one component of a by-product stream. The bioreactor system may further include connections (valves, conduits, etc.) for introducing a by-product stream (or other materials) into the at least one bioreactor and / or for removing contents (e.g., products, waste, etc.). Additional devices may be included, such as pumps, reservoirs, mixers, lines for supplying air or other materials, etc.

[0042] In some applications, the by-product stream is a stream generated in a corn ethanol plant. However, embodiments of the invention are applicable to other types of biorefineries, such as distilleries and breweries, as well as corn ethanol plants. That is, the invention can be implemented in processes and / or plants that use fermented feedstocks such as corn, sorghum (milo), barley, rye, oats, wheat, soybeans, rice, millet, sugarcane, sugar beet, grapes, agave, apples, apricots, potatoes, beets, honey, dairy, walnuts, cashews, peanuts, pecans, buckwheat, palm sap, sweet potato, ginger, triticale, cassava, guarana, coconut, cherries, blueberries, raspberries, pomegranates, pineapples, pears, plums, bananas, plantains, juniper, sunflowers, rapeseed (and other oilseeds), and the like. These and other similar feedstocks may be processed in biorefineries, including, but not limited to, biorefineries known as distilleries that produce spirits, such as brandy, gin, rum, vodka, tequila (or mezcal), and types of whiskey, such as Scotch, rye, bourbon, Irish, Canadian, and Japanese whiskey, as well as biorefineries that produce beverages, such as beer, wine, cider, mead, sake, kefir, kombucha, and the like.

[0043] In one example, the bioreactor system is used to convert stillage, particularly thin stillage, into hydrophobic products, hi another example, the bioreactor system is used to convert crude corn oil (or distillery solubles condensate) into hydrophobic products.

[0044] Essentially, "stillage" refers to the residual mash, i.e., the mixture of fermented by-products and unfermented suspended solids produced by a biorefinery. This mixture of unfermented solids may be named differently in different industries, countries, and even regions, but the embodiments described herein are applicable to any stillage by-product from the biorefineries and feedstocks mentioned above, as well as other (similar) biorefineries. FIG. 9 shows the composition of an example thin stillage by-product. Crude corn oil (DCO), also referred to herein as "corn oil," is a corn ethanol production by-product that is produced (e.g., by centrifugation, etc.) from CDS. In general, crude corn oil contains more fatty acids than many other vegetable oils and can be used as a biodiesel feedstock and poultry feed ingredient. "Distillers solubles condensate" (syrup; i.e., CDS) is a low fiber, high protein product that is rich in organic acids derived from the ethanol production process.

[0045] Each aspect of the present invention is specifically adapted for scale-up production of hydrophobic chemicals. Large-scale synthesis of hydrophobic chemicals by fermentation is difficult due to the need to separate the cell biomass from the fermentation broth, extract the product from the cells, and then separate the product from unwanted cell debris. The use of microorganisms that are tolerant to hydrophobic organic solvents allows for in situ continuous-flow product extraction. In this method, an immiscible organic solvent is pulsed into the reactor, which allows for the extraction of the hydrophobic product while leaving the biofilm intact.

[0046] Other properties may also play an important role. That is, the organisms used to practice each aspect of the invention typically exhibit some desired characteristics and / or functions, typically two or more. The organisms can form biofilms, can metabolize the major carbon components in thin stillage, crude oils, etc., and are tolerant to organic solvents. In some cases, the organisms can be engineered to have desired conversion pathways.

[0047] Examples of organisms that can be used in biofilm-type bioreactors include Marinobacter spp., Pseudomonas spp., Chromatiacea spp., Labrenzia spp., and the like. Both Marinobacter and Pseudomonas species are essentially genetically tractable (see Bird, LJ, Wang, Z., Malanoski, AP, Onderko, EL, Johnson, BJ, Moore, MH, Phillips, DA, Chu, BJ, Doyle, JF, Eddie, BJ and Glaven, SM, 2018, Development of a genetic system for Marinobacter atlanticus CP1 (sp. nov.), a wax ester producing strain isolated from an autotrophic biocathode, Frontiers in microbiology, 9, p.3176.), well known for their opportunistic feeding behavior characterized by their ability to utilize a wide variety of feedstocks (carbon sources), as well as their tolerance to a variety of stressors, including solvent exposure (Ramos-Gonzalez, MI, Ben-Bassat, A., Campos, MJ and Ramos, JL, 2003, Genetic engineering of a highly solvent-tolerant Pseudomonas putida strain for biotransformation of toluene to p-hydroxybenzoate, Applied and environmental microbiology, 69(9), pp.5120-5127 and Klein, B., Bouriat, P., Goulas, P. and Grimaud, R., 2010, Behavior of Marinobacter hydrocarbonoclasticus SP17 cells during initiation of biofilm formation at the alkane-water interface, Biotechnology and bioengineering, 105(3), pp.461-468.

[0048] In some embodiments, organisms such as Marinobacter spp., Pseudomonas spp., etc., are used as biofilm-forming hydrocarbon-degrading microorganisms, which may also be referred to as "hydrocarbon-degrading" microorganisms (e.g., bacteria, etc.) or "oil-degrading" microorganisms (e.g., bacteria, etc.). Specific examples include Marinobacter atlanticus, M. psychrophilus (Zhang, DC, Li, HR, Xin, YH, Chi, ZM, Zhou, PJ and Yu, Y., 2008, Marinobacter psychrophilus sp. nov., a psychrophilic bacterium isolated from the Arctic, International Journal of Systematic and Evolutionary Microbiology, 58(6), pp.1463-1466), Marinobacter genus LV10R520-4, Marinobacter genus LV10MA510-1, Marinobacter genus ELB17 (Cooper, ZS, Rapp, JZ, Shoemaker, A., Anderson, RE, Zhong, ZP and Deming, JW, 2022, Evolutionary divergence of Marinobacter strains in Cryopeg brines as revealed by pangenomics, Examples of Marinobacter species include Marinobacter species such as Marinobacter sp. (see Frontiers in Microbiology, p. 1883).

[0049] In some cases, the organism naturally possesses all or some of the genes necessary to produce a desired product, e.g., retinal, retinol, etc. Alternatively, the organism may naturally ingest carbon building blocks, such as acetyl Co-A, from other sources, such as isoprenoids, carotenoids, and The organisms may be engineered to have pathways for conversion to more complex chemical products such as retinoids. US 2022 / 0340949 A1 by Magyar et al. (published October 27, 2022; incorporated herein by reference in its entirety) describes engineered organisms with synthetic operons for the production of isoprenoids, carotenoids, and retinoids optimized for use in hydrocarbon degrading organisms, and methods for the synthesis and extraction of isoprenoids in biofilm bioreactors comprising the engineered organisms. In some embodiments of the present invention, the principles described in US 2022 / 0340949 A1 are applied to convert low-value by-products from corn ethanol processes and distillery / brewery wastes into useful chemicals.

[0050] In some applications, biofilm-forming microbial communities are used as microbial catalysts, with different microbial species metabolizing different components in thin stillage or fermentation by-products or catalyzing different steps in product formation.

[0051] Although alcohol (e.g., ethanol) is the product of a biorefinery, such as a corn ethanol plant, the processes involved in producing alcohol generate various by-product streams. Embodiments of the present invention relate to the production of chemicals using such by-product streams.

[0052] As known in the art, corn ethanol plants can employ any number of process types. Figure 1 shows an example schematic of a dry-mill corn ethanol plant that takes corn from U.S. farmers and combines it with yeast, water and enzymes to convert the starch present in the corn into sugars. The following steps result in ethanol and other by-products:

[0053] The dry milling plant of FIG. 1 includes a grinding stage 110 where corn is dry-ground using, for example, a hammer mill. A slurry is formed in a slurrying stage 112. A liquefaction stage 114 combines the corn with water and enzymes (e.g., yeast amylolytic enzymes). A saccharification stage 116 where the enzymes convert starches into sugars and dextrins. In a fermentation stage 118, the product from the saccharification stage is fermented by the addition of yeast and nutrients. In a distillation stage 120, alcohol is distilled in a series of distillation columns. The remaining by-product, called "whole stillage" (124), typically contains water, fiber, protein, and oil.

[0054] From the distillation column, the alcohol (e.g., ethanol, etc.) is directed to a rectification stage 122 to produce concentrated alcohol. Total stillage 124, which contains both liquid and suspended solids remaining from the fermentation, enters a stillage separation stage 130, which may separate the solids and thin stillage by centrifugation. The extract wet cake from the separation stage 130 is either collected as DDG or sent to a blending means 152. The thin stillage from the separation stage 130 is directed to an evaporation stage drying means (multi-stage drying means 132) to form a distillation solubles condensate (syrup, or CDS). The distillation solubles condensate is passed through a centrifuge means 140 to extract crude corn oil (line 150). The remaining thin stillage (depleted of oil) from the centrifuge means 140 is combined with the wet cake in a blending means 152. The resulting mixture is passed to a final drying stage 160 to produce a condensate and distiller's dried with solubles (DDGS). In another process mode, the residual liquid after extraction of the crude corn oil in the centrifuge means 140 is dried (e.g., by evaporation) to produce distiller's solubles which are sent to a drying means to produce (reduced fat) DDGS. Alternative plant / process configurations can be used depending on desired co-products and other considerations.

[0055] In some embodiments of the invention, at least one biofilm bioreactor may be incorporated into a corn ethanol plant (as shown in FIG. 1), distillery, brewery, or other biorefinery. Specifically, the at least one biofilm bioreactor may be inserted into a co-product (also referred to herein as "by-product") line (the "product" of a biorefinery, such as a corn ethanol plant, brewery, or distillery, is alcohol).

[0056] In one example, the by-product is thin stillage, a complex mixture of components (glycerol and lactic acid accounting for 30%) (Kim, Y., Mosier, NS, Hendrickson, R., Ezeji, T., Blaschek, H., Dien, B., Cotta, M., Dale, B. and Ladisch, MR, 2008, Composition of corn dry-grind ethanol by-products: DDGS, wet cake, and thin stillage, Bioresource technology, 99(12), pp.5165-5176.). The thin stillage mixture can be metabolized in a biofilm bioreactor by platform organisms as described above. The biofilm state allows microorganisms to tolerate components of thin stillage (e.g., acetate and glycerol) that can inhibit non-biofilm-forming organisms such as E. coli (Pinhal, S., Ropers, D., Geiselmann, J. and de Jong, H., 2019, Acetate metabolism and the inhibition of bacterial growth by acetate, Journal of bacteriology, 201(13), pp. e00147-19.).

[0057] FIG. 2 shows a biofilm bioreactor 205 as described in Glaven. It has become a robust platform for the production of chemicals, classically petrochemicals. Biofilms are communities of microorganisms that naturally attach to each other and to the surface of beads 206 within the reactor 205, protecting them from the surrounding environment. Biofilm-forming organisms such as M. atlanticus (or other genera and species mentioned above) have a flexible metabolism and robustness that allows them to utilize a wide variety of feedstocks that are inaccessible to traditional yeast or E. coli fermentation, such as ethanol plant by-products like thin stillage.

[0058] In many embodiments, multiple (two or more) continuous-flow biofilm bioreactors 205 connected in parallel are used to reduce the threat of one reactor contaminating the entire train. The parallel configuration also ensures a more even distribution of nutrients. Essentially, the effluent or waste stream from the biofilm bioreactors can be 1) returned to the ethanol plant pipeline at the point of takeoff [from by-product line 154 (see FIG. 3 ; described in more detail below)], 2) returned to the final gas drying means (drying stage 160) that leads to the DDGS product line at the end of the thin stillage / syrup process, or 3) disposed of as waste in a landfill without being returned to the ethanol plant. As described in more detail below, each effluent management process has advantages and disadvantages.

[0059] In many applications, a biofilm bioreactor assembly or array (comprising one, and typically two or more bioreactors, e.g., in a parallel configuration) is inserted into the line where the product is extracted, e.g., downstream of the distillation / rectification stage 120 of the ethanol plant shown in Figure 1. For example, it may be inserted downstream of the stillage separation stage 130.

[0060] By-products such as thin stillage, distillery concentrated syrup, crude corn oil, etc. are fed into the bioreactor from the bottom and flow upwards against gravity. It is passed through the bioreactor several times by a recycle system that may be located near each modular bioreactor. Culture medium and / or nutrients and / or salts and / or other materials are added to support the microorganisms in the biofilm. Exhausted The thin stillage or syrup is finally returned to the ethanol pipeline or sent to a landfill. During or after this process, the hydrophobic molecules of interest are "harvested" from the continuous-flow biofilm bioreactor by circulating an organic solvent through the reactor. The molecules of interest, including the solvent, are then passed through a separation process such as column chromatography or nanofiltration to purify the molecules of interest and regenerate the organic solvent (which can be returned to the reactor or discarded). In certain embodiments, the organic solvent hexane or crude corn oil, which has already been refined and stored in the ethanol plant, is used. In the case of the organic solvent hexane used in small-scale systems, it is also possible to evaporate the molecules of interest from the hexane. Heptanes, dodecane, other carbohydrates, vegetable oils (e.g., corn oil, etc.), other hydrophobic organic solvents, solvent mixtures, etc. may also be used.

[0061] Typically, a continuous-flow biofilm bioreactor assembly is connected to a high-pressure by-product line, e.g., a thin stillage or syrup line, which draws an initial flow of nutrient-rich liquid into a recirculating medium containing buffered pH, salts, and micronutrients for the hydrocarbon-degrading organisms. This flow can be pump-assisted, or a well-placed, well-timed one-way valve can be used to drive both the initial flow and the recirculating nutrient stream with only high pressure from the by-product line. The recirculation system replenishes the consumed nutrients by slowly "pouring" or "dripping" the connected ethanol plant by-product line, e.g., thin stillage or syrup, with each "cycle" of fluid. This is one of the key advantages of incorporating a continuous-flow bioreactor in an ethanol plant.

[0062] 3 shows one embodiment in which a parallel connected array 210 of continuous flow biofilm type bioreactors 205 containing hydrocarbon degrading organisms (e.g., as described above) is inserted in line 154 between the corn oil extraction and final evaporation step 132 and the final drying means (drying stage 160). This arrangement has the advantage that the most concentrated liquid nutrient stream can be "poured" or "trickled" as in the process described above, as well as reducing the potential for damage to ethanol plant equipment due to reinsertion of spent media.

[0063] In the approach shown in FIG. 3, the only equipment that would handle the salts and other micronutrients added to the growth medium of the hydrocarbon degrading organisms is the gas drying means in the drying stage 160. Moreover, its location after the oil extraction process avoids the impact on the high-value by-products, either by reducing the amount of by-products (which could happen if a bioreactor were inserted upstream, due to the lack of reinjection of the waste from the bioreactor) or by reducing the quality of the by-products (by reducing the oil extraction efficiency and the equipment "run time" of the extraction centrifuge means, due to the lack of reinjection of the waste and the addition of salts and nutrients). In addition, the hydrocarbon degrading organisms could directly metabolize the corn oil, which could result in partial consumption of the high-value by-products.

[0064] In the embodiment of FIG. 3, a sampling (feed) valve in line 154 (see, for example, valve 236 in FIG. 6 and FIG. 7, described in detail below) introduces the thin stillage feed from the centrifugation means 140 into a pre-prepared medium (see, for example, FIG. 7, described in detail below) or into a film-type bioreactor after mixing with salts, etc. (see, for example, FIG. 6, described in detail below). In some embodiments, the sampling valve is controlled by a sensor (e.g., located within or along the bioreactor) that monitors the level of metabolizable components, such as lactate, glycerol, etc., in the medium. The valve can be programmed to introduce a condensed nutrient stream into the recirculating medium when the sensor detects that the amount of carbon source falls below a critical threshold. In some embodiments, the feed to the array 210 is crude corn oil (from line 150). An emulsification means 156 can introduce droplets of corn oil into the reactor as a carbon source.

[0065] FIG. 4 shows another embodiment in which a continuous-flow biofilm bioreactor array 210 containing hydrocarbon degrading organisms in bioreactors 205 is inserted between any steps in the multi-stage evaporation means 132 after (centrifugal) separation of thin stillage from whole stillage (stage 130) and before centrifugal extraction of corn oil (stage 140). This allows the concentration of the thin stillage to be fine-tuned to best suit the nutrient mixture being recirculated. Many dry grind ethanol plants have six or more continuous-flow evaporators, which change the thin stillage from around 90% moisture before treatment to about 60% moisture. The ability to insert the array 210 at any point in the train allows the concentration of the thin stillage to be fine-tuned. In some applications, the by-product used in the array 210 is corn distillers syrup (CDS). However, this configuration may have a negative effect on the oil extraction process as described above. However, the oil molecules present before corn oil extraction can also serve as a carbon source for the organisms.

[0066] Figure 5 shows a further embodiment in which a continuous flow biofilm bioreactor array 210 containing hydrocarbon degrading organisms is inserted between the centrifugation of the thin stillage from the whole stillage and the multi-stage evaporation means. This reduces the amount of energy consumed by the ethanol plant to evaporate the thin stillage, but may have a negative impact on oil extraction. It also provides the most dilute nutrient source, which can complicate the "pour-in" or "trickle-in" application methods described herein and can result in long downtimes for cleaning all downstream equipment when re-injecting the effluent waste. In some variations of this embodiment, necessary salts, minerals, and other medium components are added to the diluted thin stillage by a mixing means (described in more detail below) before the medium enters the bioreactor.

[0067] The biofilm-based bioreactor array and its operation are described in detail below.

[0068] FIG. 6 shows an example of an application of a continuous-flow biofilm bioreactor array 210. As shown in FIG. 6, each bioreactor 205 is divided into individual loops 215-1 to 215-4. Specifically, each loop 215-1 to 215-4 includes a circulation pump 220 controlled by a control means 260 with a return pipe 222. A loop valve 224 controlled by the control means 260 allows for the introduction of fresh material into each loop and for the harvesting of material from the loop. The array may include one or more loops, for example, in the range of about 1 to about 100, such as 1 to 5, 10, 20, 30, 40, 50, 60, 70, 80, 90. In one example, 10 loops are used.

[0069] The growth medium storage means 230 and the solvent storage means 232 supply the growth medium and the solvent via the respective valves controlled by the control means 260 to the loops 215-1 to 215-4 via the manifold line 234. The feed valves 236 controlled by the control means 260 supply the raw material to the manifold line 234 to allow for the periodic introduction of a by-product, e.g. thin stillage. The mixing means 212 controlled by the control means 260 may supply medium components, e.g. salts, minerals, etc., to the by-product, e.g. diluted thin stillage, before the medium enters the bioreactor. The sensors 252-1 to 252-4 allow the control means 260 to monitor the amount of carbon source. The control means uses the sensors to determine when to open the valves 224-1 to 224-4 to inject the thin stillage. The sensors may be located inside each bioreactor or in each recirculation line (i.e. loops 215-1 to 215-4).

[0070] The air may be supplied from any suitable source. In one application, the air is supplied by a compressor. 250 is produced and introduced in a line into the bottom of each bioreactor 205.

[0071] In this application, the bioreactor status is continuously monitored by the control means using sensors 252-1 to 252-4 (within or along each bioreactor column). These sensors are typically electrochemical and may measure pH, dissolved oxygen, dissolved CO2, a particular carbon source such as glycerol or fatty acids, or the concentration of a product, e.g., vitamin A. In some cases, the sensors are optical sensors that measure cell density by light diffusion, total product by UV-Vis absorption, or molecular profiling by Raman or infrared spectroscopy. In some cases, the sensors measure electrical impedance, the spectrum of which provides a fingerprint to assess the status of the bioreactor and may provide information about cell health, the presence of feedstock, or product accumulation.

[0072] Waste is removed from the manifold line under control of waste valve 238. Product is removed (collected) under control of product valve 240. In one example, the product is retinol and / or retinaldehyde and / or retinoic acid.

[0073] In a specific embodiment, the bioreactor system is controlled by a control means 260, such as a microcontroller, computer system, microprocessor, etc., which monitors the bioreactor conditions (specifically, sensors as described herein) and maintains reactor productivity by opening and closing individual valves. The control means implements an algorithm that keeps the feed recirculated through each column. Dissolved oxygen is detected (by a sensor) at the outlet of each column, and the air flow (supplied from an appropriate source) can be adjusted (at the command of the control means) to compensate. Too much dissolved oxygen indicates that cell metabolism is decreasing. The control means responds to such an assessment by opening the supply valve 236.

[0074] The reactor condition may be monitored by characterizing the electrical impedance inside the bioreactor or by evaluating the associated sensors. If this condition evaluation indicates low carbon, the control means opens the feed valve. If the control means determines that inhibitory by-products have accumulated, the medium is flushed to waste and fresh medium is introduced into the reactor. If the control means determines that sufficient products have accumulated for extraction, exposure of the biomass to an organic solvent is performed. Once this process is complete, the control means causes the reactor to perform a closed loop recirculation in the column.

[0075] FIG. 6A is an exemplary flow diagram illustrating the operation, monitoring, and control of the process performed by the control means 260 in the arrangement of FIG. 6. In the process 600, feedstock is recirculated (step 605). Parameters of the operation are evaluated (by one or more suitable sensors located within the bioreactor 205 or in any of the loops 215-1 to 215-4). This information is sent to the control means 260, which may maintain the status quo or may command changes to operational performance such as valve and pump activation, flow rate, etc. For example, the dissolved oxygen status is evaluated (step 610). If the measured oxygen level is low, the control means commands an increase in air flow rate (step 620). If the oxygen level is high, the control means typically commands the column feed valve to be opened (step 630) and feedstock (e.g. thin stillage, etc.) to be added. The reactor status may be evaluated by suitable sensors (step 640). If the carbon content is determined to be low, the control means may command the column feed valve 236 to be opened (step 630). If the product becomes saturated, the control means will typically recover or extract the product (step 650). The remainder is returned to the feed being recirculated in the column (any of loops 215-1 to 215-4). If inhibitory by-products accumulate, the control means may command a flush of the existing medium and the addition of new medium to the feed being recirculated in the column (any of loops 215-1 to 215-4) (step 660).

[0076] FIG. 7 shows another embodiment of the configuration and operation of a continuous flow biofilm type bioreactor array 210.

[0077] Here, by-products, such as thin stillage, are introduced into a medium storage means 230 provided with a mixing means 212 for mixing salts, minerals, etc. with the thin stillage (see valve 236) and are introduced into the bioreactor array 210 under the control of the control means 260.

[0078] A single pump 220 supplies solvent (from storage means 232) and medium (from storage means 230) to the parallel connected bioreactors 205. Separate input valves 224-1 through 224-4 control the flow rates from input manifold 244 to the bioreactors 205. An output manifold 242 connects the bioreactors to waste and product outputs (via valves 238, 240, respectively).

[0079] Air may be supplied from any suitable source. In one application, air is introduced into the bottom of each bioreactor 205 by a line fed from a compression means 250.

[0080] In this application too, the bioreactor condition is continuously monitored by sensors in the bioreactor column or along the reactor outlet (as described with reference to Figure 6), allowing feedback control by the control means.

[0081] As in the embodiment of FIG. 6, the bioreactor system is controlled by a control means 260 that monitors the bioreactor status and maintains the reactor productivity by opening and closing individual valves. The control means implements an algorithm that keeps the feedstock fed into the reactor system. The control means responds to the evaluation of each parameter in much the same way as described above. Dissolved oxygen is detected at the outlet of each column and the airflow is adjusted (under the control of the control means 260) to compensate. For example, if there is too much dissolved oxygen, this determination indicates that the cell metabolism is decreasing and the control means takes steps to increase the feed rate. The reactor status is detected by characterizing the electrical impedance inside the bioreactor or by evaluating the attached sensors. If this evaluation of the reactor status indicates that carbon is low, the evaluation triggers the control means 260 to control the opening of the common valve 236. If it is determined that inhibitory by-products are accumulating, the medium is flushed to waste and new medium is introduced into the reactor (both processes under the control of the control means 260). Once sufficient product has been assessed to have accumulated for extraction, the control means exposes the biomass to an organic solvent. Once this process is complete, the control means again provides continuous flow of feed to the reactor.

[0082] FIG. 7A is a flow chart of the process described with reference to FIG. 7. In process 700, the feedstock passing through any bioreactor 205 (step 705) is evaluated (by suitable sensors for dissolved oxygen located within or along any bioreactor 205) (step 710). If the control means determines that the oxygen level is low, the air flow rate is increased (step 720). If the oxygen level is high, the medium feed from the storage means 230 is increased (step 730). The reactor condition is evaluated (step 740). If the carbon level is determined to be low, the feed rate may be increased (step 730). If the product becomes saturated, product extraction is typically performed (step 750). The remainder is returned to the feedstock being recycled. If inhibitory by-products accumulate, a flush of the existing medium and the addition of new medium to the feedstock being circulated is performed (step 760). At least a part (generally, all) of the above determinations and / or subsequent processing are controlled by the control means 260.

[0083] When a biofilm bioreactor array configuration like that of Figures 6 and 7 is incorporated into a corn ethanol plant, brewery, distillery, etc., it can be connected to a byproduct conduit (generally designated 310 in Figures 6 and 7) using techniques and equipment (e.g., pipes, valves, flow meters, sensors, etc.) known to those of skill in the art.

[0084] In the embodiment described, for example, with reference to FIG. 3, line 310 receives feed from the thin stillage conduit leaving the centrifuge means 140, for example, at a location upstream of the mixing stage 152. Alternatively, it can be connected to the crude corn oil line 150. In another integration arrangement (see, for example, the embodiment described with reference to FIG. 4), line 310 corresponds to or is connected to the CDS line 312 from the multi-stage evaporation means 132. In a further embodiment, line 310 can be located at the outlet of a particular evaporator of the multi-stage evaporation means 132, which allows control of the composition of the by-products fed to line 310. In the integration approach described with reference to FIG. 5, line 310 corresponds to or is connected to the conduit 314 carrying the stream leaving the stillage separation stage 130.

[0085] As previously discussed (see, e.g., Figures 6A and 7A), supplemental oxygen may be added to the recirculating fluid to improve the efficiency of the hydrocarbon degrading organisms in producing molecules of interest. In one embodiment, continuous-flow oxygenation of the biofilm bioreactor is achieved by passively pumping oxygen through the tube walls using ambient or compressed air through oxygen permeable tubing. Note that active solutions using on-line oxygen bubblers generally require a bubble trap prior to entry into the bottom of the biofilm bioreactor.

[0086] 8 shows an embodiment in which a biofilm bioreactor 205 includes oxygen permeable tubing 206 (e.g., a fluoropolymer tubing, etc.). Other oxygen permeable tubing and tubing materials that can be used include, but are not limited to, fluoroethylene propylene, low density polyethylene, and silicone.

[0087] The tubes 206 receive air through the air inlet holes 208 and extend through the reactor 205 to the air outlet holes 214. The air inlet holes 208 are constructed in the bioreactor flange 216, and the air outlet holes 214 are constructed in the bioreactor flange 218. Each flange may further include a medium inlet 252 and a medium outlet 254. In one application, the inlet and outlet holes are arranged in a staggered configuration. As a result, the tubes 206 are non-parallel to the vertical axis and the bioreactor. Other configurations may be used, such as helical tubes, coiled tubes, or multiple tubes connecting pairs of inlet and outlet holes. Oxygen gas or oxygen-enriched air may be used in addition to or instead of air. In an embodiment such as FIG. 8, the gas tubes may increase the transport of gas (e.g., air) through the bioreactor without creating bubbles. EXAMPLES

[0088] Example 1: Metabolism of glycerol and thin stillage by M. atlanticus

[0089] The two main components of thin stillage are lactic acid and glycerol (Figure 9). Marinobacter species are known to use a wide variety of organic acids, including lactic acid, as carbon sources. To examine the ability of M. atlanticus to use glycerol as a carbon source, artificial seawater media containing glycerol (concentrations: 0.8%, 4%, and 8%) as the sole carbon source were prepared. M. atlanticus grew significantly in all of these samples, indicating that it was used as a carbon source by M. atlanticus.

[0090] To further investigate the growth of M. atlanticus on thin stillage as a carbon source, metabolic assays were performed on thin stillage samples. Samples of 1 wt% thin stillage from before and after various evaporation stages were prepared in an artificial seawater medium optimized for biofilm growth. A 24-well plate was prepared with silicon dioxide beads acting as a solid support. 1 mL of ASW medium containing 1% of either thin stillage, evaporation product (Evap), or corn oil was added to each well. Each well was inoculated with 1:100 WT Marinobacter (10 μL) and sealed. The beads were seeded with biofilm by incubating the plate at 30° C. for a growth time of approximately 7.5 hours. The medium was removed and washed with carbon-free ASW.

[0091] A metabolic assay was then performed to determine the amount of biofilm formed on the beads. ASW containing succinate as a carbon source was added along with resazurin. Metabolic activity results in the reduction of resazurin to produce a fluorescent molecule. This metabolic activity correlates with total biomass, providing evidence of the relative initial biomass on the silica beads.

[0092] The fluorescence signal (excitation wavelength: 530 nanometers (nm), emission wavelength: 590 nm) was monitored by a plate reader for over 10 hours. A rapid increase in the fluorescence signal indicates an increase in initial biomass. As can be seen from the data shown in FIG. 10, more biomass was obtained for each by-product of ethanol fermentation than the control ASW medium containing organic acids (succinate). These data indicate that M. atlanticus can metabolize components of thin stillage, crude corn oil, and distillery / brewery waste and that these carbon sources may result in biofilm production with more biomass than minimal medium containing short chain organic acids.

[0093] Example 2: Production of retinoids by M. atlanticus grown on distillery corn mash waste

[0094] To confirm the ability to produce retinoids from thin stillage, a strain of M. atlanticus with the wax ester carbon storage pathway knocked out to produce retinal was engineered to produce △△M. Atlanticus (Bird, et al 2018). Then, as described in US Patent Application Serial No. 17 / 722,182, M. atlanticus was given a complete pathway for retinoid synthesis by genetic engineering with two plasmids. Corn mash waste obtained from a small distillery was mixed with artificial seawater medium (corn mash = 6% by volume, 20% by volume). In some embodiments, thin stillage from an ethanol plant is used instead of corn mash. Iron citrate was added to the medium. In some embodiments, corn mash can be used in any volume amount from 1% to 100% by volume. In some embodiments, glycerol is added to the corn mash. If the corn mash is thickened, salts and minerals are added in concentrated or solid form.

[0095] The medium was inoculated with a 1:100 dilution of a starter culture and grown for at least several hours with an overlay of a hydrophobic solvent such as dodecane, heptane, hexane, or vegetable oil, which allowed the retinoids to be extracted into the solvent layer where they could be separated by nanofiltration.

[0096] As can be seen from the data shown in Figure 11, retinoid production with the retinal absorbance signature peak (368 retinoid production) occurs over a wide range of corn mash concentrations with and without added glycerol. These data indicate that M. atlanticus can produce industrially sufficient amounts of retinoids from distillery / brewery waste and thin stillage components.

[0097] While the present invention has been particularly shown and described with reference to preferred embodiments, it will be apparent to those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as encompassed by the appended claims.

Claims

1. A biofilm-type bioreactor containing an organism capable of metabolizing at least one component of the byproduct flow, The system includes means for connecting to the byproduct flow for introducing the byproduct flow into the bioreactor, A bioreactor system in which the byproduct flow is from a corn ethanol plant, brewery, or distillery.

2. A bioreactor system according to claim 1, wherein the byproduct stream is a dilute distillation waste liquid.

3. A bioreactor system according to claim 1, wherein the organism is M. atlanticus.

4. In the bioreactor system according to claim 1, A bioreactor system in which the pressure from the byproduct flow becomes the pressure that propels the culture medium within the bioreactor.

5. In the bioreactor system according to claim 1, further, A recirculation pump for reusing the culture medium in at least one of the biofilm-type bioreactors, A valve between the by-product flow and the recirculation line, which allows for the periodic introduction of raw materials from the by-product flow, A bioreactor system equipped with this system.

6. A bioreactor system according to claim 1, wherein the bioreactor system comprises an emulsifying means for introducing corn oil as a carbon source into the reactor.

7. In the bioreactor system according to claim 1, further, A mixing means for mixing the dilute distillation waste liquid with salt before introducing it into the at least one bioreactor, A bioreactor system equipped with this system.

8. In the bioreactor system according to claim 1, further, A bioreactor system comprising multiple bioreactors operating in parallel.

9. A bioreactor system according to claim 1, wherein the bioreactor system produces one of retinol, retinaldehyde, and retinoic acid.

10. A method for converting by-products from a corn ethanol plant, distillery, or brewery into hydrophobic products using a biofilm-type bioreactor system.

11. The method according to claim 10, wherein the hydrophobic product is any one of retinol, retinaldehyde, and retinoic acid.

12. A method using the bioreactor system according to claim 1 for converting dilute distillation waste liquid or crude corn oil into a hydrophobic product.

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