Methods and systems for propagating microorganisms for mitigating mycotoxin contamination, and related systems and methods

Abstract

Systems and methods for propagating at least one microorganism expressing one or more enzymes to mitigate mycotoxin contamination in a bioprocessing facility. The system is adapted to supply a composition from a culture tank at different points of a bioprocessing facility, the composition comprising a culture solution, a microorganism, a microbial lysate, an isolated enzyme, and / or a combination of these components.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This formal patent application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 400,230, filed on August 23, 2022, which is incorporated herein by reference in its entirety. Background Art

[0003] There is a continuing need for systems and methods for mitigating mycotoxin contamination in process compositions of bioprocessing facilities. Summary of the invention

[0004] The present disclosure includes embodiments of a system for propagating at least one microorganism that expresses one or more enzymes to mitigate mycotoxin contamination. The system includes one or more propagation tanks. The system is configured to propagate at least one microorganism and form a propagation composition comprising at least one microorganism that expresses one or more enzymes to mitigate mycotoxin contamination. The system is configured to be connected to one or more points of a process in a bioprocessing facility to inoculate at least a portion of the propagation composition and / or a composition derived from the propagation composition into one or more processing compositions to mitigate mycotoxin contamination. The bioprocessing facility is configured to produce one or more bioproducts from biomass.

[0005] The present disclosure includes embodiments of a method of propagating at least one microorganism that expresses one or more enzymes to reduce mycotoxin contamination, wherein the method comprises: propagating at least one microorganism that expresses one or more enzymes to reduce mycotoxin contamination in a bioprocessing facility that produces one or more bio-products from biomass, wherein the propagation forms a propagation composition, the propagation composition comprising at least one microorganism that expresses one or more enzymes to reduce mycotoxin contamination; and inoculating at least a portion of the propagation composition and / or a composition derived from the propagation composition into one or more processing compositions of the bioprocessing facility to reduce mycotoxin contamination. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Various examples of the present disclosure will be discussed with reference to the accompanying drawings. These drawings depict only illustrative examples of the present disclosure and should not be considered to limit its scope.

[0007] Figure 1 is a process flow diagram of a non-limiting example of a system for propagating microorganisms expressing enzymes to mitigate mycotoxin contamination according to the present disclosure;

[0008] Figure 2 is a process flow diagram of another non-limiting example of a system for propagating microorganisms expressing enzymes to mitigate mycotoxin contamination according to the present disclosure; and

[0009] Figure 3is a process flow diagram of a non-limiting example of a grain to ethanol process. DETAILED DESCRIPTION

[0010] The present disclosure relates to systems and methods for propagating microorganisms that produce enzymes for mitigating mycotoxins present in biomass, wherein the propagation occurs in a bioprocessing facility that produces one or more bioproducts from the biomass.

[0011] In some embodiments, the system may be portable such that it may be transported to one or more points of use at a bioprocessing facility and connected to one or more points in the overall process of a given bioprocessing facility. By co-locating such a system within a bioprocessing facility, mycotoxins in one or more process compositions may be mitigated on a "just-in-time" basis, if desired, to align with regional and / or seasonal variations in the presence of one or more mycotoxins.

[0012] Mycotoxin contamination can be variable and depends on various conditions including weather. In some years, such as some wet years, there may be large-scale growth of mycotoxin-producing fungi in crops, resulting in serious mycotoxin contamination. In other years, there may be no mycotoxin contamination. Mycotoxin contamination may also vary from region to region. When mycotoxin contamination occurs, it can be transported to the process of bioprocessing facilities via biomass (e.g., grains such as corn). For example, in corn-ethanol processing, mycotoxin contamination may become concentrated during processing. For example, as starch is consumed and fat is separated, the mycotoxin ppm concentration in, for example, dried distillers grains and dissolved materials (DDGS) can rise to an acceptable threshold value for one or more animal species that usually consume DDGS in reasonable amounts. At this level, DDGS prices may be discounted because buyers may have to process or blend DDGS to reduce contamination to acceptable levels. Since mycotoxin mitigation may or may not be required at any given time in a bioprocessing facility (e.g., due to seasonal and geographic variations), which is different from the production of bioproducts from biomass by microorganisms (e.g., ethanol from ethanologen), it may be desirable for the system according to the present disclosure to be portable, such as by mounting the system on a portable skid. For example, mycotoxin contamination on a feedstock (e.g., feed corn to a bioprocessing facility) may be determined so that a decision can be made as to whether to use a portable skid according to the present disclosure.

[0013] In some embodiments, the system for breeding expression enzymes to reduce mycotoxin contamination according to the present disclosure is configured to be movable as a single unit between two or more bioprocessing facilities. Bioprocessing facilities include facilities configured to produce one or more bioproducts (e.g., biochemicals, biofuels, food and feed such as animal feed) from biomass using, for example, microorganisms such as bacteria and fungi. Non-limiting examples of bioproducts include one or more of alcohols (e.g., butanol, ethanol, etc.), carbon dioxide, proteins (e.g., yeast protein, grain protein, etc.), drugs, enzymes, fermented foods and beverages, distilled grains (e.g., WDG, WDGS, DDG, DDGS), and oils. Non-limiting examples of biomass include a variety of crops, such as "cellulosic" materials (e.g., grain fiber, crop waste (e.g., corn stalks), wood, urban waste, etc.) and "starchy" grains (e.g., one or more of corn, barley, rye, sorghum, triticale, and wheat). In some embodiments, the system according to the present disclosure can be made portable so that it can be deployed in a bioprocessing facility when needed. For example, the system can be mounted on a slide so that the slide systems with less than the total number of bioprocessing facilities in the work area can share the slide system as needed. The system mounted on the slide can include a platform plank, pipelines, valves, electrical connection devices, etc., to allow connection to the bioprocessing facilities described herein. The platform plank can be configured to move. For example, the platform plank can be suitable for transportation with a forklift. For example, casters can be mounted on the platform plank. Alternatively, if necessary, the slide system is a convenient unit operation package that can be permanently arranged on each bioprocessing facility. In some embodiments, the slide system that is permanently arranged can be used as a "hub" (as described below) of one or more other bioprocessing facilities located in a certain area.

[0014] The system according to the present disclosure may include one or more breeding tanks, and is configured to breed microorganisms and form a breeding composition, which includes expressing enzymes to reduce microorganisms contaminated with mycotoxins. The number and size of the breeding tanks can be selected according to a variety of factors, such as whether to use two or more different breeding schemes (e.g., different microorganisms and / or breeding culture media), process control, portability, etc. In some embodiments, the system according to the present disclosure may include 1 or more breeding tanks, 2 or more breeding tanks, 3 or more breeding tanks, or even 4 or more breeding tanks. In some embodiments, the system according to the present disclosure may include 4 or less breeding tanks, 3 or less breeding tanks, or even 2 or less breeding tanks. In some embodiments, the volume capacity of the breeding tank can be 10,000 gallons or less, 5,000 gallons or less, 3,000 gallons or less, or even 2,000 gallons or less. In some embodiments, the volume capacity range of the breeding tank can be 10 gallons to 5000 gallons, 50 gallons to 3000 gallons, 100 gallons to 2500 gallons or even 100 gallons to 500 gallons. In some embodiments, the size of the breeding tank can be conducive to breeding expression enzymes as needed ("just in time") to alleviate the microorganisms and / or scale-up (for example, from pilot facilities to commercial facilities) of mycotoxin contamination. For example, on the basis of just in time, the time that the breeding tank with relatively small size (for example, 3000 gallons or less) is used to produce the breeding composition can be relatively short. In addition, the relatively small breeding composition of volume can be conducive to temperature control, and if the breeding composition is subsequently exposed to cracking (as described below), temperature control will be carried out, which can be conducive to quality control.

[0015] The propagation composition outputs of two or more propagation tanks can be connected together and in fluid communication with one or more downstream processes (processes on a portable skid or in a bioprocessing facility). Optionally, the propagation composition output of at least one propagation tank is not connected to the propagation composition output of any other propagation output before being transferred to a downstream process. Two or more propagation tanks can be operated relative to each other, thereby breeding microorganisms in any desired manner. For example, two or more propagation tanks can be operated in series, in parallel, staggered, in stages in time, etc. relative to each other. In addition, a given propagation tank can be operated in any desired manner, such as continuously (continuous feeding and extraction), batchwise, fed-batch, etc.

[0016] A propagation tank refers to a bioreactor suitable for or configured to expose microorganisms in a propagation medium to one or more conditions (e.g., pH, time, temperature, aeration, agitation, etc.) and form a propagation composition (e.g., a propagation culture fluid). In some embodiments, a relatively small tank is easier to control relative to one or more of the above conditions.

[0017] For ventilation, one or more techniques can be used to introduce gas into the propagation medium and ventilate. For example, gas can be introduced into the head space of the propagation tank under sufficient pressure so that the gas diffuses into the propagation medium. For example, gas can be sprayed into the propagation medium at a sufficiently high rate so that the gas bubbles and passes through the propagation medium, and oxygen is transferred into the propagation medium. Alternatively, the propagation medium can be stirred or mixed to promote oxygen transfer into the propagation medium and spread over the propagation medium. For example, a continuous stirred tank reactor (CSTR) can be used to stir or mix the propagation medium. The speed (rpm) of the agitator can be adjusted according to multiple factors such as the viscosity of the propagation tank size, the propagation medium. For example, a bubble column can be used to introduce gas (for example, air and / or oxygen) into the propagation medium. In some embodiments, ventilation can provide a dissolved oxygen concentration in the propagation medium, and its range is greater than zero to 20mg dissolved oxygen from every liter of propagation medium, or even from 1mg to 5mg dissolved oxygen from every liter of propagation medium, as measured at the propagation temperature mentioned below. Alternatively, propagation can be performed anaerobically, which may necessitate the need for larger propagation tanks to achieve similar productivity as aerobic propagation.

[0018] For agitation, one or more techniques can be used to keep the contents of the propagation tank well mixed. Non-limiting examples of agitation devices include top-entry agitators, recirculation pumps, gas pulse systems, aeration, pulsair, side-entry agitators, combinations thereof, and the like.

[0019] The pH of the propagation medium can be at a pH that is conducive to replication (propagation) and production of a desired microbial population within a desired time. In some embodiments, the pH is between 4 and 7, between 4.5 and 7, or even between 4.5 and 6. Techniques for adjusting and maintaining the pH of a propagation medium for propagating microorganisms include, for example, adding one or more acidic substances and / or adding one or more alkaline substances.

[0020] With regard to temperature and time, the contents of the propagation tank (propagation medium) can be maintained at a certain temperature for a period of time to propagate the microorganisms to a desired population size. In some embodiments, the temperature of the propagation medium can be controlled in the range of 40°F to 140°F, 60°F to 120°F, or even 70°F to 100°F. In some embodiments, propagation can be carried out for a period of time, ranging from greater than zero to 72 hours, 1 hour to 48 hours, 2 hours to 48 hours, or even 10 hours to 30 hours.

[0021] The breeding tank can be used to breed the same or different microorganisms. In some embodiments, two or more breeding tanks can be used to breed the same microorganism expressing enzymes to reduce mycotoxin contamination. In some embodiments, at least one breeding tank can be used to breed two or more different microorganisms (called co-cultivation) expressing enzymes to reduce mycotoxin contamination. In some embodiments, at least one breeding tank can be used to breed microorganisms different from the microorganisms bred in the remaining one or more breeding tanks. For example, a microorganism can be a first strain of a microorganism expressing one or more enzymes, and the second microorganism can be a second strain of a microorganism expressing one or more enzymes different from at least one of the enzymes expressed by the first strain. For another example, a microorganism can be a yeast expressing one or more enzymes, and the second microorganism can be a bacterium expressing one or more enzymes different from at least one of the enzymes expressed by the yeast. Using different breeding tanks for microorganisms expressing different enzymes can be beneficial to reliability and / or quality control, thereby helping to meet the specifications of the final product (e.g., DDGS).

[0022] If desired, by breeding at least one microorganism different from other microorganisms, an "enzyme mixture" is allowed to be produced. In some embodiments, growing different microorganisms separately in different breeding tanks can more easily provide optimal growth conditions for different microorganisms, and relatively better control the ratio of various enzymes when combined to form an enzyme mixture. In some embodiments, at least a portion of the breeding composition in two or more breeding tanks can be combined in any desired ratio to make an enzyme mixture. For example, a system according to the present disclosure can be configured to take out and adjustably combine at least a portion of the contents in two breeding tanks in a volume ratio of 1: 1 or different from 1: 1. Any desired ratio can be selected according to one or more factors, such as the number of breeding tanks and the total protein amount (protein abundance) of the breeding composition. Changing the volume ratio can affect the activity of the entire enzyme mixture.

[0023] A variety of microorganisms and enzymes expressed by microorganisms can be produced using the propagation system according to the present disclosure to mitigate mycotoxin contamination. Similarly, a variety of mycotoxins can be mitigated based on one or more enzymes expressed by the microorganisms. Enzymes can mitigate mycotoxin contamination by catalyzing the conversion of mycotoxins to less toxic forms.

[0024] Non-limiting examples of mycotoxins that may be mitigated are selected from aflatoxin, ochratoxin A, patulin, T-2, HT-2, beauvericin, neosolaniol, nivalenol, deoxynivalenol (DON), 3-ADON, 15-ADON, trichothecenes, ochratoxin, zearalenone, and combinations thereof.

[0025] Non-limiting examples of enzymes that can be used to reduce mycotoxins are selected from aflatoxin oxidase, 3-O acetyltransferase, peroxidase, F420H2-dependent reductase, Ery4, UDP-glycosyltransferase, laccase, manganese peroxidase, deoxynivalenol hydroxylase, lactonohydrolase, xylanase, peroxidase, 2cys-peroxidase and combinations thereof. In some embodiments, the enzyme can be a combination of enzymes, for example, an enzyme system or mixture that works in two or more steps to remove the toxicity of mycotoxins. For example, the enzyme system can be DepA / DepB, wherein DepA converts DON into 3-keto-DON, and DepB (a kind of NADPH dependent dehydrogenase) catalyzes the reduction of 3-keto-DON to 3-epi-DON.

[0026] Non-limiting examples of microorganisms that express enzymes to mitigate mycotoxin contamination and that can be propagated in systems according to the present disclosure are described in U.S. Publication No. 2021 / 0403842 (Manning et al.), the entire contents of which are incorporated herein by reference.

[0027] The propagation medium may include at least water, one or more carbon sources, and one or more nutrient sources for the growth and propagation of the microorganisms.

[0028] As used herein, "carbon source" refers to one or more compounds that contain at least one carbon atom and can be used by microorganisms (e.g., yeast) to grow and / or reproduce to produce additional biomass. Exemplary carbon sources include, but are not limited to: monosaccharides, such as dextrose, fructose, galactose, mannose, xylose, etc.; disaccharides, such as lactose, maltose, sucrose, cellobiose, etc.; oligosaccharides; polysaccharides, such as cellulose, hemicellulose, starch, xylene, etc.; single carbon sources containing only one carbon atom, such as methanol; and polyols, such as glycerol.

[0029] In addition to the carbon source, the nutrient source of non-carbon source can also be included to help microbial reproduction. As used herein, "nutrient source" refers to one or more substances or nutrients that can be used by microorganisms to grow and / or reproduce to produce other microorganisms and are different from carbon source. In some embodiments, the nutrient source can be used as a carbon source. The nutrient source and carbon source can be obtained from a bioprocessing facility and / or transported to a bioprocessing facility from a supplier. One or more breeding tanks can be connected with one or more sources to receive the content from the source and form a breeding medium (for example, providing water, nutrients and carbon for at least one source of microbial reproduction). In some embodiments, according to the system of the present disclosure, one or more points of the process in the bioprocessing facility can be connected and fluidly communicated to receive at least a portion of the processing composition to be used as a nutrient source and / or carbon source. For example, at least one breeding tank is configured to be connected with one or more points in the bioprocessing facility to receive at least a portion of the processing composition. In some embodiments, using one or more processing compositions from a bioprocessing facility can help regulate microorganisms, (if necessary) by eliminating the need to transport this source to a bioprocessing facility to reduce the emission of carbon dioxide, etc. The processing composition from the bioprocessing facility includes the processing composition of the fermentation upstream and / or the fermentation downstream. Non-limiting examples of process compositions from bioprocessing facilities include sugar compositions (e.g., sugar slipstreams), starch compositions (e.g., starch slipstreams), distiller's grains compositions, etc. Sugar slipstreams are described in U.S. Pat. No. 10,233,466 (Redford), the entire contents of which are incorporated herein by reference. Starch slipstreams are described in U.S. Pat. No. 10,793,879 (Redford et al.), the entire contents of which are incorporated herein by reference.

[0030] As used herein, "stillers grain composition" refers to the back-end composition of the fermentation process after one or more bioproducts are separated (e.g., by distillation) from beer to form at least one target bioproduct stream (e.g., ethanol) and one or more byproduct streams (e.g., whole stillers). The stillers composition can include whole stillers, at least one stillers composition derived from whole stillers, and combinations thereof. In some embodiments, the whole stillers is derived from distilled beer in a dry-grinding corn ethanol biorefinery. Non-limiting examples of stillers compositions derived from whole stillers include stillage water, concentrated stillage water (syrup), skimmed syrup, skimmed emulsion, clarified stillage water, stillage oil, distilled stillage, distilled yeast, and the like. Skimmed syrup and skimmed emulsion are examples of stillage compositions remaining after fat (e.g., corn oil) is separated from syrup and emulsion, respectively, and may be referred to as "skimmed stillage compositions." In Figure 2In each of the drawings to FIG. 5B, non-limiting illustrations showing one or more of the above-mentioned lees compositions are shown (discussed below). Non-limiting examples of methods and systems for processing lees streams are also described in U.S. Patent No. 8,702,819 (Bootsma); U.S. Patent No. 9,061,987 (Bootsma); U.S. Patent No. 9,290,728 (Bootsma); U.S. Patent No. 10,059,966 (Bootsma); U.S. Patent No. 11,248,197 (Bootsma); and U.S. Publication No. 2020 / 0140899 (Bootsma); wherein the entire contents of each of the patent documents are incorporated herein by reference. The lees composition also includes a back-end composition of a fermentation process after separating one or more bioproducts from beer using a separation technique other than distillation (e.g., membrane separation, gas stripping, adsorption).

[0031] Alternatively, one or more inducers may be present to promote enzyme production. Non-limiting examples of inducers include IPTG (isopropyl BD-1-thiogalactopyranoside), galactose, glucose and combinations thereof. In addition, in some embodiments, the temperature may be selected to induce enzyme production. Alternatively, if desired, the use of inducers may be avoided. For example, a constitutive promoter that continuously produces an enzyme throughout the life cycle of the microorganism may be used, so that no inducer is required.

[0032] Optionally, one or more cofactors may be present for microbial growth, and / or for enzyme production and / or post-cleavage functions to mitigate mycotoxin contamination. Non-limiting examples of cofactors include ferric chloride, 5-aminolevulinic acid, NADH (nicotinamide adenine dinucleotide), and combinations thereof.

[0033] When the propagation in the propagation tank is complete, the propagation composition can be inoculated as is into one or more processing compositions of the bioprocessing facility to mitigate mycotoxin contamination, and / or further processed to form a composition derived from the propagation composition before inoculation. As used herein, the propagation composition may also be referred to as the propagation broth or full propagation broth formed in the propagation tank. In some embodiments, direct inoculation of the fermenter in the commercial bioprocessing facility with the full propagation broth from one or more propagation tanks may be beneficial to a just-in-time solution. As discussed below, examples of compositions derived from the propagation composition / propagation broth include concentrated propagation broth, lysate, concentrated lysate, enzymes separated from the propagation broth, enzymes separated from the lysate, and combinations thereof.

[0034] In some embodiments, after the desired growth is obtained, the culture can be cooled to help the microorganisms settle to the bottom of the propagation tank, whereby excess liquid can be removed to concentrate the microorganisms before the microorganisms are lysed. In some embodiments, gravity can be used to remove excess liquid without a centrifuge (e.g., sedimentation), which can be energy-efficient and maintenance-friendly.

[0035] Alternatively, the propagation culture fluid can be exposed to a lysis system to form a lysate. The lysate is formed by cracking a microorganism expressing an enzyme that alleviates mycotoxins. The cracking of a microorganism refers to the cell fragmentation that causes the outer boundary or cell membrane to rupture or destroy, so as to release intercellular material. The lysis microorganism can release the enzyme that alleviates mycotoxins, so that the enzyme is relatively more useful for alleviating the mycotoxins in the processing composition of a bioprocessing facility. In addition, the lysis microorganism has substantially killed the microorganism, which advantageously avoids competition (for example, competing to consume carbon sources, such as sugar) between the microorganism expressing the mycotoxin mitigation enzyme and any other microorganism (for example, ethanologen) in the bioprocessing facility for preparing one or more bioproducts from biomass. In some embodiments, the microorganism can release the enzyme, and thus the lysis system can be omitted.

[0036] A variety of techniques can be used to lyse microorganisms in a lysis system. There are lysis methods available at both macroscopic and microscopic scales. Lysis can be performed using one or more mechanical methods, one or more non-mechanical methods, and combinations thereof. In some embodiments, a recirculation loop can be used to recirculate the output of the lysis operation and repeatedly expose to the lysis operation until a fully lysed product is obtained.

[0037] In some embodiments, as mentioned above, the content of the breeding tank can be cooled and / or concentrated before cracking. In addition, temperature can be controlled in the whole cracking process so that temperature (lower than the fusing point of enzyme) will not exceed the temperature that makes enzyme inactivation to excessive degree thereby causing lysate invalid. Similarly, during any other process or storage period that occurs between breeding and inoculation, the temperature of enzyme can be controlled to be sufficiently low, to avoid enzyme inactivation to excessive degree. For example, a temperature control system can be included so that lysate is maintained in a frozen state until use. In some embodiments, the temperature that the system is configured to control the enzyme present in the control system is 140 ° F or lower, 130 ° F or lower, 120 ° F or lower, 110 ° F or lower or even 100 ° F or lower. System according to the present disclosure can be configured in many ways, to control the temperature in the described whole breeding and / or cracking process. In some embodiments, a portable carriage can include a fluid jacketed container that is connected with one or more cooling tower waters, heat exchangers or one or more other temperature-controlled water sources in a bioprocessing facility.

[0038] The non-limiting example of mechanical lysis includes the use of a homogenizer, a bead mill, etc. A homogenizer such as a high pressure homogenizer (HPH) is an example of a microorganism disruption device. In HPH, cells in the culture medium are forced to pass through a hole valve using high pressure, and are compressed when the cells enter the orifice and expand when discharged, resulting in cell membrane fragmentation due to the high shear force at the orifice. The lysis using HPH can involve passing the microorganism through the HPH one or more times, depending on the selected pressure and / or the microorganism involved. For example, in some embodiments, yeast can be passed through the HPH at a pressure of 1000 bar to 1200 bar, to cause a sufficient degree of lysis. In some embodiments, bacteria can be lysed at a lower pressure. For example, in some embodiments, microorganisms can be repeatedly passed through the HPH at a pressure lower than 1000 bar (e.g., from 650 bar to lower than 1000 bar), to cause a sufficient degree of lysis.

[0039] Bead milling involves disrupting cells by agitating tiny beads made of glass, steel or ceramic with a cell suspension at high speed. The beads collide with the cells, breaking the cell membrane under the action of shear forces and releasing the components within the cell.

[0040] Non-mechanical lysis methods include one or more physical lysis methods, one or more chemical lysis methods, and one or more biological lysis methods.

[0041] As used herein, physical lysis refers to a non-contact method that utilizes one or more external forces (such as heat, pressure and acoustic energy) to rupture the cell membrane. Non-limiting examples of physical lysis include thermal lysis, cavitation, osmotic shock, etc. Thermal lysis involves repeated freeze-thaw cycles, which causes ice to form on the cell membrane and helps to rupture the cell membrane. Cavitation involves the formation of cavities or bubbles and subsequent rupture. Such cavities can be formed by reducing local pressure, and reducing local pressure can be achieved by increasing speed, ultrasonic vibration, etc. The reduction in pressure causes the cavity to collapse and releases a large amount of mechanical energy in the form of a shock wave, which propagates through the culture medium. The high energy of the shock wave can cause the cell membrane to disintegrate. Ultrasonic and fluid dynamic methods can be used to produce cavitation for broken cells.

[0042] Chemical lysis refers to the use of a lysis buffer to disrupt the cell membrane. Non-limiting examples of chemical lysis include alkaline lysis and detergent lysis. Alkaline lysis can disrupt the cell membrane by changing the pH. Detergents can be added to detergent lysis to solubilize membrane proteins and disrupt cell membranes.

[0043] Non-limiting examples of biological lysis include the use of one or more enzymes, such as lysozyme, lysostaphin, digestase, cellulase, protease, or glycanase.

[0044] For systems according to the present disclosure, the lysis system can be directly or indirectly connected to one or more propagation tanks, and the lysis system is configured to form a lysate using at least a portion of the contents from the one or more propagation tanks. In some embodiments, two or more propagation tanks can be multiplexed into a single lysis system so that they are staged in time and cycled into the lysis system in a manner that produces lysate. In some embodiments, multiplexing propagation tanks with a single lysis system allows for the production of lysate in a just-in-time manner for continuous fermentation.

[0045] The system according to the present disclosure including the lysis system can customize the lysis system, thereby using one or more lysis techniques in a manner that is conducive to just-in-time delivery of the lysate and / or allows the lysate to be provided in a relatively simple manner. For example, in some embodiments, the lysis system can include one or more lysis techniques that are relatively gentle and / or avoid additional processing of the lysate before inoculating the processing composition with the lysate. Non-limiting examples of lysis techniques that are relatively gentle and / or avoid additional processing of the lysate include mechanically and / or physically lysing microorganisms to form microbial lysates.

[0046] In some embodiments, the lysis systems and methods according to the present disclosure do not include chemical lysis and / or biological lysis. For example, not using chemical lysis can avoid having to subsequently process the lysate to change one or more properties before inoculating the processing composition. For example, in some enzyme facilities, more aggressive lysis techniques use chemical lysis agents. The lysate is then typically processed to remove components that would be harmful to, for example, fermentation.

[0047] Alternatively, the system according to the present disclosure may include (connected to and fluidically communicated with) one or more enzyme stabilizers and / or one or more protease inhibitors. The system may be configured to combine one or more protease inhibitors and / or one or more stabilizers with a propagation composition and / or a composition derived from a propagation composition at one or more points in the system. Protease inhibitors include one or more chemicals that block or inhibit the action of corresponding proteases (e.g., endogenous proteases), and once an organism is lysed, the protease can contact all proteins inside and outside the cell. In some embodiments, protease inhibitors may be added during lysis and / or before storage to maintain the functionality of the enzyme that mitigates mycotoxins and not degrade the enzyme by the natural (endogenous) protease function of the microorganism. Non-limiting examples of protease inhibitors include 4-benzenesulfonyl fluoride hydrochloride (AEBSF), aprotinin, benstatin, trans-epoxysuccinyl-L-leucylamido (4-guanidino) butane (E64), leupeptin, pepstatin A and combinations thereof.

[0048] Enzyme stabilizers include one or more chemicals that help maintain the function of the enzyme that reduces mycotoxins, especially when the enzyme is present in the lysate. In some embodiments, stabilizers can be added during lysis and / or prior to storage to maintain the function of the enzyme that reduces mycotoxins. Non-limiting examples of stabilizers include glycerol, one or more sugars, one or more dextran, polyethylene glycol, one or more amino acids and their derivatives, one or more salts, one or more surfactants, and combinations thereof.

[0049] Because the system according to the present disclosure can be co-placed in a bioprocessing facility, it is possible to avoid the need for transportation to the bioprocessing facility, thereby requiring little or no protease inhibition and / or stabilization, especially if the microorganisms that produce enzymes for reducing mycotoxins are propagated according to a just-in-time program. In some embodiments, the system according to the present disclosure located in a bioprocessing facility can serve as a "hub" for one or more other bioprocessing facilities located in a relatively local area. For example, if each fermentation reactor of a bioprocessing facility located in a relatively local area requires a relatively small dosage, it may make economic sense to utilize a system according to the present disclosure located in another bioprocessing facility. In some embodiments, a relatively small dosage can include 150 gallons or less, 100 gallons or less, or even 50 gallons or less per fermenter. Containers with corresponding capacities can be filled and transported to one or more other bioprocessing facilities located in a relatively local area.

[0050] In some embodiments, if desired, optional enzyme stabilizers and / or protease inhibitors are simple short-term inputs (relatively less inhibitors and / or stabilizers) because long-term storage can be avoided. In some embodiments, the system is configured to combine one or more protease inhibitors with the lysate in a weight ratio of 1:10,000 to 1:10, 1:10,000 to 1:500, or even 1:10,000 to 1:1000. In some embodiments, the system is configured to combine one or more enzyme stabilizers (e.g., glycerol) with the propagation composition and / or a composition derived from the propagation composition in an amount greater than zero to 15% by weight.

[0051] Alternatively, a system according to the present disclosure may include (being connected to and being in fluid communication with) a system configured to separate and / or concentrate enzymes from a composition (e.g., lysate) of a propagation composition or a composition derived from a propagation composition. Concentrated enzymes may contribute to meeting the requirements of "generally recognized as safe" (GRAS). In some embodiments, a filtration system may be configured to separate and concentrate enzymes from lysate fragments. The non-limiting example of a filtration system may include two or more gradually decreasing filter openings to separate and concentrate enzymes from lysate. For example, a filtration system may include a first stage filter having a molecular weight cutoff of 100K Dalton's membrane, so that when filtering the lysate with the membrane, free soluble proteins may flow through the membrane to form a permeate, and most of the cell debris remain in the retentate. A filtration system may include a second stage filter having a 10KDa membrane, so that when filtering the permeate from the first stage filter with the membrane, the retentate contains the enzyme of interest in a concentrated form, and the permeate will mainly contain soluble small proteins, minerals and / or the like.

[0052] Optionally, the system according to the present disclosure may include (connected to and in fluid communication with) a storage system to temporarily store the propagation composition or a composition derived from the propagation composition. The storage system may facilitate the formation of an enzyme mixture by using a single storage tank to combine the contents of two or more propagation tanks (as described herein); and / or the storage system temporarily stores the propagation composition or a composition derived from the propagation composition before inoculating the processing composition in a bioprocessing facility. If desired, a dedicated storage tank may be used for each propagation tank.

[0053] Systems according to the present disclosure can be connected to one or more points in a process in a bioprocessing facility and in fluid communication with these one or more points to inoculate at least a portion of a propagation composition and / or a composition derived from a propagation composition (e.g., a lysate) into one or more processing compositions in the bioprocessing facility to reduce mycotoxin contamination. Non-limiting examples of processing compositions in a bioprocessing facility include feedstock compositions, milling compositions, soaking compositions, propagation compositions (e.g., containing ethanologen), saccharification compositions, fermentation compositions, lees compositions (see above), and combinations thereof. For example, a propagation composition comprising a microorganism that produces an enzyme for reducing mycotoxins can be introduced into a fermentable composition that also includes a microorganism that ferments sugars to produce a bioproduct such as ethanol (also known as co-culture or chimeric fermentation). The following will be combined with Figure 3 Non-limiting examples of such processing compositions in bioprocessing facilities are further discussed.

[0054] Systems according to the present disclosure may include a control system including one or more controllers in electrical communication with one or more sensors, valves, switches, etc. The controllers may send and receive signals in one or more processes in a bioprocessing facility and execute program instructions to prepare a propagation composition or a composition derived from the propagation composition and deliver it to one or more process compositions in the bioprocessing facility.

[0055] In some embodiments, the presence of mycotoxins in the incoming grain and / or one or more processing compositions in a bioprocessing facility can be determined, and if the presence of mycotoxins is determined, then a system according to the present disclosure can be used to produce a propagation composition or a composition derived therefrom. If the absence of mycotoxins is determined, the enzyme production system according to the present disclosure can be idled, shut down, or sent to another bioprocessing facility where the presence of mycotoxins is determined. For example, the concentration of one or more mycotoxins in the incoming grain and / or one or more processing compositions in a bioprocessing facility can be determined. A variety of methods can be used to detect the presence of mycotoxins or to determine their concentration. For example, a system that can be obtained from NEOGEN purchased by the company TM VERATOX TM DON 2 / 3 kit to determine the presence of DON.

[0056] In some embodiments, mycotoxin levels can be monitored and communicated to a controller that can be used to control the propagation of microorganisms expressing enzymes that mitigate mycotoxin contamination. Mycotoxin levels can be monitored at any point in a bioprocessing facility. Alternatively, monitoring can be omitted and microorganisms expressing enzymes that mitigate mycotoxin contamination can be propagated based on expected values ​​of mycotoxins. When no toxins are present, the propagation system can be idle to reduce costs. For example, grain can be tested before entering a bioprocessing facility, and the propagation system can be employed in crop years and regions where mycotoxin levels are determined to exceed a threshold.

[0057] The following discussion refers to Figures 1 to 3 . Schematic diagrams are for illustration purposes only and are not necessarily drawn to scale.

[0058] Figure 1 1 is a schematic diagram showing a non-limiting embodiment of a system 100 for propagating one or more microorganisms expressing enzymes to mitigate mycotoxin contamination. The system 100 includes three propagation tanks 101, 102, 103. The system 100 is configured to propagate microorganisms in each propagation tank and form a propagation composition. The microorganisms and / or enzymes that mitigate mycotoxin contamination may be the same or different in each propagation tank 101, 102, 103.

[0059] Lysis system 120 is connected to each of the propagation tanks 101, 102, 103 and receives the contents of propagation tank 101 via stream 104, the contents of propagation tank 102 via stream 105, and the contents of propagation tank 103 via stream 106. Lysis system 120 is configured to expose the contents of each of the propagation tanks 101, 102, 103 to one or more lysis techniques and form a lysate in stream 107. As shown, during the lysis process, at least a portion of the stream is recycled to each of the propagation tanks 101, 102, 103 via streams 108, 109, 110, respectively. In addition, as shown, during the lysis process, one or more protease inhibitors 130 may be introduced into streams 108, 109, 110, respectively, via streams 131, 132, 133. In addition, one or more protease inhibitors 130 may be introduced into lysate stream 111 via stream 134 after lysis and prior to storage system 140.

[0060] As shown, system 100 is configured to be connected to inoculate at least a portion of the lysate from storage system 140 into one or more process compositions in a bioprocessing facility (e.g., into a fermentation broth or upstream of a fermentation broth) via stream 141. Figure 3 Non-limiting examples of such one or more processing compositions are discussed further.

[0061] Figure 22 is a process flow diagram showing a non-limiting embodiment of a system 200 for propagating microorganisms expressing enzymes to mitigate mycotoxin contamination. The system 200 includes a propagation tank 230. When valve 236 is closed, one or more components 202 can be introduced into the propagation tank 230 to produce a propagation medium. For example, the one or more components can include an inoculum, a nutrient source and / or a carbon source (e.g., vinasse), process water, etc. The inoculum can include one or more microorganisms expressing enzymes to mitigate mycotoxin contamination. The system 200 is configured to propagate microorganisms in the propagation tank 230 and form a propagation composition 235. The propagation tank 230 can include a stirring mechanism 232 driven by a motor 234. A homogenizer 238 is located downstream of the propagation tank 230, wherein a recirculation line 248 returns a homogenizer output 240 to the propagation tank 230 when valve 246 is closed and valve 244 is opened. The recirculation loop 248 can be maintained until it is necessary to break the microorganisms and produce an enzyme lysate 250. After the desired amount of recirculation, valve 244 can be actuated closed and valve 246 between propagation tank 230 and lysate storage tank 260 can be actuated open to allow lysate 250 to flow to storage tank 260 and then to one or more process compositions in a bioprocessing facility (e.g., yeast propagation and / or fermentation vessel) via stream 256. Lysate storage tank 260 can include a stirring mechanism 252 driven by motor 254.

[0062] The system 200 adopts a relatively gentler mechanical lysis method through a homogenizer 238 to avoid the subsequent cleaning system that may be required for a chemical lysis method. Chemical lysis includes adding one or more chemicals to help loosen or lyse the membrane of the microorganism. The non-limiting example of this chemical includes one or more cell wall degrading enzymes and / or one or more chemicals that can dissolve the membrane / cell wall. Since the added chemical can have the same potential for lysis to be used for the microorganism (e.g., yeast) that is propagated and / or fermented in a bioprocessing facility (e.g., for ethanol production), the lysate formed by chemical lysis can be "cleaned" before being added to downstream propagation and / or fermentation processes. Otherwise, the presence of chemical substances may have an undue impact on propagation and / or fermentation (e.g., causing fermentation to stagnate or never start). If desired, a filtration system can be used to perform separation of chemical lysis compounds from the lysate, similar to that discussed above about separation and concentration enzymes from the lysate. However, due to the potential harmful effects of the chemical substances downstream, the presence of any residual chemical substances after cleaning can be monitored, which may increase complexity and cost.

[0063] also, Figure 2 The system 200 may avoid relatively long term storage and transportation and thus require little or no stabilization.

[0064] The propagation tank 230 and the lysate storage tank 260 are jacketed containers that allow the flow of temperature-controlled water to control the temperature of the contents. Figure 2 As shown, when valve 210 is open, a portion 206 of the temperature-controlled water 204 can flow into the jacket portion of the propagation tank 230; and when valve 212 is open, a portion 208 of the temperature-controlled water 204 can flow into the jacket portion of the lysate storage tank 260. In addition, when valve 218 is open, water 214 can flow out of the jacket portion of the propagation tank 230; and when valve 220 is open, water 216 can flow out of the jacket portion of the lysate storage tank 260. Streams 214, 216 combine to form stream 222.

[0065] Protease inhibitors can be introduced by actuating a valve open 242. Protease inhibitors are a simple short term stabilizing input and can be added during and / or after lysis.

[0066] Figure 3Flow chart of the conversion process of grain to ethanol, depicting an ethanol biorefining operation 300 that produces animal feed as a byproduct. Grains 301 contaminated by toxins are used as raw materials for the production of ethanol in a biorefinery. The size of grain 301 is reduced in a milling system 303. The milled grain 305 is mixed with water and further treated (e.g., heat treated and / or enzymatically treated) to convert starch and fiber into fermentable sugars in a saccharification system 306. The resulting slurry 310 is combined with ethanologen (e.g., yeast) to convert sugar into ethanol in a fermentation system 312. In some embodiments, saccharification and fermentation can occur simultaneously in a single system (e.g., in an SSF fermentation system). Fermentation product or beer 322 comprises ethanol, water, oil, dissolved solids, toxins, protein, yeast, and residual carbohydrates (including starch, sugar, and fiber). Before further processing, beer 316 can be collected in a beer storage system 318. Beer 322 is distilled in distillation system 324 to separate ethanol 325 from other components of beer 322 (called whole grains 328). Whole grains 328 are processed into cake 334 and lees water 332 in lees separation system 330. Cake 334 contains more solid particulate matter (including fiber, protein, yeast and residual solid starch) from beer 322 and some liquid (including water, oil and dissolved solids). Lees water 332 contains more liquid from beer 322, including water, oil and dissolved solids. Toxins are distributed in cake 334 and lees water 332. However, because this toxin is soluble in water, more toxins are contained in lees water 332. Lees water 332 is concentrated in evaporation system 342 to form syrup 344. Before further processing, syrup 344 can be collected in syrup storage system 348. Syrup 350 and cake 334 may be combined and dried in drying system 354 to produce distillers dried grains with lysates (DDGS) 358. Examples of biorefinery operations are described in U.S. Pat. No. 7,842,484, U.S. Pat. No. 8,409,640, U.S. Pat. No. 7,919,291, U.S. Pat. No. 8,470,550, U.S. Pat. No. 8,748,141, U.S. Pat. No. 8,679,793, U.S. Pat. No. 8,597,919, U.S. Pat. No. 8,702,819, U.S. Pat. No. 4,092,434, and U.S. Pat. No. 4,316,956, all of which are incorporated herein by reference.

[0067] As described herein, at least a portion of a propagation composition and / or a composition derived from a propagation composition may be used to inoculate one or more processing compositions to mitigate mycotoxin contamination. Figure 3In an example of, a propagation composition (and / or a composition derived therefrom) can be introduced into one or more process compositions of a biorefining operation. One or more enzymes introduced into the process composition react with the toxin (e.g., change the ring structure of the toxin) to form a less toxic or non-toxic compound. Because toxins enter the biorefinery with the grain, the toxins are present in many biorefinery process streams, and the propagation composition (and / or a composition derived therefrom) can be introduced into any of these process streams / locations to react with the toxins. For example, the propagation composition (and / or a composition derived therefrom) can be introduced into: stream 302 mixed with grain 301 before or during milling; stream 304 mixed with milled grain 305 before or during saccharification 306; stream 308 mixed with slurry 310 before or during fermentation 312; stream 314 mixed with beer 316 before or during distillation 324 or stream 320 mixed with beer 322; in stream 326 mixed with whole grains 328 before or during grain separation 330; in stream 338 mixed with grain water 332 before or during evaporation 342; in stream 346 mixed with syrup 344 or stream 352 mixed with syrup 350 before or during drying 354; in stream 340 mixed with cake 334 before or during drying 354; and / or in stream 356 mixed with DDGS 358.

[0068] Although according to the present disclosure, toxins can be repaired by introducing into any one or more of logistics 302, 304, 308, 314, 320, 326, 338, 340, 346, 352 and 356, certain factors enhance the effectiveness of repair. It has been found that mixing in an aqueous environment, relatively long residence time and / or suitable enzyme reaction conditions are conducive to reaction. Similarly, if a logistics in which toxins are concentrated is selected, a lesser volume of breeding composition (and / or a composition derived therefrom) may be required. In ethanol operations, the toxin level in DDGS can be concentrated three to five times. Almost all of one or more toxins are concentrated by the process flow of vinasse to syrup. A portion of vinasse 336 is typically used as recirculating water, returned to previous process steps, such as saccharification 306 and / or fermentation 312.

[0069] The following are exemplary numbered implementations of the present disclosure:

[0070] 1. A system for propagating at least one microorganism expressing one or more enzymes to mitigate mycotoxin contamination, wherein the system comprises one or more propagation tanks, wherein the system is configured to propagate at least one microorganism and form a propagation composition comprising at least one microorganism expressing one or more enzymes to mitigate mycotoxin contamination, and wherein the system is configured to be connected to one or more points of a process in a bioprocessing facility to inoculate at least a portion of the propagation composition and / or a composition derived from the propagation composition into one or more processing compositions to mitigate mycotoxin contamination, wherein the bioprocessing facility is configured to produce one or more bioproducts from biomass.

[0071] 2. A system according to embodiment 1, wherein the composition derived from the propagation composition is selected from a concentrated propagation culture fluid, a lysate of at least one microorganism, a concentrated lysate of at least one microorganism, an enzyme isolated from a propagation culture fluid of at least one microorganism, an enzyme isolated from a lysate of at least one microorganism, and combinations thereof.

[0072] 3. The system according to any of the preceding embodiments, wherein the system is configured to be movable as a single unit between two or more bioprocessing facilities, wherein each bioprocessing facility is configured to produce one or more bioproducts from biomass.

[0073] 4. The system according to any of the preceding embodiments, wherein each propagation tank has a volume capacity of 10,000 gallons or less.

[0074] 5. The system according to any of the preceding embodiments, wherein each propagation tank has a volume capacity of 3000 gallons or less.

[0075] 6. The system according to any of the preceding embodiments, wherein the one or more propagation tanks comprises two or more propagation tanks, wherein each propagation tank comprises at least one microorganism expressing one or more enzymes to reduce mycotoxin contamination, and wherein at least one of the two or more propagation tanks comprises at least one microorganism expressing one or more enzymes to reduce mycotoxin contamination that is different from the at least one microorganism expressing one or more enzymes to reduce mycotoxin contamination present in another propagation tank.

[0076] 7. The system according to embodiment 6, wherein the system is configured to remove and adjustably combine at least a portion of the contents of at least two propagation tanks at a volume ratio of 1:1 or different from 1:1.

[0077] 8. The system according to any of the preceding embodiments, wherein at least one propagation tank is configured to connect to one or more points in a bioprocessing facility to receive a composition selected from the group consisting of a nutrient source, a carbon source, and combinations thereof.

[0078] 9. The system according to any of the preceding embodiments, further comprising a storage system in fluid communication with at least one breeding tank, wherein the storage system is configured to receive at least a portion of the contents directly or indirectly from at least one breeding tank, wherein the storage system comprises one or more storage containers, wherein each storage container has a capacity of 150 gallons or less.

[0079] 10. The system according to any of the preceding embodiments, further comprising a source of one or more protease inhibitors, wherein the system is configured to combine the one or more protease inhibitors with the propagation composition and / or a composition derived from the propagation composition at one or more points in the system.

[0080] 11. The system of embodiment 10, wherein the system is configured to combine the one or more protease inhibitors with the lysate at a weight ratio of 1:10000 to 1:10.

[0081] 12. The system of embodiment 10, wherein the system is configured to combine the one or more protease inhibitors with the lysate at a weight ratio of 1:10000 to 1:1000.

[0082] 13. The system according to any of the preceding embodiments, further comprising a source of one or more stabilizers, wherein the system is configured to combine the one or more stabilizers with the propagation composition and / or a composition derived from the propagation composition at one or more points in the system.

[0083] 14. The system according to any of the preceding embodiments, further comprising a lysis system in fluid communication with the one or more propagation tanks, wherein the lysis system is configured to form a lysate using at least a portion of the propagation composition from the one or more propagation tanks.

[0084] 15. The system according to any of the preceding embodiments, wherein the system is configured to control the temperature of the enzymes present in the system to 110°F or less.

[0085] 16. A method for propagating at least one microorganism that expresses one or more enzymes to reduce mycotoxin contamination, wherein the method comprises: propagating at least one microorganism that expresses one or more enzymes to reduce mycotoxin contamination in a bioprocessing facility that produces one or more bioproducts from biomass, wherein the propagation forms a propagation composition, the propagation composition comprising at least one microorganism that expresses one or more enzymes to reduce mycotoxin contamination; and inoculating at least a portion of the propagation composition and / or a composition derived from the propagation composition into one or more processing compositions of the bioprocessing facility to reduce mycotoxin contamination.

[0086] 17. The method according to any of the preceding embodiments, further comprising lysing at least one microorganism expressing one or more enzymes to reduce mycotoxin contamination to form a lysate, wherein the lysing comprises mechanically and / or physically lysing the microorganism expressing the enzyme to reduce mycotoxin contamination to form a lysate.

[0087] 18. The method of embodiment 17, wherein lysing does not include chemically lysing at least one microorganism expressing one or more enzymes to mitigate mycotoxin contamination to form a lysate.

[0088] 19. The method according to any of the preceding embodiments, wherein the biomass comprises one or more cereals.

[0089] 20. The method according to any of the preceding embodiments, wherein the one or more processing compositions are selected from the group consisting of a feedstock composition, a milling composition, a steeping composition, a propagation composition, a saccharification composition, a fermentation composition, a distiller's grains composition, and combinations thereof.

[0090] 21. The method according to any of the preceding embodiments, further comprising: determining whether one or more mycotoxins are present in the incoming grains and / or one or more processing compositions of the bioprocessing facility, wherein the determination comprises measuring the concentration of the one or more mycotoxins in the incoming grains and / or one or more processing compositions of the bioprocessing facility; and if it is determined that one or more mycotoxins are present in the incoming grains and / or one or more processing compositions of the bioprocessing facility, then breeding at least one microorganism expressing one or more enzymes to reduce mycotoxin contamination in the bioprocessing facility, wherein if it is determined that one or more mycotoxins are not present in the incoming grains and / or one or more processing compositions of the bioprocessing facility, then not breeding at least one microorganism expressing one or more enzymes to reduce mycotoxin contamination in the bioprocessing facility.

[0091] 22. The method according to any of the preceding embodiments, further comprising: determining the presence or absence of one or more mycotoxins in the incoming grains and / or one or more processing compositions of different bioprocessing facilities, wherein the determination comprises measuring the concentration of one or more mycotoxins in the incoming grains and / or one or more processing compositions of different bioprocessing facilities; and if it is determined that one or more mycotoxins are present in the incoming grains and / or one or more processing compositions of different bioprocessing facilities, then breeding at least one microorganism expressing one or more enzymes to reduce mycotoxin contamination in the bioprocessing facility, wherein if it is determined that one or more mycotoxins are not present in the incoming grains and / or one or more processing compositions of different bioprocessing facilities, then not breeding at least one microorganism expressing one or more enzymes to reduce mycotoxin contamination in the bioprocessing facility.

Claims

1. A system for propagating two or more microorganisms expressing one or more enzymes to mitigate mycotoxin contamination, wherein the system comprises two or more propagation tanks, wherein each of the two or more propagation tanks comprises at least one microorganism expressing one or more enzymes to mitigate mycotoxin contamination, wherein at least one of the two or more propagation tanks comprises at least one microorganism expressing one or more enzymes to mitigate mycotoxin contamination, the at least one microorganism being different from the at least one microorganism expressing one or more enzymes to mitigate mycotoxin contamination present in another propagation tank, wherein the system is configured to propagate the at least one microorganism present in each of the two or more propagation tanks and form a propagation composition comprising the at least one microorganism expressing one or more enzymes to mitigate mycotoxin contamination, and wherein the system is configured to connect to one or more points of a process in a bioprocessing facility to inoculate at least a portion of the propagation composition and / or a composition derived from the propagation composition from each of the two or more propagation tanks into one or more process compositions to mitigate mycotoxin contamination, wherein the bioprocessing facility is configured to produce one or more bioproducts from biomass.

2. The system of claim 1 , wherein the composition derived from the propagation composition in each of the two or more propagation tanks is selected from a concentrated propagation broth, a lysate of the at least one microorganism, a concentrated lysate of the at least one microorganism, an enzyme isolated from a propagation broth of the at least one microorganism, an enzyme isolated from a lysate of the at least one microorganism, and combinations thereof.

3. The system of claim 1, wherein the system is configured to be movable as a single unit between two or more bioprocessing facilities, wherein each bioprocessing facility is configured to produce one or more bioproducts from biomass.

4. The system of claim 1, wherein each of the two or more propagation tanks has a volume capacity of 10,000 gallons or less.

5. The system of claim 1, wherein each of the two or more propagation tanks has a volume capacity of 3,000 gallons or less.

6. The system of claim 1 , wherein the two or more propagation tanks include three or more propagation tanks, wherein each of the three or more propagation tanks contains at least one microorganism that expresses one or more enzymes to reduce mycotoxin contamination, wherein at least one of the three or more propagation tanks contains at least one microorganism that expresses one or more enzymes to reduce mycotoxin contamination, the at least one microorganism being different from the at least one microorganism that expresses one or more enzymes to reduce mycotoxin contamination present in the other propagation tanks, wherein the system is configured to propagate the at least one microorganism present in each of the three or more propagation tanks and form a propagation composition comprising the at least one microorganism that expresses one or more enzymes to reduce mycotoxin contamination, and wherein the system is configured to connect to one or more points of a process in a bioprocessing facility to inoculate at least a portion of the propagation composition and / or a composition derived from the propagation composition from each of the three or more propagation tanks into one or more processing compositions to reduce mycotoxin contamination, wherein the bioprocessing facility is configured to produce one or more bioproducts from biomass.

7. The system of claim 1, wherein the system is configured to remove and adjustably combine at least a portion of the contents of at least two propagation tanks at a volume ratio of 1:1 or different from 1:

1.

8. The system of claim 1, wherein at least one of the two or more propagation tanks is configured to connect to one or more points in the bioprocessing facility to receive a composition selected from the group consisting of a nutrient source, a carbon source, and combinations thereof.

9. The system of claim 1 , further comprising a storage system in fluid communication with at least one of the two or more breeding tanks, wherein the storage system is configured to receive at least a portion of the contents directly or indirectly from at least one of the two or more breeding tanks, wherein the storage system comprises one or more storage containers, wherein each storage container has a capacity of 150 gallons or less.

10. The system of claim 1, further comprising a lysis system in fluid communication with at least one of the two or more propagation tanks, wherein the lysis system is configured to form a lysate using at least a portion of the propagation composition from at least one of the two or more propagation tanks.

11. The system of claim 1, further comprising a source of one or more protease inhibitors, wherein the system is configured to combine the one or more protease inhibitors with the lysate.

12. The system of claim 11, wherein the system is configured to combine the one or more protease inhibitors with the lysate at a weight ratio of 1:10000 to 1:

10.

13. The system of claim 11, wherein the system is configured to combine the one or more protease inhibitors with the lysate at a weight ratio of 1:10000 to 1:1000.

14. The system of claim 1, further comprising a source of one or more stabilizers, wherein the system is configured to combine the one or more stabilizers with at least one propagation composition and / or a composition derived from the at least one propagation composition at one or more points in the system.

15. The system of claim 1, wherein the system is configured to control the temperature of enzymes present in the system to 110°F or less.

16. A method of using two or more propagation tanks to propagate two or more microorganisms expressing one or more enzymes to mitigate mycotoxin contamination, wherein the method comprises: propagating at least one microorganism expressing one or more enzymes to mitigate mycotoxin contamination in a first propagation tank of a bioprocessing facility for producing one or more bioproducts from biomass, wherein the propagation forms a propagation composition comprising the at least one microorganism expressing one or more enzymes to mitigate mycotoxin contamination; propagating at least one microorganism expressing one or more enzymes to reduce mycotoxin contamination in a second propagation tank of the bioprocessing facility for producing one or more bioproducts from biomass, wherein the propagation forms a propagation composition comprising the at least one microorganism expressing one or more enzymes to reduce mycotoxin contamination, wherein the at least one microorganism expressing one or more enzymes to reduce mycotoxin contamination present in the first propagation tank is different from the at least one microorganism expressing one or more enzymes to reduce mycotoxin contamination present in the second propagation tank; At least a portion of the propagation composition and / or a composition derived from the propagation composition is inoculated into one or more process compositions of the bioprocessing facility to mitigate mycotoxin contamination.

17. The method of claim 16, further comprising lysing the at least one microorganism expressing one or more enzymes to mitigate mycotoxin contamination in at least one of the two or more propagation tanks to form a lysate, wherein lysing comprises mechanically and / or physically lysing the microorganism expressing enzymes to mitigate mycotoxin contamination to form the lysate.

18. The method of claim 17, wherein the lysing does not comprise chemically lysing the at least one microorganism expressing one or more enzymes to mitigate mycotoxin contamination to form the lysate.

19. The method of claim 16, wherein the biomass comprises one or more grains, and wherein the bioprocessing facility is a facility for producing ethanol from the grains.

20. The method of claim 16, wherein the one or more processing compositions are selected from the group consisting of a feedstock composition, a milling composition, a steeping composition, a propagation composition, a saccharification composition, a fermentation composition, a distiller's grains composition, and combinations thereof.

21. The method of claim 16, further comprising: determining the presence or absence of one or more mycotoxins in an incoming grain and / or one or more processing compositions of the bioprocessing facility, wherein the determining comprises measuring the concentration of the one or more mycotoxins in the incoming grain and / or the one or more processing compositions of the bioprocessing facility; and If the one or more mycotoxins are determined to be present in the feed grains and / or the one or more processing compositions of the bioprocessing facility, the two or more propagation tanks are used for propagation in the bioprocessing facility, wherein if the one or more mycotoxins are determined not to be present in the feed grains and / or the one or more processing compositions of the bioprocessing facility, the two or more propagation tanks are not used for propagation in the bioprocessing facility.

22. The method of claim 16, further comprising: determining the presence or absence of one or more mycotoxins in incoming grains and / or one or more processing compositions of different bioprocessing facilities, wherein said determining comprises measuring the concentration of said one or more mycotoxins in said incoming grains and / or said one or more processing compositions of said different bioprocessing facilities; and If the one or more mycotoxins are determined to be present in the feed grains and / or the one or more processing compositions of the different bioprocessing facilities, the two or more propagation tanks are used for propagation in the bioprocessing facilities, wherein if the one or more mycotoxins are determined not to be present in the feed grains and / or the one or more processing compositions of the different bioprocessing facilities, the two or more propagation tanks are not used for propagation in the bioprocessing facilities.

23. A method of propagating at least one microorganism expressing one or more enzymes to mitigate mycotoxin contamination, wherein the method comprises: propagating the at least one microorganism expressing one or more enzymes to mitigate mycotoxin contamination in a bioprocessing facility for producing one or more bioproducts from biomass, wherein the propagation forms a propagation composition comprising the at least one microorganism expressing one or more enzymes to mitigate mycotoxin contamination; lysing the at least one microorganism expressing one or more enzymes to reduce mycotoxin contamination in the propagation composition to form a lysate, wherein lysing comprises mechanically and / or physically lysing the at least one microorganism expressing one or more enzymes to reduce mycotoxin contamination in the propagation composition to form the lysate; as well as At least a portion of the lysate is inoculated into one or more process compositions of the bioprocessing facility to mitigate mycotoxin contamination.

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