JP2015523088A - Method and system for production of fermentation products - Google Patents

Abstract

The present invention relates to a method and system for the production of fermentation products such as alcohol. The present invention also provides a method for separating feed stream components for improved biomass processing and productivity.

Description

  This application is based on US Provisional Patent Application No. 61 / 674,607, filed July 23, 2012; US Provisional Patent Application No. 61 / 699,976, filed September 12, 2012; US Provisional Patent Application No. 61 / 712,385, filed on October 11, US Patent Application No. 13 / 828,353, filed March 14, 2013; and filed on March 15, 2013 No. 13 / 836,115, the entire contents of each of which are hereby incorporated by reference.

  The sequence listing relevant to this application is submitted in electronic form via EFS-Web, which is incorporated herein by reference in its entirety.

  The present invention relates to a method and system for the production of fermentation products such as alcohol. The present invention also provides a method for separating feed stream components for improved biomass processing productivity.

  Alcohols have a variety of industrial and scientific uses such as fuels, reagents, and solvents. For example, butanol is an important industrial chemical with a variety of uses including as a fuel additive, as a raw chemical in the plastics industry, and as a food grade extractant in the food and fragrance industry. Accordingly, there is a high demand for efficient and environmentally friendly production methods including alcohols such as butanol and, for example, fermentation processes and the use of biomass as a feedstock for these processes.

  The production of alcohol by fermentation is one such environmentally friendly production method. Some microorganisms that produce alcohol in high yields also have a low toxicity threshold, so the alcohol needs to be removed from the fermentor as it is produced. One method, in situ product removal (ISPR), may be used to remove alcohol from the fermentor as it is produced, thereby allowing the microorganism to produce alcohol in high yield. An example of ISPR described in the technical field is liquid-liquid extraction (see, for example, Patent Document 1). Because it is technically and economically feasible, liquid-liquid extraction is the contact of the extractant with the fermentation broth for efficient mass transfer; phase separation of the extractant from the fermentation broth; efficient extraction agent Recovery and reuse; and minimal degradation and / or contamination of the extractant over long periods of operation.

  When the water stream entering the fermentor contains undissolved solids from the raw material, the undissolved solids can interfere with liquid-liquid extraction, and the extraction method becomes technically and economically not feasible, for example, capital This can lead to increased costs and operating costs. The presence of undissolved solids during extractive fermentation reduces the mass transfer coefficient, impedes phase separation, causes oil accumulation from undissolved solids in the extractant, and can lead to a decrease in extraction efficiency over time. Yes, because the extractant is trapped in a solid and eventually removed as solubles-containing dry cereal distillers (DDGS), it may increase the loss of extractant, and extractant droplets from the fermentation broth May be delayed and / or fermenter volumetric efficiency may be reduced. Thus, solids removal provides an efficient means for producing and recovering alcohol from the fermentation process.

  In addition to solids removal, the removal of oil from the feedstock can also provide a beneficial effect on alcohol production as well as commercial benefits. For example, some oils, such as corn oil and soybean oil, can be used as biodiesel feedstock and can therefore be an additional source of income for alcohol manufacturers. In addition, oil removal is necessary to save production plant energy through more efficient fermentation, reduce fouling through oil removal, improve fermenter volumetric efficiency, and dry energy requirements such as cereal distillers Can result in significant energy reduction.

US Patent Application Publication No. 2009/030305370

  There is a continuing need to develop more efficient methods and systems for producing alcohols such as ethanol and butanol. The present invention meets this need and provides a method for producing alcohol, including methods and systems for separating raw stream components prior to fermentation and controlling the amount of undissolved solids and / or oils in the fermentation process. Methods and systems are provided.

  The present invention relates to a method and system for separating feed stream components and controlling the amount of undissolved solids and / or oil in the fermenter feed stream during the production of fermentation products. The separated components provide a mechanism for improving biomass processing productivity, including improving the alcohol fermentation byproduct profile. By separating the feed stream into specific components comprising (1) a water stream containing a fermentable carbon source, (2) a feed stream containing oil, and (3) a feed stream containing undissolved solids, these components are Can be recombined in a controlled manner, or removed from the system for other uses. This provides a mechanism for developing by-product compositions for various market needs, such as animal feed markets that require higher protein or higher fat feeds.

  The present invention also relates to a method and system for removing oil from a fermenter feed stream during the production of a fermentation product. In certain embodiments, undissolved solids and oils can be removed from the fermenter feed stream.

  The present invention is a method for producing a product alcohol comprising providing a raw slurry comprising a fermentable carbon source, an undissolved solid, and an oil; and removing a portion of the undissolved solid and oil from the raw slurry. Separating, thereby forming an aqueous solution containing a fermentable carbon source, a wet cake containing solids and an oil stream; and adding the aqueous solution to a fermentation broth containing microorganisms, thereby producing a product alcohol And a method including the steps. In certain embodiments, the method further comprises recovering the oil stream. In certain embodiments, the method further comprises washing the wet cake to obtain a water stream comprising carbohydrates. In certain embodiments, the method further comprises adding a water stream to the fermentation broth. In certain embodiments, the aqueous solution contains no more than about 5% by weight of undissolved solids. In certain embodiments, the oil is corn oil and includes one or more of triglycerides, fatty acids, diglycerides, monoglycerides, and phospholipids. In certain embodiments, the method further comprises combining a portion of the wet cake and a portion of the oil to produce a wet cake comprising triglycerides, free fatty acids, diglycerides, monoglycerides, and phospholipids. In certain embodiments, the method further comprises combining the aqueous solution with a portion of the wet cake to form a mixture of the aqueous solution and the wet cake, and adding the mixture to the fermentation broth. In certain embodiments, the step of separating the raw slurry is a single step process. In certain embodiments, the undissolved solids and oils are decanter bowl centrifuge, three-phase centrifuge, disk stack centrifuge, filtration centrifuge, decanter centrifuge, filtration, vacuum filtration, belt filter, pressure filtration, membrane filtration, Cross-flow filtration, filtration with sieve, sieving separation, grating, porous grid, flotation, hydroclone, filter press, screw press, gravity settling, vortex separator, or By their combination, they are separated from the raw slurry. In certain embodiments, one or more control parameters of the separation device are adjusted to improve the separation of the raw slurry. In some embodiments, the one or more control parameters are selected from differential speed, bowl speed, flow rate, impeller position, cough position, scroll pitch, dwell time, and discharge volume. In certain embodiments, the product alcohol is selected from ethanol, propanol, butanol, pentanol, hexanol, and fusel alcohol. In certain embodiments, the microorganism comprises a butanol biosynthetic pathway. In certain embodiments, the butanol biosynthetic pathway is a 1-butanol biosynthetic pathway, a 2-butanol biosynthetic pathway, or an isobutanol biosynthetic pathway. In certain embodiments, real-time measurements are used to monitor the separation of the raw slurry. In certain embodiments, the separation is performed by Fourier transform infrared spectroscopy, near infrared spectroscopy, Raman spectroscopy, high pressure liquid chromatography, viscometer, density meter, tension meter, droplet size analyzer, particle analyzer, or Monitored by their combination.

  The invention includes providing a raw slurry comprising a fermentable carbon source and an undissolved solid; and separating at least a portion of the undissolved solid from the raw slurry, thereby including an aqueous solution and a solid comprising the fermentable carbon source. Contacting the wet cake with a liquid to form a wet cake mixture; separating at least a portion of undissolved solids from the wet cake mixture, thereby producing a fermentable carbon source; A second aqueous solution containing and a step of producing a second wet cake containing solids. In certain embodiments, the liquid is selected from fresh water, backset, cooking water, process water, distilled water, evaporating water, or combinations thereof . In certain embodiments, the contacting and separating steps can be repeated.

  The present invention provides a raw slurry comprising a fermentable carbon source, an undissolved solid, and an oil; separating at least a portion of the oil and the undissolved solid from the raw slurry, thereby including the fermentable carbon source Producing a wet cake comprising an aqueous solution, an oil stream; and a solid; contacting the wet cake with a liquid to form a wet cake mixture; and removing at least a portion of the undissolved solid and oil from the wet cake mixture; Separating a second aqueous solution comprising a fermentable carbon source, a second oil stream; and a second wet cake comprising a solid. In certain embodiments, the step of separating the raw slurry is a single step process. In certain embodiments, the undissolved solids and oils are decanter bowl centrifuge, three-phase centrifuge, disk stack centrifuge, filtration centrifuge, decanter centrifuge, filtration, vacuum filtration, belt filter, pressure filtration, membrane filtration, Separated from raw slurry by cross-flow filtration, filtration through sieve, sieving separation, grid, porous grid, flotation method, hydroclone, filter press, screw press, gravity settler, vortex separator, or combinations thereof The In certain embodiments, one or more control parameters of the separation device are adjusted to improve the separation of the raw slurry. In some embodiments, the one or more control parameters are selected from differential speed, bowl speed, flow rate, impeller position, cough position, scroll pitch, dwell time, and discharge volume. In certain embodiments, real-time measurements are used to monitor the separation of the raw slurry. In certain embodiments, the separation is performed by Fourier transform infrared spectroscopy, near infrared spectroscopy, Raman spectroscopy, high pressure liquid chromatography, viscometer, density meter, tension meter, droplet size analyzer, particle analyzer, or Monitored by their combination.

  The present invention provides a raw slurry comprising a fermentable carbon source, an undissolved solid, and oil; and separating the raw slurry, thereby (i) a first aqueous solution comprising a fermentable carbon source, (ii) A) a first wet cake containing solids, and (iii) a stream comprising a water stream comprising oil, solids, and a fermentable carbon source is formed; adding the first aqueous solution to the fermentation broth containing microorganisms; Thereby producing a fermentation product. In certain embodiments, the method separates a stream comprising an oil, a solid, and a water stream comprising a fermentable carbon source, thereby (ii) a second aqueous solution comprising a fermentable carbon source, (ii) a solid. A second wet cake comprising, and (iii) a step in which an oil stream is formed. In certain embodiments, the first and second aqueous solutions may be combined prior to addition to the fermentation broth. In certain embodiments, the second aqueous solution can further comprise an oil. In certain embodiments, the second aqueous oil or portion thereof may be treated to produce an extractant. In certain embodiments, the oil can be treated chemically or enzymatically. In certain embodiments, the separation is performed by decanter bowl centrifugation, three-phase centrifugation, disk stack centrifugation, filtration centrifugation, decanter centrifugation, filtration, vacuum filtration, belt filter, pressure filtration, filtration using a sieve, sieving. Separation, grids, porous grids, flotation methods, hydroclones, filter presses, screw presses, gravity settlers, vortex separators, or combinations thereof can be performed.

  The present invention provides a raw slurry comprising a fermentable carbon source, an undissolved solid, and an oil; separating the raw slurry, thereby (i) a first aqueous solution comprising a fermentable carbon source and a solid; (Ii) a first wet cake comprising solids, and (iii) a first oil stream is formed; adding oil to said first aqueous solution, thereby providing an oil layer and fermentable carbon source comprising solids A second aqueous solution containing is formed. In certain embodiments, the oil layer comprising solids is separated into (i) a second oil stream, (ii) a second wet cake comprising solids, and (iii) a third aqueous solution comprising a fermentable carbon source. Can be formed. In certain embodiments, the second aqueous solution and the third aqueous solution may be added to a fermentation broth containing microorganisms, thereby producing a fermentation product. In certain embodiments, the second aqueous solution and the third aqueous solution may further comprise an oil. In certain embodiments, the second aqueous solution and the third aqueous solution may be combined, and the oil of the second aqueous solution and the third aqueous solution, or a portion thereof, is treated to produce an extractant. In certain embodiments, the oil can be treated chemically or enzymatically. In certain embodiments, the separation is performed by decanter bowl centrifugation, three-phase centrifugation, disk stack centrifugation, filtration centrifugation, decanter centrifugation, filtration, vacuum filtration, belt filter, pressure filtration, filtration using a sieve, sieving. Separation, grids, porous grids, flotation methods, hydroclones, filter presses, screw presses, gravity settlers, vortex separators, or combinations thereof can be performed.

  The present invention includes one or more liquefaction units configured to liquefy a raw material to produce a raw slurry, including an inlet for receiving the raw material; and an outlet for discharging the raw slurry And a liquefaction unit in which the raw slurry includes a fermentable carbon source, oil, and non-dissolved solids; an aqueous solution, oil stream, and non-dissolved containing the fermentable carbon source by removing the oil and non-dissolved solids from the raw slurry One or more separation units configured to produce a wet cake containing a portion of solids, wherein the centrifuge has an inlet for receiving the raw slurry; a first outlet for discharging the aqueous solution; It also relates to a system comprising a second outlet for discharging the wet cake; and a separation unit including a third outlet for discharging the oil. In certain embodiments, the system is one or more wash systems configured to recover fermentable carbon source from wet cake, the wash comprising one or more mixing units; and one or more separation units Further includes a system. In certain embodiments, the separation unit is decanter bowl centrifuge, three-phase centrifuge, disc stack centrifuge, filtration centrifuge, decanter centrifuge, filtration, vacuum filtration, belt filter, pressure filtration, membrane filtration, crossflow filtration. , Filtration through a sieve, sieving separation, grid, porous grid, flotation method, hydroclone, filter press, screw press, gravity settler, vortex separator, and combinations thereof. In certain embodiments, the system is one or more fermenters configured to ferment an aqueous solution to produce product alcohol, an inlet for receiving the aqueous solution and / or wet cake; and production It further includes a fermentation apparatus including an outlet for discharging the fermentation broth containing the product alcohol. In certain embodiments, the system further includes an online measurement device. In certain embodiments, the online measurement device is a particle size analyzer, Fourier transform infrared spectrometer, near infrared spectrometer, Raman spectrometer, high pressure liquid chromatography, viscometer, density meter, tension meter, droplet size analyzer, pH Selected from a total, a dissolved oxygen probe, or a combination thereof.

  The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate the invention and, together with the description, explain the principles of the invention and allow those skilled in the relevant art to understand the invention. And make it available for use.

Figure 2 schematically illustrates an exemplary method and system of the present invention where undissolved solids are removed by separation after liquefaction and prior to fermentation. 1 schematically illustrates an exemplary alternative method and system of the present invention in which raw materials are ground. Fig. 3 schematically illustrates another exemplary alternative method and system of the present invention in which undissolved solids and oil are removed by separation. Fig. 4 schematically illustrates another exemplary alternative method and system of the present invention in which a wet cake is subjected to one or more wash cycles. FIG. 4 schematically illustrates another exemplary alternative method and system of the present invention in which undissolved solids and oil are removed by separation and the wet cake is subjected to one or more wash cycles. Fig. 4 schematically illustrates another exemplary alternative method and system of the present invention in which an aqueous solution and a wet cake are combined and sent to fermentation. Fig. 4 schematically illustrates another exemplary alternative method and system of the present invention in which an aqueous solution is saccharified prior to fermentation. Fig. 4 schematically illustrates another exemplary alternative method and system of the present invention in which the raw slurry is saccharified prior to separation. Figure 3 schematically illustrates an exemplary alternative method and system of the present invention in which an additional separation unit is used to remove undissolved solids and oil. Figure 3 schematically illustrates an exemplary alternative method and system of the present invention in which an additional separation unit is used to remove undissolved solids and oil. Figure 3 schematically illustrates an exemplary alternative method and system of the present invention in which an additional separation unit is used to remove undissolved solids and oil. Figure 3 schematically illustrates an exemplary alternative method and system of the present invention in which an additional separation unit is used to remove undissolved solids and oil. Fig. 3 schematically illustrates an exemplary fermentation process that uses online, in-line, at-line, and / or real-time measurements to monitor the fermentation process. 1 schematically illustrates an exemplary fermentation process of the present invention including downstream processing. Transfer of i-BuOH from an aqueous solution of liquefied corn starch to a dispersion of oleyl alcohol droplets flowing upward through a bubble column when dispensing oleyl alcohol using a nozzle having an inner diameter of 2.03 mm total volume mass transfer coefficient, indicating the effect of the presence of undissolved corn mash solids for k L _a. Transfer of i-BuOH from an aqueous solution of liquefied corn starch to a dispersion of oleyl alcohol droplets flowing upward through a bubble column when dispensing oleyl alcohol using a nozzle having an inner diameter of 0.76 mm total volume mass transfer coefficient, indicating the effect of the presence of undissolved corn mash solids for k L _a. (Gravity) Indicates the position of the liquid-liquid interface in the fermentation sample tube according to the sedimentation time. Run time: Phase separation data shown for run times of 5.3 hours, 29.3 hours, 53.3 hours, and 70.3 hours. Sample data from extraction-fermentation where solids were removed from the mash feed and oleyl alcohol was the solvent. The position of the liquid-liquid interface of the final fermentation broth according to the (gravity) sedimentation time is shown. Data from extraction-fermentation where solids were removed from the mash feed and oleyl alcohol was the solvent. The concentration of glucose in the aqueous phase of the slurry as a function of time for Batch 1 and Batch 2 is shown. The concentration of glucose in the aqueous phase of the slurry as a function of time for Batch 3 and Batch 4 is shown. Figure 5 shows the effect of whether enzyme loading and +/- high temperature steps were applied at some point during liquefaction on starch conversion. The influence of the three-phase centrifugation conditions on the separation of the raw slurry is shown. The influence of the three-phase centrifugation conditions on the separation of the raw slurry is shown. The influence of the three-phase centrifugation conditions on the separation of the raw slurry is shown. The influence of the three-phase centrifugation conditions on the separation of the raw slurry is shown. The influence of the three-phase centrifugation conditions on the separation of the raw slurry is shown.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application, including definitions, will control. Also, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. All publications, patents, and other references mentioned herein are hereby incorporated by reference in their entirety for all purposes.

  In order to further define the invention, the following terms and definitions are provided herein.

  As used herein, “comprises”, “comprising”, “includes”, “including”, “has”, “having” , “Contains”, or “containing”, or any other conjugation thereof, includes the integer or group of integers described, but any other integer or It will be understood that it means not to exclude groups of integers. For example, a composition, mixture, process, method, article or device that includes an enumeration of elements is not necessarily limited to only those elements, but is not explicitly recited or such a composition, It may include mixtures, processes, methods, articles, or other elements specific to the device. Further, unless expressly stated to the contrary, “or” refers to an inclusive “or” rather than an exclusive “or”. For example, condition A or B is satisfied by one of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) And B is true (or exists), both A and B are true (or exist).

  Also, the indefinite articles “a” and “an” preceding an element or component of the invention are not intended to be limited regarding the number of instances, ie, the number of occurrences of the element or component. Thus, “a” or “an” should be read to include one or at least one, and the singular form of an element or component is plural unless the number is explicitly meant to be singular. Including.

  The terms “invention” or “invention” as used herein are non-limiting terms and are not intended to refer to any one embodiment of a particular invention, Includes all possible embodiments described.

  As used herein, the term “about”, which modifies the amount of the inventive component or reactant used, is typical of, for example, used to make concentrates or solutions in the real world. Due to physical measurements and liquid processing procedures; due to inadvertent errors in these procedures; the quantity of components that can occur due to differences in the manufacture, source, or purity of the components used to make the composition or perform the method Refers to fluctuations. The term “about” encompasses different amounts due to different equilibrium conditions for compositions obtained from a particular initial mixture. Whether or not modified by the term “about”, the claims include equivalents to the quantities. In one embodiment, the term “about” means within 10% of the reported numerical value, or within 5% of the reported numerical value.

  “Biomass” as used herein refers to corn, sugarcane (or tow), wheat, cellulosic or lignocellulosic materials and cellulose, hemicellulose, lignin, starch, oligosaccharides, disaccharides and / or monosaccharides. , And natural products containing hydrolyzable polysaccharides that provide fermentable sugars, including any sugars and starches, derived from natural resources such as materials including mixtures thereof. Biomass can also include additional components such as proteins and / or lipids. The biomass may be derived from one source, or the biomass may include a mixture derived from two or more sources. For example, the biomass may comprise a mixture of corn cobs and corn stover, or a mixture of grass and leaves. Biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, paper sludge, garden waste, wood waste and forestry waste (eg, forest thinning) It is done. Examples of biomass include, but are not limited to, corn kernels, corn cobs, corn hulls, crop residues such as corn stover, grass, wheat, rye, wheat straw, spelled wheat, rye wheat , Barley, barley straw, oat, pasture, rice, rice straw, switchgrass, potato, sweet potato, cassava, chrysanthemum, sugar cane pomace, sorghum, sugar cane, sugar beet, feed beet, soy, palm, coconut, rape , Safflower, sunflower, millet, eucalyptus, persimmon, waste paper, ingredients obtained from grinding cereals, trees, branches, roots, leaves, wood chips, sawdust, shrubs and shrubs, vegetables, fruits, flowers, animal manure, and them Of the mixture. Any mash, juice, molasses, or hydrolyzate known in the art for treating biomass for fermentation, such as grinding, (eg, enzymatic, chemical) processing, and / or liquefaction. Depending on the method, it can be formed from biomass. The biomass to be treated can include fermentable sugars and / or water. Cellulosic and / or lignocellulosic biomass can be treated to obtain a hydrolyzate containing fermentable sugars by any method known to those skilled in the art. For example, low ammonia pretreatment is disclosed in US Patent Application Publication No. 2007/0031918 A1, the entire contents of which are incorporated herein by reference. Enzymatic saccharification of cellulosic and / or lignocellulosic biomass typically utilizes an enzyme mixture to hydrolyze cellulose and hemicellulose to produce a hydrolyzate containing sugars including glucose, xylose, and arabinose To do. Saccharifying enzymes suitable for cellulosic and / or lignocellulosic biomass are described in Lynd, et al. (Microbiol. Mol. Biol. Rev. 66: 506-577, 2002).

  A “fermentable carbon source” or “fermentable carbon substrate” as used herein refers to a carbon source that can be metabolized by a microorganism. Suitable fermentable carbon sources include, but are not limited to, monosaccharides such as glucose or fructose; disaccharides such as lactose or sucrose; oligosaccharides; polysaccharides such as starch or cellulose; one carbon substrate; Of the mixture.

  As used herein, “fermentable sugar” refers to one or more sugars that can be metabolized by a microorganism for the production of fermentation products such as alcohol.

  “Raw material” as used herein refers to a raw material in a fermentation process. The feedstock may include a fermentable carbon source and may include undissolved solids and / or oils. Where applicable, the feedstock may include a fermentable carbon source either before or after the fermentable carbon source has been released from starch or obtained from hydrolysis of a complex saccharide by further processing, such as by liquefaction, saccharification, or other processes. The feedstock can include or be derived from biomass. Suitable raw materials include, but are not limited to, rye, wheat, corn, corn mash, sugar cane, sugar cane mash, barley, cellulosic material, lignocellulosic material, or mixtures thereof. When referring to “feed oil”, it will be understood that this term encompasses oil produced from a given feed.

As used herein, “fermentation broth” refers to water, fermentable carbon sources (eg, sugars, starch), dissolved solids, optionally microorganisms that produce fermentation products, and optionally fermentation products ( For example, a fermentation apparatus in which a fermentation product is made by metabolism of a fermentable carbon source by a microorganism to form a product alcohol), and optionally an undissolved solid, fermentation product, water, and carbon dioxide (CO ₂ ). Refers to a mixture with other components of the material retained therein. In some cases, the terms “fermentation medium” and “fermented mixture” as used herein may be used interchangeably with “fermentation broth”.

  A “fermentor” or “fermentor” as used herein undergoes a fermentation reaction whereby fermented products (eg, product alcohols such as ethanol or butanol) are made from fermentable carbon sources. Refers to the tank, unit, or tank to be used. A fermenter may also refer to a tank, unit, or tank in which microbial growth occurs. In some cases, both microbial growth and fermentation can occur in the fermenter. The term “fermenter” may be used synonymously with “fermentor”.

  As used herein, a “saccharification tank” refers to a tank, unit, or tank in which saccharification (ie, hydrolysis of oligosaccharides to monosaccharides) takes place. When fermentation and saccharification are performed simultaneously, the saccharification tank and the fermentation apparatus may be the same tank.

  As used herein, “saccharifying enzyme” refers to one or more polysaccharides and / or oligosaccharides such as glycogen or starch that can hydrolyze α-1,4-glucoside bonds. Refers to an enzyme. Saccharifying enzymes may also include enzymes capable of hydrolyzing cellulosic or lignocellulosic materials.

  A “liquefaction tank” as used herein refers to a tank, unit, or tank in which liquefaction takes place. Liquefaction is a process in which starch is hydrolyzed, for example, by an enzymatic process to obtain oligosaccharides. In embodiments where the feedstock is corn, the oligosaccharide is hydrolyzed from the corn starch content during liquefaction.

  “Sugar” as used herein refers to oligosaccharides, disaccharides, monosaccharides, and / or mixtures thereof. The term “sugar” may also include starch, dextran, glycogen, cellulose, pentosan, and carbohydrates including sugars.

  As used herein, “non-dissolved solid” refers to non-fermentable portions of the raw material that are not dissolved in the liquid or aqueous phase, eg, germs, fibers, and gluten. The non-fermentable portion of the feed includes the portion that remains as a solid of the feed and can absorb liquid from the fermentation broth. In some cases, the term “non-dissolving solid” as used herein may be used interchangeably with “solid” or “suspended solid”.

  “Extractant” as used herein refers to a solvent used to extract a fermentation product. In some cases, the term “extractant” as used herein may be used interchangeably with “solvent”.

  As used herein, “in situ product removal (ISPR)” refers to biological, such as fermentation, to control product concentration in a biological process as the product is produced. Refers to the selective removal of product from the process.

“Product alcohol” as used herein refers to any alcohol that can be produced by a microorganism in a fermentation process that uses biomass as the fermentable carbon source. Product alcohols include, but are not limited to, C ₁ -C ₈ alkyl alcohols. In certain embodiments, the product alcohol is an alkyl alcohol of _C 2 -C _8. In other embodiments, the product alcohol is an alkyl alcohol of C ₂ -C _5. It will be appreciated that C ₁ -C ₈ alkyl alcohols include, but are not limited to, methanol, ethanol, propanol, butanol, pentanol, hexanol, and isomers thereof. Similarly, the alkyl alcohol C ₂ -C _8, but are not limited to, ethanol, propanol, butanol, pentanol, hexanol, and isomers thereof. The term “alcohol” can also be used herein in relation to the product alcohol.

  “Butanol” as used herein refers to butanol isomers: 1-butanol (1-BuOH), 2-butanol (2-BuOH), tertiary-butanol (tert-BuOH), and / or Isobutanol (iBuOH, i-BuOH, or I-BUOH) is referred to individually or as a mixture thereof.

  “Propanol” as used herein refers to the propanol isomers: isopropanol or 1-propanol, individually or as mixtures thereof.

  “Pentanol” as used herein refers to pentanol isomers: 1-pentanol, 3-methyl-1-butanol, 2-methyl-1-butanol, 2,2-dimethyl-1-propanol. , 3-pentanol, 2-pentanol, 3-methyl-2-butanol, or 2-methyl-2-butanol, individually or as a mixture thereof.

  “Effective titer” as used herein refers to the total amount of a particular fermentation product (eg, product alcohol) produced by fermentation. In certain embodiments where the fermentation product is a product alcohol, the effective titer refers to the alcohol equivalent of the alcohol ester produced by alcohol esterification per liter of fermentation medium.

  As used herein, “water immiscible” or “insoluble” is a chemical component such as an extractant or solvent that cannot be mixed with an aqueous solution such as a fermentation broth to form one liquid phase. Point to.

  “Aqueous phase” as used herein refers to the aqueous phase of a mixture of at least two phases obtained by contacting a fermentation broth with an extractant, eg, a water-immiscible organic extractant. In one embodiment of the process described herein that includes a fermentation extraction, the term “fermentation broth” in this case refers to the aqueous phase in a biphasic fermentation extraction.

  As used herein, “aqueous phase titer” refers to the concentration of fermentation (eg, product alcohol) in the aqueous phase.

  “Organic phase” as used herein refers to the non-aqueous phase of a mixture of at least two phases obtained by contacting a fermentation broth with an extractant, eg, a water-immiscible organic extractant.

  A “portion” as used herein with respect to a process stream is a stream that retains the composition of the stream, including the entire stream, as well as any one or more components of the stream, including all components of the stream. Refers to any fractional part.

  A “byproduct” or “byproduct” as used herein refers to a product that is produced during the production of another product. In some cases, the term “byproduct” may be used interchangeably with the term “byproduct”. By-products include, for example, oil recovered from the raw slurry, wet cake, and DDGS. By-products may also include oil, wet cake, and DDGS modifications to enhance value and / or for the production of other products, such as biodiesel from oil.

  “Distillation byproduct” as used herein refers to a byproduct from the product alcohol production process that can be isolated before or after fermentation. Distillation by-products include non-fermentable products that remain after the product alcohol has been removed from the fermented mash and solids isolated from the mash. Distillation by-products as used herein can be used in a variety of animal and non-animal feed applications. Examples of distillation by-products include, but are not limited to, fatty acids from hydrolysis of oils, lipids from evaporation of thin distillation waste, syrups, cereal distillers, soluble distillers containing cereal distillers, Solids from the mash before fermentation, and solids from whole distillation waste after fermentation, biodiesel, and acylglycerides.

  “Distillation by-product for animal feed” as used herein refers to a distillation by-product suitable for use in or as animal feed. Examples of distillation by-products for animal feed include, but are not limited to, fatty acids from hydrolysis of oils, lipids from evaporation of dilute distillers, syrups, cereal distillers, soluble distillers containing cereal distillers, Solids from the mash before fermentation and solids from the total distillation waste after fermentation.

  As used herein, “cereal distiller” or “DG” refers to the non-fermentable product that remains after the product alcohol is removed from the fermented mash. Dried cereal distillers are known as “dry cereal distillers” or “DDG”. Undried cereal distillers are known as “wet cereal distillers” or “WDG”.

  "Soluble-containing cereal distillers" or "DGS" as used herein is a non-fermentable product that remains after the product alcohol is removed from the fermented mash mixed with soluble material Point to. The dried soluble matter-containing cereal distillation lees are known as “soluble matter-containing cereal distillers” or “DDGS”. Soluble-containing cereal distillers that have not been dried are known as "soluble matter-containing cereal distillers" or "WDGS".

  “Soluble-containing dry cereal distillers” (DDGS) as used herein are by-products or by-products from fermentation of raw materials or biomass (eg, fermentation of cereals or cereal mixtures that produce product alcohol). Refers to the product. In certain embodiments, DDGS may also refer to animal feed produced from the process of making product alcohol.

  As used herein, a “lipid” is a heterogeneous form of fat and fat-like substances, including fatty acids, neutral fats, waxes, and steroids, that are water insoluble and soluble in nonpolar solvents. Refers to any group. Examples of lipids include monoglycerides, diglycerides, triglycerides, and phospholipids.

  “Lipid from evaporation” as used herein with respect to a process stream refers to a lipid by-product produced by evaporation and centrifugation of dilute waste liquor after fermentation in the product alcohol production process.

  “Syrup” or “condensed distillers solubles” (CDS) as used herein with respect to process streams, is obtained by evaporation of dilute distillation waste after fermentation in the product alcohol production process Refers to the by-product produced.

  A “process stream” as used herein refers to any by-product or by-product formed by a fermentation product production process. Examples of process streams include, but are not limited to, COFA, lipids from evaporation, syrup, DG, DDG, WDG, DGS, DDGS, and WDGS. Another example of a process stream is a solid that is removed from the mash (eg, by centrifugation) prior to fermentation in the fermentation product production process (eg, a solid that is removed from corn mash before fermentation). These solids may be referred to as “wet cakes” if they are not dried, and may be referred to as “dry cakes” if they are dried. Another example of a process stream is a solid that is removed from the total distillation waste liquor (eg, by centrifugation) after fermentation in the fermentation product production process. These solids may be referred to as “WS wet cakes” if they are not dried, and may be referred to as “WS dry cakes” if they are dried. In some cases, the term “process stream” as used herein may be used interchangeably with “stream”.

The present invention provides processes and methods for producing fermentation products such as product alcohol using fermentation. Other fermentation products that can be produced using the processes and methods described herein include acids such as propanediol, butanediol, acetone, lactic acid, acetic acid, butyric acid, and propionic acid; hydrogen, methane, and dioxide Gases such as carbon; amino acids; biotin, vitamin B ₂ (riboflavin), vitamin B ₁₂ (eg, cobalamin), ascorbic acid (eg, vitamin C), vitamin E (eg, a-tocopherol), and vitamin K (eg, Vitamins such as menaquinone); antibiotics such as erythromycin, penicillin, streptomycin, and tetracycline; and other products such as citric acid, invertase, sorbitol, pectinase, and xylitol.

  As an example of the processes and methods provided herein, the feed may be liquefied to produce a feed slurry that includes a fermentable carbon source (eg, soluble sugar) and undissolved solids. In some cases, the terms “raw slurry” and “mash” may be used interchangeably. In certain embodiments, the raw slurry includes soluble sugars, undissolved solids, and oils. If the raw slurry is fed directly to the fermenter, undissolved solids and / or oils can prevent efficient removal and recovery of fermentation products such as product alcohol. For example, when liquid-liquid extraction is used to extract product alcohol from a fermentation broth, the presence of undissolved solids prevents the product alcohol from being extracted into the extractant by preventing contact between the extractant and the fermentation broth. Reducing the mass transfer rate; producing an emulsion in the fermentor, thereby preventing phase separation of the extractant and fermentation broth; slowing the withdrawal of the extractant from the fermentation broth; extractant and product At least a portion of the alcohol is “trapped” by the solid, thus reducing the efficiency of recovering and reusing the extractant; oil contamination reduces the life of the extractant; and in the fermenter May cause system inefficiencies, including but not limited to, reducing fermentor volumetric efficiency. These effects can increase capital and operating costs. In addition, extractants that are “trapped” in non-dissolved solids used to produce soluble matter-containing dry cereal distillers (DDGS) can deviate from DDGS values and requirements for selling as animal feed. Thus, to avoid and / or minimize these problems, at least a portion of the undissolved solids can be removed from the raw slurry prior to adding the raw slurry to the fermentor. The extraction activity and efficiency of product alcohol production is extracted in a fermentation broth containing an aqueous solution from which undissolved solids have been removed as compared to the extraction performed in a fermentation broth containing aqueous solutions from which undissolved solids have not been removed. Can be increased if

  The method and system of the present invention will be described with reference to the figures. In certain embodiments, for example, as shown in FIG. 1, the system includes a liquefaction 10 configured to liquefy the raw material to produce a raw slurry.

  For example, the raw material 12 can be introduced into the inlet of the liquefaction 10. Raw material 12 is any containing fermentable carbon source such as starch, including but not limited to barley, oats, rye, sorghum, wheat, rye wheat, spelled wheat, millet, sugarcane, corn, or combinations thereof. Any suitable biomass material. Water can also be introduced into the liquefaction 10.

  The process of liquefying the raw material 12 includes hydrolyzing the raw material 12 to produce a water-soluble sugar. Any known liquefaction process used in the art may be used, including but not limited to acid processes, enzyme processes, or acid-enzyme processes. Such processes can be used alone or in combination. In certain embodiments, an enzymatic process can be used, and a suitable enzyme 14, such as α-amylase, is introduced into the inlet of liquefaction 10. Examples of α-amylases that can be used in the methods and systems of the present invention are US Pat. No. 7,541,026; US Patent Application Publication No. 2009/0209026; US Patent Application Publication No. 2009/0238923. US Patent Application Publication No. 2009/0252828; US Patent Application Publication No. 2009/0314286; US Patent Application Publication No. 2010/02278970; US Patent Application Publication No. 2010/0048446 And U.S. Patent Application Publication No. 2010/0021587, the entire contents of each of which are incorporated herein by reference.

  In certain embodiments, an enzyme for liquefaction and / or saccharification may be produced by a microorganism (eg, microorganism 32). Examples of microorganisms that produce such enzymes are US Pat. No. 7,498,159; US Patent Application Publication No. 2012/0003701; US Patent Application Publication No. 2012/0129229; PCT International International Publication No. 2010/096562 pamphlet; and PCT International Publication No. 2011/153516 pamphlet, the entire contents of each of which are incorporated herein by reference.

  The process of liquefying the raw material 12 produces a raw slurry 16 containing a fermentable carbon source and undissolved solids. In certain embodiments, feed slurry 16 may include a fermentable carbon source, oil, and undissolved solids. The undissolved solid is a non-fermentable part of the raw material 12. In certain embodiments, feedstock 12 can be corn, such as dry-milled, unfractionated corn kernels, and the undissolved solid can include germs, fiber, and gluten. In some embodiments, the raw material 12 is corn or corn seed and the raw slurry 16 is a corn mash slurry. The raw slurry 16 can be discharged from the outlet of the liquefaction 10 and sent to the separation 20.

  In certain embodiments, nutrients such as amino acids, nitrogen, minerals, trace elements, and / or vitamins may be added to the raw slurry 16 or the fermentation 30. For example, the following: biotin, pantothenate, folic acid, niacin, aminobenzoic acid, pyridoxine, riboflavin, thiamine, vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, inositol, potassium (eg, potassium phosphate), Boric acid, calcium, chloride, chromium, copper (eg, copper sulfate), iodide (eg, potassium iodide), iron (eg, ferric chloride), lithium, magnesium (eg, magnesium sulfate), manganese ( For example, one or more of manganese sulfate), molybdenum, calcium chloride, phosphorus, potassium, sodium chloride, vanadium, zinc (eg, zinc sulfate), yeast extract, soybean peptone, etc. are added to the raw slurry 16 or fermentation 30. Can be. Examples of amino acids include essential amino acids such as histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine and alanine, arginine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine, glycine, hydroxylysine, hydroxy Other amino acids such as proline, ornithine, proline, serine, and tyrosine.

  Separation 20 via the inlet can be configured to remove undissolved solids from the raw slurry 16. Separation 20 may also be configured to remove oil or remove both oil and undissolved solids. Separation 20 can be any device capable of separating solids and liquids. For example, the separation 20 can be any conventional centrifuge used in the industry, including, for example, a decanter bowl centrifuge, a three-phase centrifuge, a disk stack centrifuge, a filtration centrifuge, or a decanter centrifuge. Can be a machine. In certain embodiments, the separation is performed by filtration, vacuum filtration, belt filter, membrane filtration, crossflow filtration, pressure filtration, sieving filtration, sieving separation, grate or lattice, porous lattice, flotation method , Hydroclone, filter press, screw press, gravity settler, vortex separator, or any method or separation device that can be used to separate solids and liquids.

  The raw slurry 16 that is sent to the separation 20 can be separated to form a liquid phase, water stream, or aqueous solution 22 (also known as a low viscosity mash) and a solid phase, solid stream, or wet cake 24. The aqueous solution 22 can include, for example, sugars in the form of oligosaccharides, and water. In certain embodiments, the aqueous solution 22 may include at least about 10% by weight oligosaccharide, at least about 20% by weight oligosaccharide, or at least about 30% by weight oligosaccharide. In certain embodiments, the aqueous solution 22 may be discharged from an outlet located near the top of the separation 20. In certain embodiments, the aqueous solution 22 can have a viscosity of less than about 20 centipoise (cP). In certain embodiments, the aqueous solution 22 may include less than about 20 g / L monomer glucose, less than about 10 g / L monomer glucose, or less than about 5 g / L monomer glucose. Suitable methods for determining the amount of monomeric glucose such as high performance liquid chromatography (HPLC) are well known in the art.

  The wet cake 24 can be discharged from the separation 20. In certain embodiments, wet cake 24 may be discharged from an outlet located near the bottom of separation 20. The wet cake 24 may include undissolved solids. In certain embodiments, wet cake 24 may also include a portion of sugar and water. After the aqueous solution 22 is drained from the separation 20, the wet cake 24 can be washed with additional water using the separation 20. In certain embodiments, the wet cake 24 may be washed with additional water using an additional separation device. By washing the wet cake 24, sugars or sugar sources (eg, oligosaccharides) present in the wet cake are recovered, and the recovered sugar and water can be reused in the liquefaction 10. After washing, the wet cake 24 can be processed to form DDGS using any suitable known method. There are several advantages to forming DDGS from wet cake 24. For example, because undissolved solids are not sent to the fermenter, they are not subjected to the conditions of the fermentor, so the undissolved solids do not come into contact with microorganisms present in the fermentor, such as product alcohol Other components such as fermentation products or extractants are not trapped in the undissolved solid. Because DDGS does not contain microorganisms or other components of fermentation broth (eg, product alcohol, extractant), these effects provide advantages for subsequent processing and use of, for example, DDGS as animal feed.

  In certain embodiments, the undissolved solids are separated from the raw slurry and are separated from two product streams, for example, an aqueous solution of oligosaccharides containing a lower concentration of solids compared to the raw slurry, and compared to the raw slurry. A wet cake containing a higher concentration of solids can be formed. In addition, a third stream containing oil can be produced. Thus, several product streams can be produced using different separation techniques or combinations thereof. As an example, the raw slurry 16 can be separated using a three-phase centrifuge. A three-phase centrifuge allows for three-phase separation resulting in two liquid phases (eg, a water stream and an oil stream) and one solid stream (eg, a solid or wet cake) (eg, Flottweg Tricanter®) , Flottweg AG, Vilsibiburg, Germany). The two liquid phases may be separated or decanted from the bowl via, for example, two drainage systems to prevent cross-contamination, and the solid stream may be removed via separate drainage systems.

  In certain embodiments using corn as feedstock 12, a three-phase centrifuge can be used to simultaneously remove solids and corn oil from feed slurry 16 (eg, liquefied corn mash). Solids are undissolved solids that remain after starch is hydrolyzed to soluble oligosaccharides during liquefaction, and corn oil is free oil removed from the germ during grinding and / or liquefaction. In certain embodiments, a three-phase centrifuge may have one feed stream and three outlet streams. The feed stream can consist of liquefied corn mash produced during liquefaction. The mash can consist of an aqueous solution of liquefied starch (eg, oligosaccharides); an undissolved solid composed of insoluble non-starch components derived from corn; and corn oil composed of glycerides and free fatty acids. The three outlet streams from the three-phase centrifuge consist of a wet cake containing undissolved solids from the mash (ie, wet cake 24); a heavy centrifuge stream containing liquefied starch from the mash. And a light centrate stream containing corn oil from mash. In certain embodiments, the light centrifuge liquid stream (ie, oil 26) may be sent to a storage tank or any tank suitable for storing oil. In certain embodiments, the oil can be sold as a by-product, converted to another by-product, or used in processes such as when converting corn oil to corn oil fatty acids. In certain embodiments, a heavy centrifuge stream (ie, aqueous solution 22) can be used for fermentation. In certain embodiments, to recover soluble starch in the liquid phase of the cake, wet cakes such as the evaporator condensate and / or backflow described herein can be washed with process reuse water.

  In certain embodiments, the wet cake 24 is a composition formed from the raw slurry 16 and is at least about 50 wt.% Undissolved solid present in the raw slurry, at least about 55 wt.% Present in the raw slurry. Non-dissolved solid, at least about 60 wt.% Undissolved solid present in the raw slurry, at least about 65 wt.% Non-dissolved solid present in the raw slurry, at least about 70 wt.% Non-dissolved present in the raw slurry. Solids, at least about 75 wt.% Undissolved solids present in the raw slurry, at least about 80 wt.% Undissolved solids present in the raw slurry, at least about 85 wt.% Undissolved solids present in the raw slurry, At least about 90% by weight of undissolved solids present in the raw slurry, at least about 9 present in the raw slurry. May comprise at least about 99% by weight of undissolved solids are present by weight% of undissolved solid or as a raw material slurry.

  In some embodiments, the aqueous solution 22 formed from the raw slurry 16 is about 50 wt.% Or less non-dissolved solid present in the raw slurry, about 45 wt.% Non-dissolved solid present in the raw slurry, or raw slurry. About 40% by weight or less of undissolved solids present in the raw material slurry, about 35% by weight or less of undissolved solids present in the raw material slurry, about 30% by weight or less of undissolved solids in the raw material slurry About 25 wt% or less of undissolved solid present, about 20 wt% or less of non-dissolved solid present in the raw slurry, about 15 wt% or less of non-dissolved solid present in the raw slurry, present in the raw slurry About 10 wt% or less undissolved solids, about 5 wt% or less undissolved solids present in the raw slurry, or about 1 wt% undissolved solid present in the raw slurry

  A fermentation 30 configured to ferment the aqueous solution 22 to produce a fermentation product, such as product alcohol, has an inlet for receiving the aqueous solution 22. Fermentation 30 may include a fermentation broth. In certain embodiments, a microorganism 32 selected from the group consisting of bacteria, cyanobacteria, filamentous fungi, and yeast may be introduced into the fermentation 30 to be included in the fermentation broth. In certain embodiments, the microorganism 32 may be a bacterium such as Escherichia coli. In certain embodiments, the microorganism 32 may be Saccharomyces cerevisiae. In certain embodiments, the microorganism 32 consumes sugar in the aqueous solution 22 and produces butanol. In certain embodiments, the microorganism 32 may be a recombinant microorganism.

  In certain embodiments, the microorganism 32 can be modified to include a biosynthetic pathway. In certain embodiments, the biosynthetic pathway can be a butanol biosynthetic pathway. In certain embodiments, the butanol biosynthesis pathway can be a 1-butanol biosynthesis pathway, a 2-butanol biosynthesis pathway, or an isobutanol biosynthesis pathway. In certain embodiments, the biosynthetic pathway converts pyruvate into a fermentation product. In certain embodiments, the biosynthetic pathway converts pyruvate as well as amino acids into a fermentation product. In certain embodiments, the biosynthetic pathway comprises at least one heterologous polynucleotide encoding a polypeptide that catalyzes substrate-product conversion of the biosynthetic pathway. In certain embodiments, each substrate-product conversion of the biosynthetic pathway is catalyzed by a polypeptide encoded by a heterologous polynucleotide. Examples of production of product alcohols by microorganisms including biosynthetic pathways are described, for example, in US Pat. No. 7,851,188, as well as US Patent Application Publication No. 2007/0092957; No. 2007/0292927; No. 2008/0182308; No. 2008/0274525; No. 2009/0155870; No. 2009/0305363; 2009/0305370, the entire contents of each of which are hereby incorporated by reference.

  In certain embodiments, the microorganism 32 can also be immobilized by adsorption, covalent bonding, crosslinking, confinement, encapsulation, and the like. Methods for confining cells are known in the art, such as US Patent Application Publication No. 2011/0306116, the entire contents of which are hereby incorporated by reference.

  In certain embodiments of the methods and systems described herein, in situ product removal (ISPR) can be used to remove fermentation products such as product alcohol (eg, butanol) from the fermentation broth. In certain embodiments, as the fermentation product is produced by a microorganism, ISPR can be performed in fermentation 30 or outside fermentation 30 using, for example, liquid-liquid extraction. A method for producing and recovering product alcohol from fermentation broth using extractive fermentation is described in US Patent Application Publication No. 2009/0305370; US Patent Application Publication No. 2010/0221802; US Patent Application Publication. No. 2011/0097773; U.S. Patent Application Publication No. 2011/0312044; U.S. Patent Application Publication No. 2011/0312043; and U.S. Patent Application Publication No. 2012/0156738; The entire contents of each are hereby incorporated by reference.

In certain embodiments, the fermentation 30 may have an inlet for receiving the extractant 34. In certain embodiments, the extractant 34 can be added to the fermentation broth outside the fermentation 30. In certain embodiments, the extractant 34 may be added to an external extractor or an external extraction loop. An alternative means of adding the extract 34 to or outside the fermentation 30 is represented by a dotted line. In certain embodiments, the extractant 34 can be an immiscible organic solvent. In certain embodiments, the extractant 34 can be a water-immiscible organic solvent. In certain embodiments, the extractant 34 can be an organic extractant selected from the group consisting of saturated, monounsaturated, polyunsaturated compounds, and mixtures thereof. In certain embodiments, the extraction agent _34, C 12 fatty _alcohols, fatty acids C 12 _{-C 22} of _{-C 22,} esters of fatty acids of C 12 _{-C 22,} fatty aldehydes of _C 12 _{~C 22,} C 12 _~C It can be selected from the group consisting of ₂₂ fatty amides, and mixtures thereof. In certain embodiments, the extraction agent 34 _also, fatty _alcohols, fatty acids C 4 _{-C 28} in C 4 _{-C 22,} esters of fatty acids of C 4 _{-C 28,} fatty aldehydes of C 4 _{-C 22,} and their It can be selected from the group consisting of mixtures. In certain embodiments, the extraction agent _34, C 12 fatty _alcohols, fatty acids C 12 _{-C 22} of _{-C 22,} esters of fatty acids of C 12 _{-C 22,} fatty aldehydes of _C 12 _{~C 22,} C 12 _{~C 22} fatty amides, and first extraction agent selected from mixtures thereof; fatty _alcohols, fatty acids C 7 _{-C 11} and of _C 7 _{-C 11,} esters of fatty acids of _C 7 ~C _11, C 7 ~ fatty aldehydes of C _11, and may include a second extraction agent selected from mixtures thereof. In certain embodiments, the extractant 34 can be a carboxylic acid. In certain embodiments, the extractant 34 can be corn oil fatty acid (COFA) or soybean oil fatty acid (SOFA). In certain embodiments, the extractant 34 is oleyl alcohol, behenyl alcohol, cetyl alcohol, lauryl alcohol, myristyl alcohol, stearyl alcohol, oleic acid, lauric acid, myristic acid, stearic acid, methyl myristate, methyl oleate, 1-nonanol. , 1-decanol, 2-undecanol, 1-nonanal, 1-undecanol, undecanal, laurinaldehyde, 20-methylundecanal, and mixtures thereof. In the methods and systems described herein and shown in the figures, an extractant may be added to the fermentation apparatus or an external extractor.

In certain embodiments, the extractant may be selected based on specific characteristics. For example, the extractant can have a high _Kd . K _d refers to the partition coefficient of the fermentation product (eg, product alcohol) between the extractant phase (eg, organic phase) and the aqueous phase. In certain embodiments, the extractant can have high selectivity. For example, selectivity refers to the relative amount of product alcohol to water taken up by the extractant.

  In certain embodiments, the extractant can be biocompatible. In certain embodiments, biocompatibility refers to a measure of the ability of a microorganism to utilize a fermentable carbon source in the presence of an extractant. In certain embodiments, the extractant can be a mixture of a biocompatible extractant and a non-biocompatible extractant. In certain embodiments, a non-biocompatible extractant refers to an extractant that interferes with the ability of a microorganism to utilize a fermentable carbon source. For example, in the presence of a non-biocompatible extractant, the microorganism does not utilize a fermentable carbon source at a rate greater than about 50% of the rate in the absence of the extractant. In certain embodiments, in the presence of a non-biocompatible extractant, the microorganism does not utilize a fermentable carbon source at a rate greater than about 25% of the rate in the absence of the extractant. Examples of mixtures of biocompatible and non-biocompatible extractants include, but are not limited to, oleyl alcohol and nonanol, oleyl alcohol and 1-undecanol, oleyl alcohol and 2-undecanol, oleyl alcohol and 1 -Nonanal, oleyl alcohol and decanol, and oleyl alcohol and dodecanol. Additional examples of biocompatible and non-biocompatible extractants are described in US Patent Application Publication No. 2011/0097773, the entire contents of which are hereby incorporated by reference.

  The extractant 34 comes into contact with the fermentation broth and a stream 36 is formed comprising a two-phase mixture (ie, an aqueous phase and an organic phase). If the fermentation product is a product alcohol, the product alcohol present in the fermentation broth is transferred to the extractant 34 to form an extractant rich in product alcohol (eg, organic phase). In certain embodiments, stream 36 can be discharged through the outlet of fermentation 30. The product alcohol can be separated from the extractant in stream 36 using conventional techniques. A feed stream can be added to fermentation 30. Fermentation 30 may be any suitable fermenter known in the art.

  In certain embodiments, when extractant 34 is not added to the fermentation broth, stream 36 includes the fermentation broth and product alcohol. Stream 36 or a portion thereof containing product alcohol and fermentation broth can be discharged from fermentation 30 and further processed for product alcohol recovery. In certain embodiments, the fermentation broth can be recycled to fermentation 30.

  In certain embodiments, simultaneous saccharification and fermentation (SSF) can be performed in fermentation 30. Any known saccharification process used in the art may be used, including but not limited to acid processes, enzyme processes, or acid-enzyme processes. In certain embodiments, an enzyme 38 such as glucoamylase may be introduced to hydrolyze saccharides (eg, oligosaccharides) in the raw slurry 16 or aqueous solution 22 to form monosaccharides. Examples of glucoamylases that can be used in the methods and systems of the present invention are US Pat. No. 7,413,887; US Pat. No. 7,723,079; US Patent Application Publication No. 2009/0275080. U.S. Patent Application Publication No. 2010/0267114; U.S. Patent Application Publication No. 2011/0014681; U.S. Patent Application Publication No. 2011/0020899, each of which is incorporated by reference in its entirety Incorporated herein by reference. In certain embodiments, the glucoamylase can be expressed by a recombinant microorganism that also produces a fermentation product (eg, product alcohol).

  In certain embodiments, an enzyme such as glucoamylase can be added to the liquefaction. By adding an enzyme such as glucoamylase to the liquefaction, the viscosity of the raw slurry or the liquefied mash can be reduced, and the separation efficiency can be improved. In certain embodiments, any enzyme capable of reducing the viscosity of the raw slurry can be used (eg, Viscozyme®, Sigma-Aldrich, St. Louis, MO). The viscosity of the raw material can be measured by any method known in the art, including the method described in Example 22.

  In certain embodiments, stream 35 can be discharged from the outlet of fermentation 30. The discharged stream 35 may contain microorganisms 32 such as yeast. The microorganism 32 can be separated from the stream 35 by, for example, centrifugation (not shown). Thereafter, the microorganism 32 can be reused for the fermentation 30, and the fermentation 30 can increase the production rate of the product alcohol over time, thereby increasing the efficiency of product alcohol production.

  When a portion of stream 35 exits fermentation 30, stream 35 is less than about 50 wt.% Undissolved solid present in the raw slurry, less than about 45 wt.% Undissolved solid present in the raw slurry, raw slurry. About 40% by weight or less of undissolved solids present in the raw material slurry, about 35% by weight or less of undissolved solids present in the raw material slurry, about 30% by weight or less of undissolved solids in the raw material slurry About 25 wt% or less of undissolved solid present, about 20 wt% or less of non-dissolved solid present in the raw slurry, about 15 wt% or less of non-dissolved solid present in the raw slurry, present in the raw slurry It may comprise up to about 10% by weight undissolved solids, up to about 5% by weight undissolved solids present in the raw slurry, or up to about 1% by weight undissolved solid present in the raw slurry.

  In certain embodiments, for example, as shown in FIG. 2, the method and system of the present invention may include a grinder 40 configured to dry grind raw material 12. Raw material 12 may enter the grinder 40 through the inlet. The pulverizer 40 can pulverize or grind the raw material 12. In some embodiments, the raw material 12 may not be fractionated. In certain embodiments, the feedstock 12 can be unfractionated corn kernels. The grinder 40 can be any suitable known grinder, such as a hammer mill. The dry-pulverized raw material 44 is discharged from the pulverizer 40 through the outlet and enters the liquefaction 10. The rest of FIG. 2 is similar to FIG. 1 and will not be described again in detail. In other embodiments, the raw material can be fractionated and / or wet milled as is known in the art instead of being fractionated and / or dry milled.

  Wet milling is a multi-step process that separates biomass into several components such as germs, seed coat fibers, starch, and gluten to gain value from each by-product separately. Using corn as a feedstock, this process produces several by-products: starch, gluten feed, gluten meal, and a corn oil stream. These streams can be recombined and processed to produce products customized for the feed industry. As an example of a wet milling process, a raw material (e.g., corn) may be sent to a dip tank, where the raw material is about 120-130 ° F (about 50-55 ° C) for about 30-50 hours, For example, it is immersed in a sodium dioxide solution. Nutrients released into the water can be collected and evaporated to produce a condensed fermentation extract (or leachate). The germ can be removed from the soaked material and further processed to recover oil and germ meal. After removal of the germ, the remaining portion of the raw material can be processed to remove the bran and produce a starch and gluten slurry. The slurry can be further processed to separate starch and gluten protein, which can be dried to form gluten meal. The starch stream may be further processed by fermentation to produce a fermentation product (eg, product alcohol) or may be used by the food, paper, or textile industry. For example, a starch stream can be used to produce a sweetener. Gluten meal and gluten feed streams that contain both protein, fat, and fiber can be used in dairy and beef cattle, poultry, pigs, livestock, horses, aquaculture, and pet feed. The gluten feed can also be used as a carrier for added micronutrients. Gluten meal can also contain, for example, methionine and xanthophyll that can be used as pigment components in poultry feed (eg, pigments provide egg yolk with a yellow color). A condensed fermented extract containing protein, growth factor, vitamin B, and minerals can be used as a high energy liquid feed ingredient. The condensed extract can also be used as a pellet binder. This process provides a purified starch stream; however, this process is costly and involves separating the biomass into its non-starch components that may be unnecessary for fermentation production.

  Fractionation removes fibers and germs containing the majority of lipids (eg, oil) present in ground whole corn, resulting in fractionated corn having a higher starch (endosperm) content. It is done. In certain embodiments, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, At least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more of the oil can be removed from the germ by fractionation. In certain embodiments, fractionation results in an undissolved solids content of the raw material that is at least about 0.5%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, or at least about 5% of the raw material. % Can be reduced.

  Dry fractionation is less expensive than wet milling because it does not separate the germ from the fiber. Advantages of fractionation include, for example, improved product yield, increased volume (ie, space) in the fermentor, smaller column diameter, lower enzyme packing and improved efficiency of saccharification, improved oil Removal, reduced equipment clogging due to the presence of oil, shutdown for less cleaning, increased protein levels in DDGS, reduced drying time of DDGS, and reduced energy consumption. However, the fraction does not remove the entire fiber or germ and does not completely remove the solid. In addition, some starch is lost during fractionation.

  Dry milling can also be used for raw material processing. The raw material can be ground, for example, using a hammer mill to produce a meal, which can then be mixed with water to form a slurry. The slurry can be subjected to liquefaction by the addition of an enzyme such as amylase to hydrolyze the starch into sugars and form a mash. The mash can be heated (“cooked”) to inactivate the enzyme and then cooled to add to the fermentation. Enzymes such as chilled mash, microorganisms, and glucoamylase can be added to the fermentation to produce a fermentation product (eg, product alcohol). After fermentation, the fermentation broth can be sent to distillation for recovery of the fermentation product. If undissolved solids have not been removed, the bottom stream of the distillation column can be collected for further processing, a total distillation waste solution containing unfermented solids (eg, distilled cereal solids), dissolved material, and water. It is. For example, the total distillation effluent can be separated into solid (eg, wet cake) and dilute distillation effluent. Separation can be accomplished by a number of means including, but not limited to, centrifugation, filtration, sieving separation, hydroclone, or any other means or separation device for separating liquids from solids. The dilute distillation effluent can be sent to evaporation to form a concentrated distillation solubles (CDS) or syrup. The dilute distillation effluent may contain soluble nutrients, grain solids (or particulates), and microorganisms. The solid (eg, wet cake) can be combined with syrup and then dried to form DDGS. Syrup contains protein, fat, and fiber and vitamins and minerals such as phosphorus and potassium; it can be added to animal feed for its nutritional value and palatability. DDGS contains protein, fat, and fiber; provides a source of bypass protein. DDGS can be used in animal feed for dairy and beef cattle, poultry, pigs, livestock, horses, aquaculture, and pets.

  In certain embodiments, for example, as shown in FIG. 3, the method and system of the present invention may include the discharge of oil 26 from the outlet of the separation 20. FIG. 3 is similar to FIG. 1 except that the oil stream 26 exits the separation 20 and will not be described again in detail.

  The raw slurry 16 sent to the separation 20 is directed to a first liquid phase or aqueous solution 22 containing fermentable sugars, a second liquid phase containing oil 26, and a solid phase or wet cake 24 containing undissolved solids. And can be separated. Any suitable separation device, such as a three-phase centrifuge, can be used to drain the aqueous solution 22 (or water stream), wet cake 24 (or solid stream), and oil 26 (or oil stream). In certain embodiments, feedstock 12 is corn and oil 26 is corn oil (eg, free corn oil). The term free corn oil as used herein refers to corn oil free of corn germ. In certain embodiments, oil 26 may be sent to a storage tank or any tank suitable for storing oil. In certain embodiments, when the raw material is corn, some of the oil from raw material 12, such as corn oil, remains in wet cake 24.

  In certain embodiments, the fermentation broth in fermentation 30 includes a reduced amount of corn oil as oil 26 is removed from feed 12 via separation 20. In certain embodiments, the fermentation broth has no more than about 25% by weight undissolved solids, the fermentation broth has no more than about 15% by weight undissolved solids, and the fermentation broth has no more than about 10% by weight non-dissolved solids. Having dissolved solids, the fermentation broth has no more than about 5 wt% undissolved solids, the fermentation broth has no more than about 1 wt% undissolved solids, or the fermentation broth is about 0.5 wt% % Of undissolved solids.

  In certain embodiments, the method and system of FIG. 2 may be modified to include draining an oil stream from the separation 20 described herein in connection with the method and system of FIG.

  As shown in FIG. 4, the oil can be removed along with the wet cake 24 if the oil is not drained separately. When the wet cake 24 is removed via the separation 20, in some embodiments, if the feed is corn, some of the oil from the feed 12 such as corn oil remains in the wet cake 24. Wet cake 24 may be sent to mixing 60 and combined with water or other solvent to form wet cake mixture 65. In certain embodiments, the water can be fresh water, reflux, cooked water, process water, distilled residual water, evaporated water, or any water source available at a fermentation treatment facility, or any combination thereof. The wet cake mixture 65 can be sent to the separation 70 to produce a fermentable sugar recovered from the wet cake 24 and a washed centrifuge 75 containing the wet cake 74. Washed centrifuge liquid 75 can be reused for liquefaction 10. The remaining part of FIG. 4 is the same as FIG. 1 and will not be described again in detail.

  In some embodiments, the separation 70 may be, for example, decanter bowl centrifugation, three-phase centrifugation, disk stack centrifugation, filtration centrifugation, decanter centrifugation, filtration, vacuum filtration, belt filter, membrane filtration, crossflow filtration, processing Separation of solids and liquids, including pressure filtration, sieving filtration, sieving separation, grid, porous grid, flotation, hydroclone, filter press, screw press, gravity settler, vortex separator, or combinations thereof It can be any separation device that can.

  In certain embodiments, the wet cake may be subjected to one or more cleaning cycles or cleaning systems. For example, the wet cake 74 can be further processed by sending the wet cake 74 to a second cleaning system. In certain embodiments, the wet cake 74 may be sent to the second mixing 60 'to form a wet cake mixture 65'. The wet cake mixture 65 'can be sent to the second separation 70' to produce a wash centrifuge 75 'and a wet cake 74'. Wash centrifuge 75 'may be reused for liquefaction 10 and / or wash centrifuge 75' may be combined with wash centrifuge 75, and the combined wash centrifuge may be It can be reused for liquefaction 10. The wet cake 74 'can be combined with the wet cake 74 for further processing as described herein. In certain embodiments, separation 70 ′ can be, for example, decanter bowl centrifugation, three-phase centrifugation, disk stack centrifugation, filtration centrifugation, decanter centrifugation, filtration, vacuum filtration, belt filter, membrane filtration, crossflow filtration, Solids and liquids, including pressure filtration, sieving filtration, sieving separation, grid, porous grid, flotation, hydroclone, filter press, screw press, gravity settler, vortex separator, or combinations thereof It can be any separation device capable of separation. In certain embodiments, the wet cake may be subjected to one, two, three, four, five, or more cleaning cycles or cleaning systems.

  The wet cake 74 can be combined with solubles and then dried to form DDGS through any suitable known process. There are several advantages to forming DDGS from wet cake 74. For example, because undissolved solids are not added to the fermentor, the undissolved solids are not subjected to the conditions of the fermentor, so the undissolved solids do not come into contact with microorganisms present in the fermentor, such as product alcohol Other components such as fermentation products or extractants are not trapped in the undissolved solid. Because DDGS does not contain microorganisms or other components of fermentation broth (eg, product alcohol, extractant), these effects provide advantages for subsequent processing and use of, for example, DDGS as animal feed.

  As shown in FIG. 4, the oil is not drained separately from the wet cake, and the oil is included as part of the wet cake and is ultimately present in the DDGS. When corn is used as a raw material, corn oil contains triglycerides, diglycerides, monoglycerides, fatty acids, and phospholipids, which provide a source of metabolic energy for animals. The presence of oil in the wet cake and ultimately in the DDGS provides the desired animal feed, for example an animal feed with a high fat content.

  In certain embodiments, the oil can be separated from DDGS and converted to ISPR extractant for subsequent use in the same or different fermentation processes. Methods for obtaining extractants from biomass are described in US Patent Application Publication No. 2011/0312043, US Patent Application Publication No. 2011/0312044, US Patent Application Publication No. 2012/0156738, and PCT International. Publications of International Publication No. 2011/159998; the entire contents of each are incorporated herein by reference. The oil can be separated from DDGS using any suitable known process including, for example, a solvent extraction process. In one embodiment of the invention, DDGS is filled into an extraction tank and washed with a solvent such as hexane to remove the oil. Other solvents that can be used include, for example, petroleum distillates such as isobutanol, isohexane, ethanol, petroleum ether, or mixtures thereof. After oil extraction, DDGS can be treated to remove any residual solvent. For example, the DDGS can be heated to evaporate any residual solvent using any method known in the art. After removing the solvent, the DDGS can be subjected to a drying process to remove any residual water. Treated DDGS can be used as supplementary feed for animals such as dairy and beef cattle, poultry, pigs, livestock, horses, aquaculture, and pets.

  After extraction from DDGS, the resulting oil and solvent mixture can be collected to separate the oil from the solvent. In one embodiment, the oil / solvent mixture may be processed by evaporation so that the solvent can be evaporated, collected and reused. The recovered oil can be converted to ISPR extractant for subsequent use in the same or different fermentation processes.

  Since the oil present in the fermenter can be hydrolyzed to fatty acids and glycerin, removal of the raw oil component is advantageous for production. Glycerin can accumulate in water and reduce the amount of water available for reuse throughout the fermentation system. Thus, removal of the feed oil component increases the efficiency of production by increasing the amount of water that can be recycled through the system. In addition, oil removal is necessary to save production plant energy through more efficient fermentation, reduce fouling through oil removal, improve fermenter volumetric efficiency, and dry energy requirements such as cereal distillers Can result in significant energy reduction.

  As shown in FIG. 5, the oil can be removed at various points during the process described herein. The raw slurry 16 is obtained by using, for example, a three-phase centrifuge, a first liquid phase or aqueous solution 22 (or water stream), a second liquid phase (or oil stream) containing oil 26, and a solid phase or wet cake. Can be separated into 24 (or solid streams). The wet cake 24 can be further processed to recover fermentable sugar and oil. Wet cake 24 may be sent to mixing 60 and combined with water or other solvent to form wet cake mixture 65. In certain embodiments, the water can be backflow, cooking water, process water, distilled residual water, water collected from evaporation, or any water source available at a fermentation treatment facility, or any combination thereof. The wet cake mixture 65 may be sent to a separation 70 (eg, a three-phase centrifuge) to produce a washed centrifuge 75 that includes fermentable sugar, oil 76, and wet cake 74. Washed centrifuge liquid 75 can be reused for liquefaction 10. Oil 76 and oil 26 can be combined and further processed for the manufacture of various consumer products. In certain embodiments, the oil (eg, oil 26, oil 76) can be further processed to produce an extractant. For example, the oil can be treated chemically or enzymatically to produce an extractant. In certain embodiments where the oil is corn oil, the corn oil may be treated chemically or enzymatically to produce fatty acids (eg, corn oil fatty acids), which can be used as an extractant. . In certain embodiments where the oil is treated enzymatically, the enzymatic reaction can be subjected to treatment (eg, heat) after conversion to inactivate the enzyme. In certain embodiments, the oil can be enzymatically treated with enzymes such as esterases, lipases, phospholipases, lysophospholipases, or combinations thereof. In certain embodiments, the oil may be chemically treated with ammonium hydroxide, anhydrous ammonia, ammonium acetate, hydrogen peroxide, toluene, glacial acetic acid, or combinations thereof. Methods for obtaining extractants from biomass are described in US 2011/0312043 and US 2011/0312044, the entire contents of each of which are hereby incorporated by reference. Incorporated. In certain embodiments in which the oil is corn oil, the feed slurry is at least about 1 wt%, at least about 2 wt%, at least about 3 wt%, at least about 4 wt% corn oil, or at least about 5 wt% Corn oil may be included. The remaining part of FIG. 5 is the same as FIG. 1 and will not be described again in detail.

  As described herein, the wet cake may be subjected to one or more cleaning cycles or cleaning systems. In certain embodiments, the wet cake 74 may be sent to the second mixing 60 'to form a wet cake mixture 65'. The wet cake mixture 65 'can be sent to the second separation 70' to produce a washed centrifuge 75 ', oil 76', and wet cake 74 '. Wash centrifuge 75 'may be reused for liquefaction 10 and / or wash centrifuge 75' may be combined with wash centrifuge 75, and the combined wash centrifuge may be It can be reused for liquefaction 10. The wet cake 74 'can be combined with the wet cake 74 for further processing as described herein. Oil 76, oil 76 ', and oil 26 can be combined and further processed for the manufacture of various consumer products. In certain embodiments, the oil (eg, oil 26, oil 76, oil 76 ') may be further processed to produce an extractant as described herein. Methods for obtaining extractants from biomass are described in US 2011/0312043 and US 2011/0312044, the entire contents of each of which are hereby incorporated by reference. Incorporated.

  As shown in FIG. 6, the aqueous solution 22 and wet cake 24 can be combined, cooled, and sent to the fermentation 30. The raw slurry 16 can be separated into a first liquid phase or aqueous solution 22, a second liquid phase containing oil 26, and a solid phase or wet cake 24 using, for example, a three-phase centrifuge. In certain embodiments, the oil 26 (or oil stream) may be sent to a storage tank or any tank suitable for storing oil. The aqueous solution 22 (or water stream) and the wet cake 24 (solid stream) can be sent to the mix 80 and reslurried to form an aqueous solution / wet cake mixture 82. Mixture 82 may be sent to cooler 90 to produce cooled mixture 92, which may be sent to fermentation 30. In certain embodiments, when oil 26 is removed from feed 12 via separation 20, mixtures 82 and 92 contain a reduced amount of corn oil. The remaining part of FIG. 6 is the same as FIG. 1 and will not be described again in detail.

  In certain embodiments, for example, as shown in FIGS. 7 and 8, saccharification is performed separately between separation 20 and fermentation 30 (FIG. 7) or between liquefaction 10 and separation 20 (FIG. 8). It can be performed in the saccharification system 50. FIGS. 7 and 8 are similar to FIG. 1 except that they contain a separate saccharification 50 and that the fermentation 30 does not accept the enzyme 38. In certain embodiments, enzyme 38 may also be added to fermentation 30.

  Any known saccharification process used in the art may be used, including but not limited to acid processes, enzyme processes, or acid-enzyme processes. Saccharification 50 can be performed in any suitable saccharification tank. In certain embodiments, an enzyme 38 such as glucoamylase may be introduced to hydrolyze saccharides (eg, oligosaccharides) in the raw slurry 16 or aqueous solution 22 to form monosaccharides. For example, in FIG. 7, the oligosaccharides present in the aqueous solution 22 that are discharged from the separation 20 and sent to the saccharification 50 through the inlet are hydrolyzed to monosaccharides. An aqueous solution 52 containing monosaccharides is discharged from the saccharification 50 through the outlet and sent to the fermentation 30. Alternatively, as shown in FIG. 8, the oligosaccharides present in the raw slurry 16 discharged from the liquefaction 10 and sent to the saccharification 50 through the inlet are hydrolyzed into monosaccharides. A raw material slurry 54 containing monosaccharides is discharged from the saccharification 50 through the outlet and sent to the separation 20.

  In certain embodiments, the systems and methods of FIGS. 1-6 can be modified to include a separate saccharification system as described herein in connection with the systems and methods of FIGS.

  In certain embodiments, for example, as shown in FIGS. 9A-9D, the systems and methods of the present invention may include a series of two or more separation devices. 9A-9D are the same as FIG. 1 except that a separation system has been added and will not be described again in detail.

  The aqueous solution 22 discharged from the separation 20 can be sent to the separation 20 '. Isolation 20 'may be the same as isolation 20 or different from isolation 20 and may operate in the same way. Separation 20 ′ removes undissolved solids and oils that are not separated from aqueous solution 22, and (i) an aqueous solution similar to aqueous solution 22 except that it contains a reduced amount of undissolved solids and oil compared to aqueous solution 22. 22 ′, (ii) wet cake 24 ′ similar to wet cake 24, and (iii) oil 26 ′ similar to oil 26. The aqueous solution 22 ′ can then be introduced into the fermentation 30. In certain embodiments, one or more additional separation devices may be present after separation 20 '.

  In certain embodiments, separation 20 ′ can be, for example, decanter bowl centrifugation, three-phase centrifugation, disk stack centrifugation, filtration centrifugation, decanter centrifugation, filtration, vacuum filtration, belt filter, membrane filtration, crossflow filtration, Solids and liquids, including pressure filtration, sieving filtration, sieving separation, grid, porous grid, flotation, hydroclone, filter press, screw press, gravity settler, vortex separator, or combinations thereof It can be any separation device capable of separation.

  In certain embodiments, the systems and methods of FIGS. 1-9 include additional separation devices for removing undissolved solids as described herein in connection with the systems and methods described herein. It can be modified to include.

  In certain embodiments, stream 35 can be discharged from the outlet of fermentation 30. The absence or minimal of undissolved solids exiting fermentation 30 via stream 35 has several additional advantages. For example, the need for units and operations in downstream processing, such as a beer column or distillation column, can be reduced or eliminated, thereby increasing the efficiency of production. Also, as a result of less undissolved solids in the stream exiting the fermenter, some or all of the total distillation waste centrifuge can be omitted.

  Referring to FIG. 9B, the aqueous solution 22 discharged from the separation 20 may be sent to the separation 20 '. The separation 20 ′ may be the same as the separation 20 or may be different from the separation 20. Separation 20 'may operate to include a separation additive 28 such as an extractant or flocculant. Separation additive 28 may assist in the removal of oil or solids. Separation 20 ′ removes undissolved solids and oils that are not separated from aqueous solution 22, and (i) an aqueous solution similar to aqueous solution 22 except that it contains a reduced amount of undissolved solids and oil compared to aqueous solution 22. 22 ', and (ii) may be similar to the combined stream of oil 26 and wet cake 24, and may produce a stream 23 that may also contain a separation additive 28. Stream 23 may be sent to separation 20 ″ and may produce a stream containing separation additive 28 ′ and wet cake 24 ′. Separation 20 ″ is identical to separation 20 and separation 20 ′. Or may be different from separation 20 and separation 20 ′. Aqueous solution 22 ′ can be introduced into fermentation 30.

  In certain embodiments, separation 20, separation 20 ′, and separation 20 ″ can be, for example, decanter bowl centrifugation, three-phase centrifugation, disk stack centrifugation, filtration centrifugation, decanter centrifugation, filtration, vacuum filtration, belt filter , Membrane filtration, cross flow filtration, pressure filtration, sieving filtration, sieving separation, grid, porous grid, flotation method, hydroclone, filter press, screw press, gravity settler, vortex separator, or those It can be any separation device capable of separating solids and liquids, including combinations.

  Referring to FIG. 9C, the raw slurry 16 can be discharged from the liquefaction 10 and sent to the separation 20. The raw slurry 16 may be separated to produce a stream 25 comprising the following stream: (i) an aqueous solution 22, (ii) a wet cake 24, and (iii) a water stream comprising oil, solids, and a fermentable carbon source. . In certain embodiments, stream 25 solids may be light solids. In certain embodiments, the light solids may be solids that are less dense than water but denser than oil. In certain embodiments, light solids can be coated in oil and become a lower density solid than water. In certain embodiments, the solid can have lipophilicity and / or hydrophilicity. In certain embodiments, stream 25 solids may include one or more of the following: germ, fiber, starch, and gluten. In certain embodiments, the solids of stream 25 may include particulates. In certain embodiments, the stream 25 solids include germ, gluten, and fiber. In certain embodiments, an aqueous solution 22 comprising a fermentable carbon source can be sent to the fermentation 30 for production of the fermentation products described herein. In certain embodiments, wet cake 24 may be further processed as described herein, eg, to form DDGS.

  Stream 25 discharged from separation 20 may be sent to separation 20 '. The separation 20 ′ may be the same as the separation 20 or may be different from the separation 20. In certain embodiments, separation 20 and separation 20 ′ can be, for example, decanter bowl centrifugation, three-phase centrifugation, disk stack centrifugation, filtration centrifugation, decanter centrifugation, filtration, vacuum filtration, belt filter, membrane filtration, cloth Solids including flow filtration, pressure filtration, sieving filtration, sieving separation, grids, porous grids, flotation methods, hydroclones, filter presses, screw presses, gravity settlers, vortex separators, or combinations thereof And any separation device capable of separating liquids. In certain embodiments, the separation can be a single step process.

  Stream 25 may be separated by separation 20 'to produce the following streams: (i) aqueous solution 22', (ii) wet cake 24 ', and (iii) oil 26. In certain embodiments, the aqueous solution 22 ′ may be combined with the aqueous solution 22 and the combined aqueous solution may be sent to the fermentation 30. In certain embodiments, the amount of solids in the aqueous solution 22 ′ is reduced compared to the amount of solids in the aqueous solution 22. In certain embodiments, the aqueous solution 22 'may contain oil, and the oil in the aqueous solution 22' may be further processed to produce an extractant. For example, the aqueous solution 22 'can be treated chemically or enzymatically to produce an extractant. In certain embodiments, the aqueous solution 22 'can be treated chemically or enzymatically to produce a fatty acid (eg, corn oil fatty acid) that can be used as an extractant. In certain embodiments where the aqueous solution 22 'is treated enzymatically, the enzymatic reaction can be subjected to treatment (eg, heat) after conversion to inactivate the enzyme. By converting oil in aqueous solution 22 'having a relatively small flow volume to fatty acid, the capital cost of producing extractant via enzymatic or chemical conversion can be reduced. Methods for obtaining extractants from biomass are described in US 2011/0312043 and US 2011/0312044, the entire contents of each of which are hereby incorporated by reference. Incorporated.

  In certain embodiments, wet cake 24 'may be combined with wet cake 24, and the combined wet cake may be further processed as described herein. In certain embodiments, the wet cake 24 'may include oil, and the oil-enriched wet cake may be combined with the wet cake 24 to provide a source of metabolic energy for animal feed. Wet cakes with a (eg increased triglyceride content) can be produced. In certain embodiments, this wet cake with increased fat content can be combined with syrup to produce a high triglyceride, high protein, low carbohydrate DDGS. In certain embodiments, the wet cake 24 'containing oil may be further processed as described herein, for example, to produce DDGS.

  In certain embodiments, oil 26 may be sent to a storage tank or any tank suitable for storing oil. In some embodiments, oil 26 or a portion thereof may be combined with raw slurry 16 (dotted line, FIG. 9C). The addition of oil to the feed slurry can improve the removal of solids via stream 25 by increasing the amount of solids trapped.

  In certain embodiments, oil 26 may be further processed to produce an extractant as described herein. Capital cost and energy requirements to produce extractant via enzymatic or chemical conversion by conversion of oil 26, which has a relatively small flow volume and may have a reduced flow rate compared to feed slurry 16 The amount can be reduced. In certain embodiments, the flow rate of the oil 26 may be about 1% to about 10% of the flow rate of the raw slurry 16. Methods for obtaining extractants from biomass are described in US 2011/0312043 and US 2011/0312044, the entire contents of each of which are hereby incorporated by reference. Incorporated.

  In certain embodiments, the stream 25 may be generated by adjusting one or more parameters of the separation device. For example, the stream 25 can be generated by adjusting the centrifuge (or immersion) centrifuge, such as a decanter centrifuge or a three-phase centrifuge.

  As described herein, since solids can interfere with liquid-liquid extraction, using extraction methods may not be technically and economically feasible. During the extraction process, a lag layer may form at the interface between the aqueous and organic phases, and a lag layer composed of solids (eg, light solids) can accumulate and prevent phase separation There is sex. To alleviate the formation of the lag layer, the removal of solids via the pre-fermentation stream 25 can eliminate the formation of the lag layer, thereby improving the processing downstream of the fermentation broth and the recovery of the fermentation product.

  In another embodiment that mitigates the formation of a lag layer, oil may be added to the aqueous solution as a means for selectively capturing solids that form the lag layer. Referring to FIG. 9D, the raw slurry 16 can be discharged from the liquefaction 10 and sent to the separation 20. The raw slurry 16 can be separated to produce the following streams: (i) an aqueous solution 22, (ii) a wet cake 24, and (iii) an oil 26. In certain embodiments, wet cake 24 may be further processed as described herein, eg, to form DDGS. In certain embodiments, oil may be added to the aqueous solution 22 and the resulting mixture may be sent to a tank 80 where the mixture settles or separates and (i) a solid. An oil layer containing 86 and (ii) an aqueous solution 22 ′ are formed. In certain embodiments, the aqueous solution 22 'can be sent to the fermentation 30 for production of a fermentation product as described herein. In certain embodiments, the amount of solids in the aqueous solution 22 ′ is reduced compared to the amount of solids in the aqueous solution 22. In certain embodiments, the solid enriched oil layer 86 can be removed (eg, removed from the mixture) and filtered to remove solids from the oil layer. In certain embodiments, the solid-enriched oil layer 86 may be sent to a separation 20 ′ and separated into the following streams: (i) an aqueous solution 22 ″, (ii) a wet cake 24 ′, and (iii) oil. 26 ′ (oil with less solids) may be produced.In certain embodiments, the oil added to the aqueous solution 22 may be oil 26, oil 26 ′, and / or an external oil source separated from the raw slurry 16. .

  In certain embodiments, wet cake 24 'may be combined with wet cake 24, and the combined wet cake may be further processed as described herein. In certain embodiments, the wet cake 24 ′ may include oil, and the oil-enriched wet cake may be combined with the wet cake 24 to produce a wet cake having an increased fat content; Further processing can be performed as described herein. In certain embodiments, the wet cake 24 'containing oil may be further processed as described herein.

  In certain embodiments, the aqueous solution 22 ″ may be combined with the aqueous solution 22 ′, and the combined aqueous solution may be sent to the fermentation 30. The amount of solids in the combined aqueous solution is compared to the aqueous solution 22. In some embodiments, the aqueous solution 22 ′ and the aqueous solution 22 ″ may include an oil, and the oil may contain an extractant. It can be further processed to produce. For example, aqueous solution 22 ′ and aqueous solution 22 ″ may be combined and treated chemically or enzymatically (dotted line in FIG. 9D) to produce an extractant as described herein. Methods for obtaining extractants from biomass are described in US 2011/0312043 and US 2011/0312044, the entire contents of each of which are hereby incorporated by reference. Incorporated.

  In certain embodiments, separation 20 and separation 20 ′ can be, for example, decanter bowl centrifugation, three-phase centrifugation, disk stack centrifugation, filtration centrifugation, decanter centrifugation, filtration, vacuum filtration, belt filter, membrane filtration, cloth Solids including flow filtration, pressure filtration, sieving filtration, sieving separation, grids, porous grids, flotation methods, hydroclones, filter presses, screw presses, gravity settlers, vortex separators, or combinations thereof And any separation device capable of separating liquids. In certain embodiments, the separation can be a single step process. In certain embodiments, the tank 80 can be a static mixer, a mixer settler, a decanter, a gravity settler, and combinations thereof. In certain embodiments, the process may be maintained at a temperature that minimizes contamination (eg, 70-110 ° C.).

  In certain embodiments, for example, as shown in FIG. 10, the system and method of the present invention may include means for online, in-line, at-line, and / or real-time measurements (circles represent measurement devices) The dotted line represents the feedback loop). FIG. 10 is similar to FIG. 5 except that a measurement device for on-line, in-line, at-line, and / or real-time measurement has been added and will not be described in detail again.

  The processes described herein include, for example, the concentration and other physics of various streams generated during fermentation (eg, raw slurry, aqueous solution, oil stream, wet cake, wet cake mixture, wash centrifuge, etc.). It can be an integrated fermentation process using on-line, in-line, at-line, and / or real-time measurement of mechanical properties. These measurements can be used, for example, in a feedback loop to adjust and control fermentation conditions and / or conditions of the fermentation apparatus, liquefaction unit, separation unit, and mixing unit. In certain embodiments, the concentration of fermentation products and / or other metabolites and substrates in the fermentation broth can be measured using any suitable measurement device for online, in-line, at-line, and / or real-time measurements. . In certain embodiments, the measurement device is: Fourier transform infrared spectrometer (FTIR), near infrared spectrometer (NIR), Raman spectrometer, high pressure liquid chromatography (HPLC), viscometer, density meter, tensiometer. , A droplet size analyzer, a particle analyzer, a pH meter, a dissolved oxygen (DO) probe, and the like. In certain embodiments, off-gas released from the fermenter can be analyzed, for example, by an in-line mass spectrometer. Measurement of off-gas released from the fermenter can be used as a means to identify chemical species present during the fermentation reaction. The concentration of fermentation products and other metabolites and substrates can also be measured using the techniques and devices described herein.

  In certain embodiments, the measured input may be sent to a controller and / or control system, such as a fermentation device (temperature, pH, nutrient, enzyme and / or substrate concentration), liquefaction unit, separation unit, and mixing The conditions in the unit can be modified to maintain various stream concentrations or concentration profiles. With such a control system, process parameters can be maintained to improve overall factory productivity and economic goals. In certain embodiments, real-time control of fermentation may be performed by varying concentrations of components (eg, biomass, sugars, enzymes, nutrients, microorganisms, etc.) in the fermentation apparatus, liquefaction unit, separation unit, and mixing unit. In certain embodiments, an automated system can be used to regulate separation and mixing conditions, flow rates to and from separation and mixing operations, solids and oil removal, and sugar and starch recovery.

  During the fermentation process, the unit of operation can function at a sub-optimal level over time. In order to maintain overall factory productivity, it may be necessary to adjust flow rates, mixing rates, equipment and the like for operations such as liquefaction and separation. The methods and systems described herein are online, in-line, at-line, to monitor the concentration and other physical characteristics of fermentation streams such as feed slurry 16, aqueous solution 22, oil 26, and wet cake 24. And / or can be integrated using real-time measurements. These measurements can be used, for example, to adjust and control feed slurry fermentation, separation conditions, and wash cycle performance in a feedback loop. Online, in-line, at-line, and / or real-time measurements may be used to provide immediate feedback and adjustment of process conditions, resulting in an overall improved fermentation process. For example, the amount of fermentable carbon source (eg, starch, sugars), oil, and solids can be monitored in feed slurry 16, aqueous solution 22, and streams 65 and 75 using, for example, FTIR or NIR. By monitoring these parameters, the enzyme concentration and residence time for liquefaction and saccharification may be adjusted to improve the preparation of the raw slurry 16, and the separation and mixing conditions may include, for example, the aqueous solution 22 and the flow. It can be adjusted to increase the amount of fermentable carbon source, oil, and / or solids in 65 and 75.

  As another example, washing wet cake 24 allows for the recovery of sugars, starch, and oil in the wet cake, minimizing the yield loss of these fermentable carbon sources and oils. By monitoring the moisture, sugar, starch, and oil content of streams 24, 65, 74, and 75, the cleaning performance can be tuned to improve sugar, starch, and oil recovery. For example, real-time measurements of sugar, starch, and oil content of these streams may be made by FTIR and NIR, which includes immediate feedback of mixing (60) and separation (20, 70) conditions and Allows adjustment. Separation device differential speed, feed speed, bowl speed, scroll differential speed, impeller position, cough position, scroll pitch, dwell time, and discharge volume are the moisture, sugars of streams 24, 65, 74, and 75, Starch and can be adjusted to change the oil content. Mixing (60) conditions, such as pump speed or stirrer speed, can also be adjusted to alter the moisture, sugar, starch, and oil content of streams 24, 65, 74, and 75. In addition, the wash ratio (eg, the ratio between water and wet cake) can also be adjusted to alter the moisture, sugar, starch, and oil content of streams 24, 65, and 74. FTIR measurements and droplet imaging can be used to monitor the water content in the oil stream (26, 76). In addition, the color and turbidity of the oil stream (26, 76) can be monitored to assess the quality of the oil. This real-time measurement would allow adjustment to separation (20, 70) conditions that produce a cleaner oil stream (eg, less water).

  In another embodiment of the methods and systems described herein, the moisture content of the wet cake 24 can be monitored using real-time measurements. Real-time measurements of the moisture content of the wet cake may be made by NIR, and these measurements allow for immediate feedback and adjustment of the separation (20, 70) conditions. By reducing the moisture content of the wet cake, the overall energy utilization for the production process can be improved because less energy is required to dry the wet cake. Further, starch recovery can be improved due to the lower moisture content of the wet cake.

  As another example of a process control method using real-time measurements, solids in the aqueous solution 22 and oils 26, 76 may be processed using a process particle analyzer (JM Canty, Inc., Buffalo, NY), focused beam reflectometry (FBRM ( (Registered trademark)), or particle size analysis such as particle microscopic imaging (PVM®) technique (Mettler-Toledo, LLC, Columbia OH). By monitoring solids in real time, process steps are tuned to improve solids removal, thereby minimizing the amount of solids in aqueous solutions and oil streams, maximizing solids recovery, and downstream Can improve the overall fermentation process including the treatment of

  The methods and systems disclosed in FIGS. 1-10 include removing undissolved solids and / or oils from the raw slurry 16, resulting in improved process productivity and cost effectiveness. Improved productivity includes improved efficiency of fermentation product production and / or improved extraction activity compared to methods and systems that do not remove undissolved solids and / or oils prior to fermentation.

  An exemplary fermentation process of the present invention including downstream processing is described in FIG. Several processes and flows in FIG. 11 have been identified using the same names and numbering as used in FIGS. 1-10 and are the same or similar to those described in FIGS. Represents process and flow.

  As described herein with reference to FIGS. 1-10, feed 12 may be processed to separate undissolved solids and / or oil (100). Briefly, feedstock 12 can be liquefied to produce a feed slurry that includes undissolved solids, fermentable carbon source, and oil. For example, ground grain and one or more enzymes can be combined to produce a raw slurry. This feed slurry can be heated (or cooked), liquefied, and / or flushed with flash steam to produce a “cooked” feed slurry or mash. In certain embodiments, the raw slurry can be heated to at least about 100 ° C. In certain embodiments, the raw slurry can be heated for about 30 minutes. In certain embodiments, the raw slurry can be subjected to raw starch hydrolysis (also known as low temperature cooking or low temperature hydrolysis). In the raw starch hydrolysis process, the heating (or cooking) step is omitted, and omission of this step reduces energy consumption and steam load (eg, water consumption). In certain embodiments, liquefaction and / or saccharification can occur at fermentation temperatures (eg, about 30 ° C. to about 55 ° C.). In certain embodiments, liquefaction and / or saccharification is performed using raw starch enzymes or cold hydrolyzing enzymes such as Stargen ™ (Genencor International, Palo Alto, Calif.) And BPX ™ (Novozymes, Franklinton, NC). Can be done. In certain embodiments, liquefaction and / or saccharification may be performed at a temperature less than about 50 ° C.

  The raw slurry is then subjected to separation to remove undissolved solids and an aqueous solution 22 (or centrifuge) containing wet cake 24, oil 26, and a fermentable carbon source, eg, dissolved fermentable sugar. ) May be generated. Separation: decanter bowl centrifugation, three-phase centrifugation, disc stack centrifugation, filtration centrifugation, decanter centrifugation, filtration, vacuum filtration, belt filter, membrane filtration, crossflow filtration, pressure filtration, filtration using a sieve , Sieving separation, grids, porous grids, flotation methods, hydroclones, filter presses, screw presses, gravity settlers, vortex separators, or combinations thereof may be performed by several means. This separation step may comprise at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% non-feed from the raw slurry. Dissolved solids can be removed. In certain embodiments, the aqueous solution 22 may comprise at least about 0.5%, at least about 1%, or at least about 2% undissolved solids.

  The wet cake 24 is reslurried or washed with water and subjected to separation to remove additional fermentable sugars, and the washed wet cake (eg, 74, 74 ′ described in FIGS. 1-10) is obtained. Can be generated. In certain embodiments, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, or about 15% of fermentable sugar can be recovered from the washed wet cake. The cleaning process can be repeated several times, eg, once, twice, three times, four times, five times, or more times. The water used to reslurry or wash the wet cake can be recycled water (eg, reverse flow, cooking water, process water, distilled residual water, evaporating water) produced during the fermentation process. In certain embodiments, the wet cake may be reslurried or washed with beer. The washing centrifuge liquid produced by the washing / separation process (eg, 75, 75 ′ described in FIGS. 4 and 5) is returned to the mixing step to form a slurry with the milled cereal, or Can be used for liquefaction processes. In certain embodiments, the washed centrifuge liquid can be heated or cooled prior to the mixing step.

  The aqueous solution 22 can be further processed as described herein. For example, the aqueous solution 22 can be heated using steam or process to process heat exchange. A saccharifying enzyme may be added to the aqueous solution 22, and the dissolved fermentable sugar of the aqueous solution 22 may be partially or fully saccharified. The saccharified aqueous solution 22 can be cooled by several means such as interprocess exchange, exchange with cooling water, or exchange with cold water.

  The aqueous solution 22 and the microorganism 32 may be added to the fermentation 30, where fermentable sugars are metabolized by the microorganism 32 to produce a stream 105 containing the fermentation product (eg, product alcohol). In certain embodiments, the microorganism 32 can be a recombinant microorganism capable of producing a product alcohol such as 1-butanol, 2-butanol, or isobutanol. In certain embodiments, ammonia and a recycle stream can also be added to fermentation 30. In certain embodiments, the process may include at least one fermenter, at least two fermenters, at least three fermenters, at least four fermenters, at least five fermenters, or more fermenters. In certain embodiments, carbon dioxide produced during fermentation can be vented to a scrubber to reduce atmospheric emissions (eg, alcohol atmospheric emissions) and increase product yield.

  Stream 105 containing product alcohol may be sent to beer tower 120 to produce alcohol enriched stream 122 and bottom stream 125. The alcohol enriched stream 122 may be sent to the alcohol recovery 160 for product alcohol recovery. The product alcohol can be enriched using methods known in the art, including but not limited to distillation, adsorption (eg, with a resin), separation by molecular sieve, pervaporation, gas stripping, extraction, and the like. Can be recovered from the chemical stream 122. A bottom stream 125 containing dilute distillate with most of the solids removed prior to fermentation can be concentrated by evaporation via evaporation 130 to form syrup 135. Syrup 135 may be combined with wet cake (24, 74, 74 'described herein) in mixer 140, and then the combined stream 145 of wet cake and syrup is Dried in 150, DDGS can be produced.

  In certain embodiments, stream 105 can be degassed. In certain embodiments, stream 105 may be heated prior to degassing, for example, by inter-process exchange with a hot mash. In certain embodiments, the vapor may be exhausted to a condenser and then to a scrubber. The degassed stream 105 can be further heated, for example, by interprocess heat exchange with distillation and / or other streams in the alcohol recovery zone.

  In another embodiment of FIG. 11, the aqueous solution 22, the microorganism 32, and the extractant can be added to the fermentation 30 to generate a two-phase flow. In certain embodiments, an extractant can be added to fermentation 30 via a recycling loop. In certain embodiments, the extractant can be added downstream of fermentation 30 or external to fermentation 30. A stream containing the fermentation broth and fermentation product (eg, product alcohol) can be sent to an external extractor to produce a stream containing the product alcohol and a bottoms stream. In certain embodiments, a stream comprising product alcohol may be sent to alcohol recovery 160 for product alcohol recovery. In certain embodiments, the bottoms stream can be sent to a separation device to separate the bottoms stream into dilute distillation waste and extractant. In certain embodiments, the recovered extractant can be reused for extraction of product alcohol. The dilute waste liquor containing the majority of the solids removed prior to fermentation can be concentrated by evaporation 130 to form a syrup. The syrup may be combined with the wet cake (24, 74, 74 ′ described herein) in the mixer 140, and then the combined stream of wet cake and syrup 145 is added to the dryer 150. Dried in, DDGS can be produced.

  In certain embodiments, the aqueous solution 22, the microorganism 32, and the extractant can be added to the fermentation 30 to form a single liquid phase stream. In certain embodiments, a two-phase stream or a single liquid phase stream may be withdrawn batchwise from fermentation 30 or may be withdrawn continuously from fermentation 30. In certain embodiments, an extractant can be added downstream of fermentation 30 or external to fermentation 30 to form a single liquid phase stream. In certain embodiments, the extractant can be added to an external extractor to form a single liquid phase stream.

  An exemplary process for alcohol recovery is described herein, and additional methods for recovering product alcohol from a fermentation broth are described in US Patent Application Publication No. 2009/0305370; US Patent Application Publication No. 2010/0221802; U.S. Patent Application Publication 2011/0097773; U.S. Patent Application Publication 2011/0312044; U.S. Patent Application Publication 2011/0312043; U.S. Patent Application Publication 2012 / US Patent Application Publication No. 2012/0156738; PCT International Publication No. International Publication No. 2011/159998; and PCT International Publication No. International Publication No. 2012/030374 pamphlet; The entire contents of It is incorporated herein by reference. For example, vacuum evaporation can be used to recover product alcohol from the fermentation broth. Preheated beer (e.g., water stream 22) and solvent (e.g., extractant) may enter the preflash tower, which may be a modification of a conventional dry ground fuel ethanol plant beer tower. The column is operated at a pressure below atmospheric pressure and can be driven by water vapor taken from a series of evaporators or from a mash cooking process. The top of the preflash tower can be condensed by heat exchange by several combinations of cooling water and interprocess heat exchange including heat exchange with the preflash tower feed. The liquid condensate can be sent to an alcohol / water decanter.

  The bottom of the preflash tower can be advanced to a solvent decanter. The bottoms of the preflash tower can substantially remove product alcohol. The decanter can be a still well, a centrifuge, or a hydroclone. Water can be substantially separated from the solvent phase in the decanter to produce an aqueous phase. The aqueous phase containing suspended and dissolved solids can be centrifuged to produce a wet cake and dilute distillate waste. The wet cake can be combined with other streams and dried to produce DDGS, which can be dried and sold separately from other streams that produce DDGS, or it can be sold as a wet cake . The aqueous phase can be split to provide a backflow that is partially used to reslurry the wet cake described herein. The split also provides a dilute distillation waste that can be injected into the evaporator for further processing.

  The organic phase produced in the solvent decanter can be an ester of an alcohol. The solvent can be hydrolyzed to regenerate the reactive solvent and recover additional alcohol. Alternatively, the organic phase can be filtered and sold as a product. The hydrolysis is driven by heat and can be obtained with a homogeneous catalyst or with a heterogeneous catalyst. The heat injection into this process can be a furnace, hot oil, electrical heat injection, or high pressure steam. The water added to drive hydrolysis can come from recycled water streams, fresh water, or steam.

  The cooled and hydrolyzed solvent may be injected into a solvent tower at a pressure below atmospheric pressure, where the solvent can substantially remove the product alcohol by steam. This steam may be steam from the evaporator, may be steam from the flash step of the mash process, or may be steam from a boiler (eg, the entire contents of which are hereby incorporated by reference). U.S. Patent Application Publication No. 2009/0171129, incorporated herein by reference). A rectifying tower from a conventional dry grinding ethanol plant may be suitable as a solvent tower. The rectifying tower can be modified to act as a solvent tower. The bottom of the solvent tower can be cooled, for example, by cooling water or interprocess heat exchange. The cooled bottoms may be decanted to remove residual water, which may be reused for other steps in the process or reused for liquefaction.

  The top of the solvent tower may be cooled by exchange with cooling water or by inter-process heat exchange, and the condensate is sent to an evacuated alcohol / water decanter that can be shared with the top of the preflash tower. obtain. Other mixed water and product alcohol streams, including scrubber tower bottoms and condensate from the degassing step, may be added to the decanter. Exhaust gas containing carbon dioxide may be sent to the water scrubber. The decanter aqueous layer may also be fed to the solvent column or the product alcohol may be removed in a small dedicated distillation column. The aqueous layer may be preheated by interprocess exchange with preflush tower tops, solvent tower tops, or solvent tower bottoms. This dedicated tower can be retrofitted from a conventional dry ground fuel ethanol process side stripper.

  The organic layer of alcohol / water decanter can be injected into the alcohol tower. This column may be a super-atmospheric column and may be driven by vapor condensation in the boiler. In order to reduce the energy demand for operating the tower, the feed to the tower can be heated by inter-process heat exchange. The inter-process heat exchanger may include a preflash tower partial condenser, a solvent tower partial condenser, a hydrolyzer product, water vapor from the evaporator, or an alcohol tower bottom. The alcohol tower vapor condensate may be cooled and returned to the alcohol / water decanter. The bottoms of the alcohol tower may be cooled by inter-process heat exchange, including exchange with alcohol tower feed, and can be further cooled by cooling water, filtered, and sold as product alcohol.

  The dilute distillation liquor produced from the bottom of the preflash tower as described herein can be sent to a multi-effect evaporator (eg, the United States of which is incorporated herein by reference in its entirety). (See Japanese Patent Application Publication No. 2011/0315541). The evaporator can have two, three, or more stages. The evaporator may have a four body configuration with two effects similar to the conventional design of a fuel ethanol plant, may have three bodies with three effects, or other configurations You may have. The dilute waste liquor can enter any of the plurality of utility parts. At least one of the first effect body may be heated by steam from the overpressure alcohol tower. Steam can be withdrawn from the lowest pressure utility and heat in the form of water vapor can be provided to preflash and solvent towers at pressures below atmospheric pressure. Syrup from the evaporator may be added to the cereal dryer of the still.

  Carbon dioxide emissions from fermenters, degassers, alcohol / water decanters, and other sources can be sent to a water scrubber. The water supplied to the upper part of the scrubber may be fresh water or reused water. The recycled water can be treated (eg, biologically digested) and quenched to remove volatile organic compounds. The scrubber bottoms may be sent to an alcohol / water decanter, solvent tower, or used with other recycled water to re-slurry the wet cake as described herein. Good. Condensate from the evaporator can be processed by anaerobic biological digestion or other processes to purify the water before reuse to re-slurry the wet cake.

  The oil can be separated from the process stream at any of several points. For example, a centrifuge may be operated to produce an oil stream after filtration of cooked mash, or a preflush tower water phase centrifuge may be operated to produce an oil stream. Good. The intermediate concentrated syrup or the final syrup can be centrifuged to produce an oil stream.

  In another embodiment, the multiphase material can exit the bottom of the preflash tower and be processed in a separation system as described herein. The concentrated solids can be redistributed into a water stream, and this combined stream can be separated from the liquefied mash and used to repulp and inject the washed low starch solids.

  In another exemplary process for product alcohol recovery, the extractant removes product alcohol from the fermentation broth during fermentation to maintain the product alcohol in the fermentation broth below a certain concentration. Can be used. In certain embodiments, product alcohol removal may be performed by esterification with a carboxylic acid in the presence of a catalyst to produce an alcohol ester. A description of methods and systems for extracting alcohol by formation of alcohol esters can be found in US Patent Application Publication No. 2012/0156738, the entire contents of which are hereby incorporated by reference. For example, oil separated from the raw slurry can be hydrolyzed by a catalyst such as esterase (eg, lipase), and triglycerides in the oil can be converted to fatty acids such as carboxylic acids. These fatty acids can be used as extractants for the recovery of product alcohol. In certain embodiments, the hydrolysis of the oil can be performed in the fermentor by the addition of a catalyst to the fermentor. In certain embodiments, the hydrolysis of the oil may be performed in a separate tank and the fatty acid may be added to the fermenter. For example, the raw slurry may be sent to a tank or tank, and an esterase such as lipase can be added to the tank to convert the oil present in the raw slurry to fatty acids. The raw material slurry containing the fatty acid can be sent to a fermentation apparatus.

  The product alcohol produced by fermentation can react with fatty acids to produce alcohol esters. In certain embodiments, these alcohol esters can be extracted from the fermentation broth. For example, a fermentation broth containing an alcohol ester may be separated, such as a three-phase centrifuge, to separate the fermentation broth into three streams: an undissolved solid (including microorganisms), a water stream, and an organic stream containing the alcohol ester. Can be transferred to the device. In certain embodiments, the fermentation broth can be separated into two streams: a non-dissolved solid (including microorganisms) and a two-phase mixture including an aqueous phase and an organic phase. This separation of the fermentation broth may be performed continuously during the fermentation, for example by removing a portion of the fermentation broth for separation, or batchwise, e.g. Can be removed for separation.

  In certain embodiments, the biphasic mixture may be separated into an organic phase containing an alcohol ester and an aqueous phase, the separation comprising siphoning, aspiration, decantation, centrifugation, gravity settling, It can be performed using any method known in the art, including but not limited to phase separation by membranes, hydroclone, and the like. The organic phase containing the alcohol ester can be further processed to recover the product alcohol. For example, the organic phase containing the alcohol ester may be transferred to a tank where the alcohol ester may be hydrolyzed in the presence of a catalyst to form product alcohol and fatty acid, This mixture of alcohol and fatty acid can be processed by distillation to separate the product alcohol and fatty acid.

  In certain embodiments, the fatty acids can be recycled to the fermenter or extraction tower. In certain embodiments, water streams and undissolved solids can be recycled to the fermenter.

  In certain embodiments, the extraction of product alcohol may be performed downstream of the fermenter or external to the fermenter. In certain embodiments, the fermentation system may include an external extraction system including, for example, a mixing device and a separation system. The fermentation broth may be sent to a mixing device, and the extractant may be added to the mixing device and combined with the fermentation broth to produce a two-phase mixture. The biphasic mixture may be introduced into a separation system, where separation of the biphasic mixture produces an organic phase containing alcohol and an aqueous phase. In certain embodiments, the aqueous phase or a portion thereof can be returned to the fermenter. In certain embodiments, the organic phase containing the alcohol can be sent to the extractant tower. Biomass processing productivity in these embodiments is greatly improved by separation of biomass feedstock components after liquefaction but before fermentation. In particular, by reducing the amount of undissolved solids and / or oil, improved efficiency of the external alcohol extraction system is obtained.

  In certain embodiments, the oil may be separated from the raw material or raw slurry and stored in an oil reservoir. For example, the oil can be separated from the raw material or raw slurry using any suitable means for separation including three-phase centrifugation or mechanical extraction. Oil extraction aids such as surfactants, demulsifiers, or flocculants as well as enzymes can be used to improve the removal of oil from the raw material or raw material slurry. Examples of oil extraction aids include, but are not limited to, non-polymeric liquid surfactants; talcum powder; fine talcum powder; Pectinex® Ultra SP-L, Celluclast®, and Viscozyme ( Registered trademark L (Sigma-Aldrich, St. Louis, MO), and NZ 33095 (Novozymes, Franklinton, NC); enzymes such as salt (NaOH); and calcium carbonate. Another means for improving oil removal may be pH adjustment, such as increasing or decreasing the pH. Additional benefits of oil removal include improved oil yield, improved oil quality, reduced system deposition, and reduced downtime. Furthermore, oil removal can also result in cleaner and higher quality oils.

  The remaining feed or feed slurry can then be further processed to remove any residual oil. For example, the raw material or raw material slurry after separation of oil may be sent to a tank or a tank, a catalyst such as esterase (for example, lipase) is added to the tank, and the oil present in the raw material or raw material slurry is a fatty acid. Can be converted to The removal of oil from the feedstock or feed slurry not only increases the efficiency of the enzyme, but can also reduce the amount of enzyme required for the processes described herein. The raw material or raw slurry can then be sent to a fermentor and microorganisms can also be added to the fermentor for production of product alcohol. In certain embodiments, the catalyst can be deactivated, for example, by heating. In certain embodiments, deactivation may be performed in a separate tank, eg, a deactivation tank. The deactivated raw material or raw material slurry may be sent to a fermenter and microorganisms may also be added to the fermenter for production of product alcohol. Removal of oil from raw materials or raw slurry by converting oil to fatty acids reduces production plant energy savings through more efficient fermentation, reduces fouling through oil removal, and reduces energy requirements such as cereal distillers. This can result in a reduction in the energy required to dry. After fermentation, the fermentation broth containing the product alcohol can be sent to an external tank, such as an external extractor or an external extraction loop, for product alcohol recovery. The oil removal provided herein differs from known techniques in that embodiments of the present invention separate the oil from the raw slurry so that a stable emulsion is less likely to occur by the raw stream separation process. Less likely to require the addition of a protic solvent to break up the emulsion formed with the recovery of the incorporated bio-oil (eg, US Pat. No. 7,601,858; US Pat. (See 8,192,627). In certain embodiments of the invention, an emulsion may be formed, but is easily broken down by mechanical processing or other conventional means.

If an extractant is used to recover the product alcohol, removal of the oil prior to the fermentation process reduces the amount of oil taken up by the extractant and thus an extractant for recovering the product alcohol The effectiveness of can be extended. Oil taken up by the extractant can reduce the _Kd as well as the selectivity of the extractant, which in turn can increase the operating cost of the production process. Because the extractant can be reused in the production process, each fermentation cycle exposes the extractant to more oil that is taken up by the extractant, and over time, the _Kd and selectivity of the extractant decrease significantly. Can be done. The methods and systems described herein are intended to maintain the _Kd and selectivity of the extractant by removing oil from the feed or feed slurry and / or converting the oil to fatty acids by the addition of a catalyst. Provide the means.

  The methods and systems described herein can result in improved extraction activity and / or efficiency in fermentation product (eg, product alcohol) production as a result of removal of undissolved solids. For example, extractive fermentation without the presence of undissolved solids is a result of increased mass transfer rate of product alcohol from the fermentation broth to the extractant, better phase separation, and increased extractant droplet rise rate. It can result in lower hold-up of the extractant. Also, for example, the extractant droplets that are retained in the fermentation broth during fermentation detach more quickly and more completely from the fermentation broth, thereby reducing the free extractant in the fermentation broth. Furthermore, the lower retention of the extractant can reduce the amount of extractant lost during the process. Further advantages of solids removal include, for example, the reduction of capital costs and energy use, the elimination of stirrers in the fermenter and downstream processing equipment such as beer towers and centrifuges; resulting in improved fermenter productivity, Increased fermenter volume; reduced extractant retention, increased extractant recovery and reuse, reduced extractant flow rate that reduces operating costs, and continuous fermentation or smaller, resulting in improved production efficiency The possibility of using a fermentation apparatus is mentioned. In certain embodiments, the volume of fermenter available for fermentation can be increased by at least about 5%, at least about 10%, or more.

  Examples of improved extraction efficiency include, for example, partition coefficient stabilization, improved phase separation, improved mass transfer coefficient, operation at lower titers, improved process stream reusability, improved fermentation volume efficiency, These include improved feed loading, improved product alcohol tolerance of microorganisms, water reuse, reduced energy, increased reuse of extractants, and microorganism reuse. For example, because the oil in the fermentation broth can be reduced by removing solids from the raw slurry prior to fermentation, the extractant is exposed to less oil and the oil is combined with the extractant to extract the extractant. The distribution coefficient of the can be reduced. Thus, a reduction in oil in the fermentation broth results in a more stable partition coefficient over multiple fermentation cycles. In certain embodiments, the partition coefficient may be reduced by less than about 10%, less than about 5%, or less than about 1% over 10 or more fermentation cycles. As another example of improved extraction efficiency, a higher mass transfer rate (eg, in the form of a higher mass transfer coefficient) results in improved efficiency of product alcohol production. In certain embodiments, the mass transfer coefficient can be increased by at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold.

  The improved phase separation between the fermentation broth and the extractant reduces the likelihood of emission formation, resulting in improved efficiency of product alcohol production. For example, in the absence of an emulsion, phase separation can occur faster and can be more complete. In certain embodiments, phase separation can occur when no appreciable phase separation has been previously observed. In certain embodiments, phase separation can occur in 24 hours. In certain embodiments, the phase separation is at least about 2 times (2 ×) faster and at least about 5 times (5 ×) faster than if the solids were not removed or if an emulsion was formed. Or at least about 10 times (10 ×) faster.

  As described herein, nutrients such as nitrogen, minerals, trace elements, and / or vitamins may be added to the raw slurry or fermenter. Because these added nutrients as well as the nutrients that are naturally present in the raw material can be soluble in oil, the presence of oil in the raw material or raw slurry can reduce the concentration of these nutrients in the fermentation broth. . By removing oil from the raw material or raw slurry, nutritional losses can be minimized. Furthermore, the presence of solids in the fermentation broth can reduce the concentration of nutrients. By removing solids and / or oils from the raw material or raw slurry, nutritional losses can be minimized.

  In the methods and systems described herein, the presence of oil in the fermentation process can affect the extractant partition coefficient across multiple fermentations. The removal of oil from the raw material or raw slurry can reduce the variability of the extractant partition coefficient across multiple fermentations, and thus improve the scalability of the methods and systems described herein. Scalability refers to the ability to change (eg, scale up or down) a method or system to accommodate, for example, manufacturing demand (eg, operating capacity) without sacrificing functionality. .

  In addition, the removal of solids from the raw material or raw material slurry can also affect scalability. For example, when an external extractor is used, solids reduction (eg, total suspended solids, TSS reduction) can result in improved performance and better scalability. The reduction of solids improves the mass transfer rate of product alcohol between the aqueous phase and the organic phase (eg, fermentation broth and extractant). The solid particles effectively coat the surface of the extractant droplets and reduce the area of mass transfer. Solid particles also inhibit phase separation by increasing viscosity and emulsification tendency. Since the presence of solids can interfere with the operation of the external extractor, the removal of solids from the feed or the raw slurry provides improved scalability and reliability of the external extractor.

  Thus, removal of solids and / or oils can improve the unit operation scalability of the methods and systems described herein. For example, removal of solids and / or oils can improve unit operations such as, but not limited to, extractor performance, distillation column performance, heat exchanger performance, and / or evaporator performance.

  As an example of an improved unit operation, referring to FIG. 11, stream 105 may be sent to beer tower 120 to produce alcohol-enriched stream 122 and bottom stream 125. The bottoms stream 125 containing the distillate waste liquor along with most of the solids removed prior to fermentation can be concentrated by evaporation 130. By removing the solids prior to fermentation, less solids can be sent to the evaporator, resulting in a lower feed rate to the evaporator. Lower feed rates require less energy and thus lower costs due to lower energy requirements.

  In certain embodiments, it may be necessary to remove water from the oil recovered from the feedstock or feed slurry. In certain embodiments, the water is separated by gravity, coalescing separator, centrifugation (eg, decanter), adsorption or absorption, distillation, heating, vacuum dehydration, and / or air stripping (eg, air, nitrogen). Can be removed by several methods including: Examples of adsorption media include, but are not limited to, activated alumina, bentonite clay, calcium chloride, calcium sulfate, cellulose, magnesium sulfate, molecular sieves, polymers, and / or silica gel. In certain embodiments, the adsorption medium may be continuously stirred with oil, or the oil may flow through the packed bed with the adsorption medium.

  In some cases, it may be necessary to clean and / or sterilize equipment used to produce fermentation products such as product alcohol. Examples of equipment include, but are not limited to, fermentation equipment, liquefaction tanks, saccharification tanks, holding tanks, storage tanks, heat exchangers, pipelines, equipment connections, nozzles, fixtures, and valves. Cleaning and sterilization not only reduces or eliminates microbial contamination, but can also minimize the accumulation of residues (eg, carbohydrates, sugars) in the facility. The accumulation of residues on the equipment can provide nutrients for undesirable microorganisms and lead to the growth of these microorganisms. There are several methods used to clean and / or sterilize the fermentation facility, including in-place cleaning (CIP) and in-place sterilization (SIP). CIP and SIP may be performed manually or automated systems are available. A suitable but efficient CIP or SIP can maximize the profit of the production plant, for example by minimizing the need to shut down due to microbial contamination.

  In the methods and systems described herein, the efficiency of CIP and SIP can be improved, for example, by removal of solids and oils from the feed or feed slurry prior to fermentation. The absence of solids in the fermentation facility allows for less wash water, less wash liquid, and shorter time for CIP and SIP. Thus, removal of solids can increase the efficiency of the CIP and SIP processes and reduce the cost of the CIP and SIP processes. Also, caustic agents used in CIP (eg, sodium hydroxide) can react with triglycerides in oil to form soap (ie, saponification), which can affect the efficiency of CIP. Therefore, removal of oil can reduce soap formation during CIP and improve CIP efficiency.

  During the fermentation process, fouling can affect the productivity and efficiency of the production process. Generally, fouling refers to the deposition of material or particles from the outside, such as the deposition of material on the surface of heat exchangers and distillation column reboilers. This deposition of material on the surface of the heat exchanger can interfere with heat transfer and reduce the operating capability of the heat exchanger. For example, material deposition can hinder fluid flow through the exchanger and increase flow resistance. Also, the deposition of material on the surface of a heat exchanger or other device may require further cleaning. These further cleaning needs require factory shutdowns and can reduce factory productivity. In the methods and systems described herein, for example, removal of oil and / or solids from the raw material or raw slurry prior to fermentation minimizes material sticking, and in devices such as heat exchangers The rate of material deposition on the surface can be reduced. Thus, the removal of solids and oil can reduce the rate of fouling of the heat exchanger and minimize the fouling effect on heat transfer and operating capabilities.

  In another example of one embodiment of the method and system of the present invention, the material discharged from the fermenter is processed in a separation system including devices such as a centrifuge, a settler, a hydroclone, etc., and combinations thereof. Thus, concentrated forms of microorganisms can be recovered that can be reused for re-use in subsequent fermentations either directly or after reconditioning. The ability to recycle microorganisms increases the overall rate of fermentation product production, such as product alcohol production, reduces total titer requirements, and / or reduces aqueous titer requirements, thereby healthier microorganisms and more High production rates can be achieved. This separation system can also produce an organic stream containing fermentation products (eg, product alcohol) and other by-products produced from the fermentation, as well as an aqueous stream containing a small amount of immiscible organics. This water stream can be used before or after removing the product alcohol content to wash the solids separated from the raw slurry. This has the advantage of avoiding what could otherwise be a long belt driven transport system to move these solids from the liquefaction zone to the grain drying and syrup mixing zone. In addition, the total distillation effluent produced after the product alcohol is removed needs to be separated into dilute distillation effluent and wet cake fractions using existing or new separation devices. The dilute distillation effluent can partially form a backflow that can be combined with cooking water to prepare a new batch of fermentable mash. Another advantage of this embodiment is that a portion of any residual fermentable sugar retained in the solid separated from the raw slurry can be captured and recovered through this back flow. Alternatively, the microorganisms contained in the solid stream can be redistributed into a water stream, and this combined stream is freed of any product alcohol content remaining from the fermentation by distillation. If the microorganism is non-viable, the non-viable microorganism can be further separated, for example, for use as a nutrient in a propagation process.

  In certain embodiments of the methods and systems described herein, by-products (or by-products) of the fermentation process may be further processed, for example, undissolved solids are processed to produce DDGS. obtain. Other by-products such as fatty acid esters that may have an inhibitory effect on microorganisms can be recovered from the fermentation broth and / or by-product stream to improve the yield of product alcohol. Recovery of fatty acid esters or other lipids can be performed using a solvent to extract the fatty acid esters from the byproduct stream. In certain embodiments, several by-product streams may be combined and the fatty acid ester may be recovered from the combined stream.

  In one embodiment of solvent extraction of lipids (eg, fatty acid esters), solids can be separated from the total distillation effluent (“separated solids”), which means that this stream contains the majority of fatty acid esters. Because it can. These separated solids can then be fed to an extractor and washed with a solvent. In certain embodiments, the separated solid can be washed at least twice. After washing, the resulting mixture of lipid and solvent, known as miscella, can be collected for separation of the extracted lipid from the solvent. For example, the resulting mixture of lipid and solvent can be sent to a separator or extractor for further processing. During the extraction process, the solvent not only extracts lipids into solution, but also collects particulates (“fines”). These fines are generally undesirable impurities in the miscella, and in one embodiment, the miscella is discharged from the separator or extractor and sent to a separation device that separates or scrubs the fines from the miscella. obtain.

  In order to separate the lipids and solvents contained in the miscella, the miscella can be subjected to a distillation step. In this process, the miscella can be processed, for example, by an evaporator, which is high enough to evaporate the solvent, but not high enough to adversely affect or evaporate the extracted lipids. Heat the miscella to temperature. As the solvent evaporates, the solvent can be collected, for example, in a condenser and reused for future use. Separation of the solvent from the miscella results in the accumulation of crude lipids, which can be further processed to separate water, fatty acid esters (eg, fatty acid isobutyl esters), fatty acids, and triglycerides. A solvent-based extraction system for recovering triglycerides is described in US 2010/0092603, the entire contents of which are hereby incorporated by reference.

  After lipid extraction, the solids may be sent from the extractor and sent to a stripping device (eg, a desolventizer) that removes residual solvent. Recovery of residual solvent can be important for process economics. In certain embodiments, the solid can be sent to a desolventizer in a vapor tight environment to store and collect the solvent that can temporarily evaporate from the solid. As the solid enters the desolventizer, the solid can be heated to evaporate and remove the residual solvent. To heat the solid, the desolvator may include a mechanism for distributing the solid to one or more trays, where the solid is heated directly, such as by direct contact with heated air or steam. Or may be heated indirectly, such as by heating a tray carrying solids. To facilitate the movement of solids from one tray to another, the tray carrying the solids can include openings that allow the solids to be transported from one tray to the next. From the desolvator, the solid may optionally be sent to a mixer, where the solid is mixed with other by-products before being sent to the dryer. In certain embodiments, the solid is sent to a desolventizer and the solid is contacted by steam. In certain embodiments, the vapor and solid streams in the desolvator may be reversed. In certain embodiments, the vapor exiting the desolventizer may be condensed, optionally mixed with miscella, and then fed to the decanter to form a water-enriched phase. The water enriched phase exiting the decanter may be fed to a distillation column where the solvent is removed from the water enriched stream. In certain embodiments, the solvent-depleted water enriched stream exits the bottom of the distillation column and may be recycled to the fermentation process, for example, it can be used to process the feedstock. In certain embodiments, the top and bottom products of the distillation column can be recycled to the fermentation process. For example, the lipid-enriched tower bottom can be added to the feed of the hydrolyzer. The overhead can be condensed, for example, and fed to a decanter to form a solvent-enriched stream and a water-enriched phase. The solvent-enriched stream exiting the decanter may optionally be used as a solvent feed to the extractor, and the water-enriched phase exiting the decanter may be fed to a stripping column to remove the solvent from the water.

  In certain embodiments, by-products or by-products are obtained from the raw slurry used in the fermentation process. As described herein, when corn is used as a feedstock, the corn oil can be separated from the feed slurry prior to fermentation. The advantage of removing corn oil prior to the fermentation process is that more corn oil is recovered compared to the removal of corn oil at the end of the fermentation process (eg, from syrup), corn oil is removed at the end of the fermentation process. The recovery of higher quality and thus higher value oil compared to removal, the production of corn oil as a by-product, and the conversion of corn oil to other products.

  For example, corn oil contains antioxidants such as triglycerides, diglycerides, monoglycerides, fatty acids, plant sterols, vitamin E, carotenoids (eg, β-carotene, β-cryptoxanthin, luteinzeaxanthin), phospholipids, and tocopherols. In this regard, corn oil is added to other by-products at different concentrations or rates to provide the ability to change the amount of these components in the resulting by-product. In this way, the fat content of the resulting by-product can be controlled to obtain a low-fat, high-protein animal feed that is more suitable for dairy cow needs compared to, for example, high-fat products. In another embodiment where a high fat animal feed may be desired, corn oil can be used as a component of animal feed because it can provide a source of metabolic energy due to its high triglyceride content. Furthermore, natural antioxidants in corn oil not only reduce the occurrence of acid odor but also provide a source of vitamin E.

Corn oil separated from the feedstock can be further processed to produce refined corn oil or edible oil for consumer use. For example, crude corn oil can be degummed to remove phospholipids, alkali purified to neutralize free fatty acids, decolorized to remove color bodies and trace elements, remove wax dewaxing to, and deodorized ^{(e.g., corn oil, 5 th Edition,} corn Refiners Association, Washington, D.C., see 2006) by, it may be further processed to produce a purified corn oil. Refined corn oil can be used by food manufacturers, for example, for the production of food. Free fatty acids removed by alkaline refining may be used as soap oil stock, and the wax recovered from the dewaxing process can be used in animal feed.

  Corn oil may be used in the manufacture of resins, plastics, polymers, lubricants, paints, varnishes, printing inks, soaps, and textiles; it can also be used in the pharmaceutical industry as a component of pharmaceutical formulations. Corn oil can also be used as a raw material for biodiesel or renewable diesel.

  In certain embodiments, an oil such as corn oil may be used as a raw material for the production of an extractant for extractive fermentation. For example, oil derived from biomass can be converted into an extractant that can be used to remove product alcohols such as butanol from fermentation broth. The glycerides in the oil are chemically converted into reaction products such as fatty acids, fatty alcohols, fatty amides, fatty acid alkyl esters, fatty acid glycol esters, and hydroxylated triglycerides, or mixtures thereof, which can be used as fermentation product extractants. Or it can be enzymatically converted. Using corn oil as an example, corn oil triglycerides are reacted with a base such as ammonia hydroxide or sodium hydroxide to give fatty amides, fatty acids, and glycerol. These fatty amides, fatty acids, or mixtures thereof can be used as extractants. In certain embodiments, vegetable oils such as corn oil can be hydrolyzed by enzymes such as lipases to form fatty acids (eg, corn oil fatty acids). Methods for obtaining extractants from biomass are described in US Patent Application Publication No. 2011/0312043, US Patent Application Publication No. 2011/0312044, and PCT International Publication No. WO 2011/159998. Has been. In certain embodiments, the extractant may be used in whole or in part as a component of animal feed, or the extractant may be used as a raw material for biodiesel or renewable diesel.

  In certain embodiments, corn oil can also be used as a feedstock for biodiesel or renewable diesel. In certain embodiments, oils or combinations of oils can also be used as feedstock for biodiesel or renewable diesel. Examples of oils include canola oil, castor oil, corn oil, jojoba oil, carranja oil, mahua oil, linseed oil, soybean oil, coconut oil, peanut oil, rapeseed oil, rice oil, safflower oil, and sunflower oil . Biodiesel is obtained from either transesterification or esterification of alcohols such as methanol, ethanol, and butanol with vegetable oils. For example, biodiesel may be produced by acid, alkaline, or enzyme-catalyzed transesterification or esterification (eg, transesterification of vegetable oil-derived triglycerides or esterification of vegetable oil-derived free fatty acids). Inorganic acids such as sulfuric acid, hydrochloric acid, and phosphoric acid; organic acids such as toluene sulfonic acid and naphthalene sulfonic acid; solid acids such as Amberlyst® sulfonated polystyrene resin; or zeolites that are acid-catalyzed transesterification or esterification Can be used as a catalyst for. Bases such as potassium hydroxide, potassium methoxide, sodium hydroxide, sodium methoxide, or calcium hydroxide can be used as catalysts for alkali-catalyzed transesterification or esterification. In certain embodiments, biodiesel can be produced by an integrated process, such as acid-catalyzed esterification of free fatty acids followed by base-catalyzed transesterification of triglycerides.

  Enzymes such as lipases or esterases can be used to catalyze transesterification or esterification reactions. The lipase can be derived from bacteria or fungi, such as Pseudomonas, Thermomyces, Burkholderia, Candida, and Rhizomucor. In certain embodiments, the lipase is a fluorescent bacterium (Pseudomonas fluorescens), Pseudomonas cepacia, Rhizomucor miemurai, Burkholderia cepacia (Berhardia cemocia, Burkholderia cepacia (Berkoldia cemosia). It can be derived from Candida antarctica. In certain embodiments, the enzyme can be immobilized on a soluble or insoluble carrier. Enzyme immobilization consists of 1) binding of the enzyme to a porous or non-porous carrier carrier via covalent support, physical adsorption, electrostatic binding, or affinity binding; 2) bifunctional Or cross-linking with multifunctional reagents; 3) incorporation into gel matrices, polymers, emulsions, or some form of membrane; and 4) various techniques including any combination of these methods. In certain embodiments, the lipase can be immobilized on, for example, acrylic resin, silica, or beads (eg, polymethacrylate beads). In certain embodiments, the lipase can be soluble.

  In certain embodiments, the biodiesel described herein has one of the following fatty acid alkyl esters (FAAE): fatty acid methyl esters (FAME), fatty acid ethyl esters (FAEE), and fatty acid butyl esters (FABE). May include more than one. In certain embodiments, the biodiesel described herein can include one or more of the following: myristate, palmitate, stearate, oleate, linoleate, linolenate, arachidate, and behenate.

  In certain embodiments, the oil from the fermentation process can be recovered by evaporation to form a non-aqueous stream. This non-aqueous stream may contain fatty acid esters and fatty acids, and this stream may be sent to a hydrolyzer to recover product alcohol and fatty acids. In certain embodiments, this stream can be used as a feedstock for biodiesel production.

  In certain embodiments, the biodiesel described herein meets the specifications of the American Society for Testing and Materials (ASTM) D6751. In certain embodiments, the biodiesel described herein meets the standard of European standard EN 14214.

  In certain embodiments, reactor configurations for the production of biodiesel include, for example, batch stirred tank reactors, continuous stirred tank reactors, packed bed reactors, fluidized bed reactors, expanded bed reactors, and A recirculating membrane reactor may be mentioned.

  In certain embodiments, the composition comprises at least 2% biodiesel, at least 5% biodiesel, at least 10% biodiesel, at least 20% biodiesel, at least 30% biodiesel, at least 40% biodiesel. , At least 50% biodiesel, at least 60% biodiesel, at least 70% biodiesel, at least 80% biodiesel, at least 90% biodiesel, or 100% biodiesel.

  In certain embodiments, the biodiesel described herein can be mixed with petroleum-based diesel fuel to form a biodiesel blend. In certain embodiments, the biodiesel blend comprises at least 2% biodiesel, at least 3% biodiesel, at least 4% biodiesel, at least 5% biodiesel, at least 6% biodiesel, At least 7% biodiesel, at least 8% biodiesel, at least 9% biodiesel, at least 10% biodiesel, at least 11% biodiesel, at least 12% biodiesel, at least 13% % Biodiesel, at least 14% biodiesel, at least 15% biodiesel, at least 16% biodiesel, at least 17% biodiesel Diesel may comprise at least 18 vol% biodiesel, at least 19 vol% biodiesel, or at least 20 vol% biodiesel. In certain embodiments, the biodiesel blend may comprise up to about 20% biodiesel.

  A by-product of biodiesel production is glycerol. In addition, glycerol can also be a byproduct of the production of extractants from oil and fermentation process byproducts. Biodiesel feedstock can be produced by reacting fatty acids such as COFA with glycerol. The reaction can be catalyzed by a strong inorganic acid such as sulfuric acid or by a solid acid catalyst such as an Amberlyst ™ polymer catalyst and an ion exchange resin. A high conversion is obtained by withdrawing water from the reaction mass. The reaction product may contain monoglycerides, diglycerides, and triglycerides in proportions determined by the ratio of reactants and the extent of reaction. The glyceride mixture can be used in place of the triglyceride raw material normally used to make biodiesel. In certain embodiments, glycerides can be used as surfactants or as raw materials for biodiesel.

  In certain embodiments, the solid may be separated from the raw slurry and may include triglycerides and fatty acids. These solids may be used as animal feed and can be recovered either as a drain from centrifugation or after drying. Solids may be particularly suitable as ruminant (eg, dairy) feed due to the high content of available lysine and bypass or rumen non-degradable proteins. For example, these solids can be particularly useful in high protein, low fat feeds. In certain embodiments, these solids may be used as a base, i.e. other by-products such as syrup may be added to the solids to form a product that can be used as animal feed. Good. In certain embodiments, varying amounts of other by-products are required for specific animal species (eg, dairy and beef cattle, poultry, pigs, livestock, horses, aquaculture, and pets) where the resulting product characteristics are It may be added to the solid to adjust to meet the properties.

  In certain embodiments where a low fat animal feed is desired, the oil can be removed from the feed prior to fermentation. By removing corn oil, the DDGS produced can have a low fat, high protein content. If corn oil is not removed, the oil present in the wet cake can be oxidized by a drying process. This oxidation causes a darkening action and produces darker DDGS. If the oil is removed from the raw material prior to fermentation, the color of the DDGS produced will be lighter and this lighter color DDGS may be desirable for certain animal feed products.

  The solid composition separated from the total distillation effluent can include, for example, crude proteins, fatty acids, and fatty acid esters. In certain embodiments, the composition may be used wet or dry as animal feed, in which case, for example, high protein (eg, high lysine), low fat, and high fiber content is desired. . In certain embodiments, if a higher fat, lower fiber animal feed is desired, fat may be added to the composition, for example from another byproduct stream. In certain embodiments, this higher fat, low fiber animal feed can be used in pigs or poultry. In certain embodiments, non-aqueous compositions of CDS may include other dissolved and suspended solids such as, for example, proteins, fatty acids, and fatty acid esters and salts and carbohydrates. The CDS composition may be used, for example, in a wet or dry state as animal feed, in which case a high protein, low fat, high inorganic salt feed component is desired. In certain embodiments, the composition may be used as a component of dairy cow feed.

  In certain embodiments, one or more streams produced by production of product alcohol by a fermentation process are combined to produce a composition comprising at least about 90% fatty acids that can be used as a fuel source, such as biodiesel. Can be done.

  The various streams produced by the production of product alcohol by the fermentation process can be combined in many ways to produce several by-products. For example, if crude corn from mash is used to produce a fatty acid that is used as an extractant and the lipid is extracted by an evaporator, the remaining streams are combined to produce crude protein, crude fat, triglyceride, fatty acid, And a by-product composition comprising a fatty acid ester. In another example, when an oil such as corn oil is removed from the raw slurry, the oil can be added to a cereal distiller, for example, to produce an animal feed product.

  In certain embodiments, the compositions of the methods and systems described herein comprise at least about 20-35 wt% crude protein, at least about 1-20 wt% crude fat, at least about 0-5 wt%. It may comprise triglycerides, at least about 4-10% by weight fatty acids, and at least about 2-6% by weight fatty acid esters. In certain embodiments, the composition comprises about 25% crude protein, about 10% crude fat, about 0.5% triglyceride, about 6% fatty acid, and about 4% fatty acid ester. May be included. In some embodiments, the composition comprises at least about 25-31% crude protein, at least about 6-10% crude fat, at least about 4-8% triglyceride, at least about 0-2% fatty acid. , And at least about 1-3 wt% fatty acid ester. In certain embodiments, the composition comprises about 28% crude protein, about 8% crude fat, about 6% triglyceride, about 0.7% fatty acid, and about 1% fatty acid ester. May be included. In certain embodiments, the fatty acid ester can be a fatty acid methyl ester, a fatty acid ethyl ester, a fatty acid butyl ester, or a fatty acid isobutyl ester.

  In certain embodiments, solids separated from total distillation waste and oil extracted from the raw slurry may be combined and the resulting composition comprises crude protein, crude fat, triglyceride, fatty acid, fatty acid ester, lysine, Neutral detergent fibers (NDF) and acidic detergent fibers (ADF) can be included. In certain embodiments, the composition comprises at least about 26-34% crude protein, at least about 15-25% crude fat, at least about 12-20% triglyceride, at least about 1-2% fatty acid. , At least about 2-4 wt% fatty acid ester, at least about 1-2 wt% lysine, at least about 11-23 wt% NDF, and at least about 5-11 wt% ADF. In certain embodiments, the composition comprises about 29% crude protein, about 21% crude fat, about 16% triglyceride, about 1% fatty acid, about 3% fatty acid ester, about 1% by weight. % Lysine, about 17% by weight NDF, and about 8% by weight ADF. In certain embodiments, the fatty acid ester can be a fatty acid methyl ester, a fatty acid ethyl ester, a fatty acid butyl ester, or a fatty acid isobutyl ester. The high fat, triglyceride, and lysine content and lower fiber content of this composition would be desirable for pig and poultry feed.

  As described herein, the various streams produced by the production of product alcohols by the fermentation process are many to produce compositions comprising crude proteins, crude fats, triglycerides, fatty acids, and fatty acid esters. Can be combined in the following manner. For example, a composition comprising at least about 6% crude fat and at least about 28% crude protein can be used as an animal feed product for dairy animals. A composition comprising at least about 6% crude fat and at least about 26% crude protein can be used as an animal feed product for feedlot cattle, while at least about 1% crude fat and at least about 27% crude protein. The containing composition can be used as an animal feed product for wintering cattle. Compositions comprising at least about 13% crude fat and at least about 27% crude protein can be used as poultry animal feed products. Compositions comprising at least about 18% crude fat and at least about 22% crude protein can be used as monogastric animal feed products. Various streams feed products for specific animal species (eg livestock, ruminants, cattle, dairy animals, pigs, goats, sheep, poultry, horses, aquaculture, or pets such as dogs, cats, and rabbits) Can be combined to customize.

  The DDGS produced by the method of the present invention produces a high value-added feed product customized by the addition of one or more of the following: protein, fat, fiber, ash, lipid, amino acid, vitamin, and mineral Can be modified. Amino acids include, for example, essential amino acids such as histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine, and alanine, arginine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine, glycine, hydroxylysine, hydroxy Other amine acids such as proline, ornithine, proline, serine, and tyrosine. Examples of the mineral include calcium, chloride, cobalt, copper, fluoride, iodine, iron, magnesium, manganese, phosphorus, potassium, selenium, sodium, sulfur, and zinc. Vitamins include, for example, vitamins A, C, D, E, K, and B (thiamine, riboflavin, niacin, pantothenic acid, biotin, vitamin B6, vitamin B12 and folate).

  As described herein, the methods and systems of the present invention provide several advantages that can result in improved production of product alcohols such as butanol. For example, improved mass transfer allows operation at lower aqueous titers, making microorganisms “healthy”. Better phase separation can lead to improved fermenter volumetric efficiency and the possibility of processing less reactor content through beer towers, distillation towers, and the like. Furthermore, there is less solvent loss through the solid and there is a possibility of cell reuse. The method and system of the present invention may also provide higher quality of DDGS.

  The methods and systems described herein also provide for removal of oil prior to fermentation, which may allow controlled addition of oil to the fermentation. Furthermore, removal of the oil prior to fermentation can provide flexibility in the amount of oil present in the DDGS. That is, oil may be added to DDGS in various amounts, producing DDGS with varying fat content depending on the nutritional requirements of a particular animal species.

  The methods and systems described herein can be demonstrated using computer modeling, such as Aspen modeling (see, eg, US Pat. No. 7,666,282). For example, the commercially available modeling software Aspen Plus® (Aspen Technology, Inc., Burlington, Mass.) Is used by the American Institute to create Aspen models for integrated alcohol fermentation, purification, and water management processes. of Chemical Engineers, Inc. (New York, NY) and can be used with a physical property database such as DIPPR (Design Institute for Physical Property Research). This process modeling can perform many basic engineering calculations such as mass and energy balance, gas / liquid equilibrium, and reaction rate calculations. To generate an Aspen model, information input includes, for example, experimental data, moisture content and ingredient composition, temperature for cooking and flushing mash, saccharification conditions (eg, enzyme ingredient, starch conversion, temperature, pressure) , Fermentation conditions (eg, microbial raw materials, glucose conversion, temperature, pressure), deaeration conditions, solvent towers, preflash towers, condensers, evaporators, centrifuges, and the like.

Recombinant microorganisms While not wishing to be bound by theory, the methods described herein relate to any alcohol-producing microorganism, particularly those that produce alcohol with potency above their resistance level. Therefore, it is considered useful.

  Alcohol-producing microorganisms are known in the art. For example, methanol is produced by fermentative oxidation of methane by a methane-utilizing bacterium (for example, Methylosinus trichospolium), and yeast strain CEN. Ethanol is produced by PK113-7D (CBS 8340, the Central Blue floor Scheme; van Dijken, et al., Enzyme Microb. Techno. 26: 706-714, 2000). Recombinant microorganisms that produce alcohol are also known in the art (eg, Ohta, et al., Appl. Environ. Microbiol. 57: 893-900, 1991; Underwood, et al., Appl. Environ. Microbiol. 68: 1071-1081, 2002; Shen and Liao, Metab. Eng.10: 312-320, 2008; Hahnai, et al., Appl. Environ.Microbiol.73: 7814-7818, 2007; U.S. Pat. No. 5,712,133; PCT application publication number WO 1995/028476; Feldmann, et al., Appl. Biobiol.Biotechnol.38: 354-361,1992; Zhang, et al., Science 267: 240-243, 1995; U.S. Patent Application Publication No. 2007 / 0031918A1; U.S. Patent No. 7,223,575 U.S. Patent No. 7,741,119; U.S. Patent No. 7,851,188; U.S. Patent Application Publication No. 2009 / 0203099A1; U.S. Patent Application Publication No. 2009 / 0246846A1; and PCT Application Publication No. WO 2010/075241, all of which are incorporated herein by reference).

  Furthermore, the microorganism can be modified using recombinant techniques to produce recombinant microorganisms capable of producing product alcohols such as ethanol and butanol. Microorganisms that are modified by recombinant techniques to produce product alcohols via biosynthetic pathways include Clostridium, Zymomonas, Escherichia, Salmonella, Serratia (Serratia), Erwinia, Klebsiella, Shigella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Lactobacillus, Lactobacillus (Enterococcus), Alcaligenes, Klebsiella (K ebsiella), Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Schizosaccharomyces, Schizosaccharomyces Examples include members of the genus Yarrowia, Pichia, Candida, Hansenula, Issatchenkia, or Saccharomyces. In certain embodiments, the recombinant microorganisms are Escherichia coli, Lactobacillus plantarum, Kluyveromyces lactis, Kluyveromyces marciane cerevisiae and Kluyveromyces cerevisiae. (Saccharomyces cerevisiae) may be selected. In certain embodiments, the recombinant microorganism is yeast. In certain embodiments, the recombinant microorganism is a genus Saccharomyces, Zygosaccharomyces, Schizosaccharomyces, Dekkera, Torulopis genus, Torulopsis s. Crabtree positive yeast selected from several species of the genus (Candida). Examples of crabtree positive yeast species include, but are not limited to, Saccharomyces cerevisiae, Saccharomyces kluyveri, Schizosaccharomyces pombe (Schizosaccharomyces pombe). Mikatae (Saccharomyces mikitae), Saccharomyces paradoxus (Saccharomyces paradoxus), Zygosaccharomyces rouxii, and Candida glabrata (Candrida) ata).

  Saccharomyces cerevisiae is known in the art and is the American Type of Culture Culture Collection (Rockville, MD), CentralbureCulture (Soccharomyces cerevisiae). Available from a variety of sources including, but not limited to, Ferm Solutions, North American Bioproducts, Martrex, and Lalland. Examples of Saccharomyces cerevisiae include, but are not limited to, BY4741, CEN. PK 113-7D, Ethanol Red (registered trademark) yeast, Ferm Pro (registered trademark) yeast, Bio-Ferm (registered trademark) XR yeast, Gert Strand Prestige Batch Turbo alcohol yeast, Gert Strand Pot Distillers yeast, Gert Strb yeast yeast, Gert Strb yeast FerMax ™ Green yeast, FerMax ™ Gold yeast, Thermosacc® yeast, BG-1, PE-2, CAT-1, CBS7959, CBS7960, and CBS7961.

  In certain embodiments, the microorganism can be immobilized or encapsulated. For example, microorganisms are immobilized using alginate, calcium alginate, or polyacrylamide gels, or by induction of biofilm formation on various high surface area support substrates such as diatomite, celite, diatomaceous earth, silica gel, plastic, or resin. Or it can be encapsulated. In certain embodiments, ISPR can be used in combination with immobilized or encapsulated microorganisms. This combination can improve productivity such as specific volume productivity, metabolic rate, product alcohol yield, tolerance to product alcohol. Furthermore, immobilization and encapsulation can minimize the effects of process conditions such as shear on microorganisms.

  Production of butanol using fermentation and microorganisms that produce butanol are described, for example, in US Pat. No. 7,851,188 and US Patent Application Publication No. 2007/0092957; 2007/0259410. No. 2007/0292927; No. 2008/0182308; No. 2008/0274525; No. 2009/0155870; No. 2009/0305363; 2009/0305370, the entire contents of each of which are hereby incorporated by reference. In certain embodiments, the microorganism is modified to include a biosynthetic pathway. In certain embodiments, the biosynthetic pathway is a modified butanol biosynthetic pathway. In certain embodiments, the biosynthetic pathway converts pyruvate into a fermentation product. In certain embodiments, the biosynthetic pathway converts pyruvate as well as amino acids into a fermentation product. In certain embodiments, at least one, at least two, at least three, or at least four polypeptides that catalyze each substrate-product conversion of the pathway are encoded by heterologous polynucleotides in the microorganism. In certain embodiments, all polypeptides that catalyze pathway substrate-product conversion are encoded by heterologous polynucleotides in the microorganism. In certain embodiments, a polypeptide that catalyzes substrate-product conversion from acetolactate to 2,3-dihydroxyisovaleric acid and / or a polypeptide that catalyzes substrate-product conversion from isobutyraldehyde to isobutanol comprises: Reduced nicotinamide adenine dinucleotide (NADH) can be used as a cofactor.

Biosynthetic pathways Biosynthetic pathways for the production of isobutanol that can be used include those described in US Pat. No. 7,851,188, incorporated herein by reference. In one embodiment, the isobutanol biosynthetic pathway comprises the following substrate-product conversion:
a) pyruvate to acetolactate, which can be catalyzed, for example, by acetolactate synthase;
b) 2,3-dihydroxyisovaleric acid from acetolactate, which can be catalyzed, for example, by acetohydroxy acid reductoisomerase;
c) 2,3-dihydroxyisovaleric acid to α-ketoisovaleric acid, which can be catalyzed, for example, by acetohydroxyacid dehydratase;
d) α-ketoisovaleric acid to isobutyraldehyde, which can be catalyzed, for example, by branched chain α-keto acid decarboxylase; and e) isobutyraldehyde to isobutanol, which can be catalyzed, for example, by branched chain alcohol dehydrogenase.

In another embodiment, the isobutanol biosynthetic pathway comprises the following substrate-product conversion:
a) pyruvate to acetolactate, which can be catalyzed, for example, by acetolactate synthase;
b) 2,3-dihydroxyisovaleric acid from acetolactate, which can be catalyzed, for example, by ketolate reductoisomerase;
c) 2,3-dihydroxyisovaleric acid to a-ketoisovaleric acid, which can be catalyzed, for example, by dihydroxy acid dehydratase;
d) α-ketoisovaleric acid to valine, which can be catalyzed, for example, by transaminase or valine dehydrogenase;
e) valine to isobutylamine, which can be catalyzed, for example, by valine decarboxylase;
f) Isobutylamine to isobutyraldehyde, which can be catalyzed by, for example, omega transaminase; and g) Isobutyraldehyde to isobutanol, which can be catalyzed by, for example, branched chain alcohol dehydrogenase.

In another embodiment, the isobutanol biosynthetic pathway comprises the following substrate-product conversion:
a) pyruvate to acetolactate, which can be catalyzed, for example, by acetolactate synthase;
b) 2,3-dihydroxyisovaleric acid from acetolactate, which can be catalyzed, for example, by acetohydroxy acid reductoisomerase;
c) 2,3-dihydroxyisovaleric acid to α-ketoisovaleric acid, which can be catalyzed, for example, by acetohydroxyacid dehydratase;
d) α-ketoisovaleric acid to isobutyryl-CoA, which can be catalyzed, for example, by branched-chain ketoacid dehydrogenase;
e) Isobutyryl-CoA to isobutyraldehyde, which can be catalyzed by, for example, acylated aldehyde dehydrogenase; and f) Isobutyraldehyde to isobutanol, which can be catalyzed by, for example, branched chain alcohol dehydrogenase.

Biosynthetic pathways for the production of 1-butanol that can be used include those described in US Patent Application Publication No. 2008/0182308, which is incorporated herein by reference. In one embodiment, the 1-butanol biosynthetic pathway comprises the following substrate-product conversion:
a) For example, acetyl-CoA to acetoacetyl-CoA, which can be catalyzed by acetyl-CoA acetyltransferase;
b) acetoacetyl-CoA to 3-hydroxybutyryl-CoA, which can be catalyzed, for example, by 3-hydroxybutyryl-CoA dehydrogenase;
c) 3-hydroxybutyryl-CoA to crotonyl-CoA, which can be catalyzed, for example, by crotonase;
d) Crotonyl-CoA to butyryl-CoA, which can be catalyzed, for example, by butyryl-CoA dehydrogenase;
e) butyryl-CoA to butyraldehyde, which can be catalyzed by, for example, butyraldehyde dehydrogenase; and f) butyraldehyde to 1-butanol, which can be catalyzed by, for example, butanol dehydrogenase.

Biosynthetic pathways for the production of 2-butanol that can be used include US Patent Application Publication No. 2007/0259410 and US Patent Application Publication No. 2009/0155870, which are incorporated herein by reference. What is described is mentioned. In one embodiment, the 2-butanol biosynthetic pathway includes the following substrate-product conversion:
a) α-acetolactic acid from pyruvate, which can be catalyzed, for example, by acetolactate synthase;
b) α-acetolactic acid to acetoin, which can be catalyzed, for example, by acetolactate decarboxylase;
c) Acetoin to 3-amino-2-butanol, which can be catalyzed, for example, by acetoin aminase;
d) 3-amino-2-butanol to 3-amino-2-butanol phosphate, which can be catalyzed, for example, by aminobutanol kinase;
e) 2-butanone from 3-amino-2-butanol phosphate, which can be catalyzed by, for example, aminobutanol phosphate phosphorylase; and f) 2-butanone to 2-butanol, which can be catalyzed by, for example, butanol dehydrogenase.

In another embodiment, the 2-butanol biosynthetic pathway comprises the following substrate-product conversion:
a) α-acetolactic acid from pyruvate, which can be catalyzed, for example, by acetolactate synthase;
b) α-acetolactic acid to acetoin, which can be catalyzed, for example, by acetolactate decarboxylase;
c) 2,3-butanediol from acetoin, which can be catalyzed, for example, by butanediol dehydrogenase;
d) 2,3-butanediol to 2-butanone, which can be catalyzed by, for example, diol dehydratase; and e) 2-butanone to 2-butanol, which can be catalyzed by, for example, butanol dehydrogenase.

Biosynthetic pathways for the production of 2-butanone that can be used include US Patent Application Publication No. 2007/0259410 and US Patent Application Publication No. 2009/0155870, which are incorporated herein by reference. What is described is mentioned. In one embodiment, the 2-butanone biosynthetic pathway includes the following substrate-product conversion:
a) α-acetolactic acid from pyruvate, which can be catalyzed, for example, by acetolactate synthase;
b) α-acetolactic acid to acetoin, which can be catalyzed, for example, by acetolactate decarboxylase;
c) Acetoin to 3-amino-2-butanol, which can be catalyzed, for example, by acetoin aminase;
d) 3-amino-2-butanol to 3-amino-2-butanol phosphate, which can be catalyzed by, for example, aminobutanol kinase; and e) 3-amino-2-butanol, which can be catalyzed by, for example, aminobutanol phosphate phosphorylase From phosphate to 2-butanone.

In another embodiment, the 2-butanone biosynthetic pathway comprises the following substrate-product conversion:
a) α-acetolactic acid from pyruvate, which can be catalyzed, for example, by acetolactate synthase;
b) α-acetolactic acid to acetoin, which can be catalyzed, for example, by acetolactate decarboxylase;
c) 2,3-butanediol from acetoin, which can be catalyzed by, for example, butanediol dehydrogenase; and d) 2-butanone from 2,3-butanediol, which can be catalyzed by, for example, diol dehydratase.

The terms “acetohydroxyacid synthase”, “acetolactic acid synthase”, and “acetolactic acid synthetase” (abbreviated as “ALS”) have enzymatic activity that catalyzes the conversion of pyruvate to acetolactate and CO ₂ . Used interchangeably herein to refer to a polypeptide. An example of acetolactate synthase is known by EC number 2.2.1.6 (Enzyme Nomenclature 1992, Academic Press, San Diego). These unmodified enzymes are Bacillus subtilis (GenBank number: CAB15618 (SEQ ID NO: 1), Z99122 (SEQ ID NO: 2), NCBI (National Center for Biotechnology Information) Amino acid sequence, NCBI nucleotide sequence), Klebsiella pneumoniae (GenBank number: AAA25079 (SEQ ID NO: 3), M73842 (SEQ ID NO: 4)), and Lactococcus lactis (GenBank ) Number: AAA25161 (SEQ ID NO: 5), L16975 (SEQ ID NO: 6)), but not limited to Available from several sources.

  The terms “keto acid reductoisomerase” (“KARI”), “acetohydroxy acid isomero reductase”, and “acetohydroxy acid reductoisomerase” are used interchangeably and are derived from (S) -acetolactic acid to 2,3 -Refers to a polypeptide having enzymatic activity that catalyzes the reaction to dihydroxyisovaleric acid. An example of a KARI enzyme may be classified as EC number EC 1.1.1.86 (Enzyme Nomenclature 1992, Academic Press, San Diego), Escherichia coli (GenBank number: NP — 418222 (sequence) No. 7), NC — 000913 (SEQ ID NO: 8)), Saccharomyces cerevisiae (GenBank numbers: NP — 013459 (SEQ ID NO: 9), NC — 00144 (SEQ ID NO: 10)), Methanococcus maripardis (Methanococcus Methanococus) (GenBank number: CAF30210 (SEQ ID NO: 11), BX957220 (SEQ ID NO: 12)), and Bacillus subtilis (GenBank numbers: CAB14789 (SEQ ID NO: 13), Z99118 (SEQ ID NO: 14)), available from a wide range of microorganisms including but not limited to It is. KARI includes Anaerostipes cakae KARI mutants “K9G9” and “K9D3” (SEQ ID NOS: 15 and 16, respectively). The keto acid reductoisomerase (KARI) enzyme is incorporated herein by reference, US 2008/0261230, 2009/0163376, and 2010/0197519. And PCT application publication number WO 2011/041415. Examples of KARI disclosed in these documents are Lactococcus lactis, Vibrio cholera, Pseudomonas aeruginosa PAO1, and PF5 derived from Pseudomonas fluorescens PF5. To do. In some embodiments, KARI uses NADH. In certain embodiments, KARI uses reduced nicotinamide adenine dinucleotide phosphate (NADPH).

  The terms “acetohydroxy acid dehydratase” and “dihydroxy acid dehydratase” (“DHAD”) refer to polypeptides having enzymatic activity that catalyzes the conversion of 2,3-dihydroxyisovaleric acid to α-ketoisovaleric acid. An example of acetohydroxy acid dehydratase is known by EC number 4.2.1.9. Such enzymes include Escherichia coli (GenBank number: YP — 026248 (SEQ ID NO: 17), NC000913 (SEQ ID NO: 18)), Saccharomyces cerevisiae (GenBank number: NP — 25 — (SEQ ID NO: 19), NC 001142 (SEQ ID NO: 20), M. maripaldis (GenBank number: CAF 29874 (SEQ ID NO: 21), BX957219 (SEQ ID NO: 22)), Bacillus subtilis (B. subtilis (GenBank number: CAB14105 (SEQ ID NO: 23), Z99115 (SEQ ID NO: 24)), Lactococcus lactis (L. and is available from a wide range of microorganisms including, but not limited to, N. crassa, U.S. Patent Application Publication No. 2010/0081154, and U.S. Pat. Japanese Patent No. 7,851,188 describes dihydroxy acid dehydratase (DHAD) containing DHAD derived from Streptococcus mutans.

The term “branched α-keto acid decarboxylase”, “α-keto acid decarboxylase”, “α-ketoisovalerate decarboxylase” or “2-ketoisovalerate decarboxylase” (“KIVD”) It refers to a polypeptide having enzymatic activity that catalyzes the conversion of α-ketoisovaleric acid to isobutyraldehyde and CO ₂ . An example of a branched chain α-keto acid decarboxylase is known by EC number 4.1.1.72, Lactococcus lactis (GenBank number: AAS49166 (SEQ ID NO: 25)) AY548760 (SEQ ID NO: 26); CAG34226 (SEQ ID NO: 27), AJ746364 (SEQ ID NO: 28), Salmonella typhimurium (GenBank number: NP_461346 (SEQ ID NO: 29), NC_003197 (SEQ ID NO: 30)), Clostridium acetobutylicum (GenBank number: NP — 149189 (SEQ ID NO: 31), NC — 001988 SEQ ID NO: 32)), M. caseoliticus (SEQ ID NO: 33), and L. grayi (SEQ ID NO: 34) from several sources including but not limited to Is possible.

  The term “branched alcohol dehydrogenase” (“ADH”) refers to a polypeptide having an enzymatic activity that catalyzes the conversion of isobutyraldehyde to isobutanol. An example of a branched chain alcohol dehydrogenase is known by EC number 1.1.1.265, but other alcohol dehydrogenases (especially EC 1.1.1.1 or 1.1.1.2) Can also be classified. Alcohol dehydrogenase can be NADPH-dependent or NADH-dependent. Such enzymes are Saccharomyces cerevisiae (GenBank number: NP — 010656 (SEQ ID NO: 35), NC — 00136 (SEQ ID NO: 36), NP — 014051 (SEQ ID NO: 37), NC — 00145 (SEQ ID NO: 38)), Escherichia coli (GenBank numbers: NP_417484 (SEQ ID NO: 39), NC_000913 (SEQ ID NO: 40)), C.I. A number including, but not limited to, C. acetobutylicum (GenBank numbers: NP_349892 (SEQ ID NO: 41), NC_003030 (SEQ ID NO: 42); NP_349891 (SEQ ID NO: 43), NC_003030 (SEQ ID NO: 44)) Available from these sources. US Patent Application Publication No. 2009/0269823 describes an alcohol dehydrogenase (ADH) derived from SadB, Achromobacter xylosoxidans. Alcohol dehydrogenase is also ADH from horse liver (as described in US Patent Application Publication No. 2011/0269199, incorporated herein by reference) and Beijerinckia indica ADH. Including.

  The term “butanol dehydrogenase” refers to a polypeptide (or polypeptide) having enzymatic activity that catalyzes the conversion of isobutyraldehyde to isobutanol or the conversion of 2-butanone and 2-butanol. Butanol dehydrogenase is a subset of a broad family of alcohol dehydrogenases. Butanol dehydrogenase can be NAD- or NADP-dependent. NAD-dependent enzymes are known as EC 1.1.1.1 and are available, for example, from Rhodococcus rubber (GenBank number: CAD36475, AJ491307). NADP-dependent enzymes are known as EC 1.1.1.2 and are available, for example, from Pyrococcus furiosus (GenBank number: AAC25556, AF013169). Furthermore, butanol dehydrogenase is available from Escherichia coli (GenBank number: NP 417484, NC_000913), and cyclohexanol dehydrogenase is Acinetobacter sp. (GenBank number: AAG10026, AF282240). The term “butanol dehydrogenase” also refers to an enzyme that catalyzes the conversion of butyraldehyde to 1-butanol using either NADH or NADPH as a cofactor. Butanol dehydrogenase is, for example, C.I. C. acetobutylicum (GenBank numbers: NP — 149325, NC — 001988; note: this enzyme has both aldehyde and alcohol dehydrogenase activities); NP — 349891, NC — 003030; and NP — 349892, NC — 003030 and Escherichi (GenBank numbers: NP_417-484, NC_000913).

The term “branched keto acid dehydrogenase” is usually the conversion of α-ketoisovaleric acid to isobutyryl-CoA (isobutyryl-coenzyme A) using NAD ⁺ (nicotinamide adenine dinucleotide) as the electron acceptor. Refers to a polypeptide having an enzymatic activity that catalyzes. An example of a branched chain keto acid dehydrogenase is known by EC number 1.2.4.4. Such a branched chain keto acid dehydrogenase is composed of four subunits, and the sequences from all the subunits are Bacillus subtilis (GenBank number: CAB14336 (SEQ ID NO: 45), Z99116. (SEQ ID NO: 46); CAB14335 (SEQ ID NO: 47), Z99116 (SEQ ID NO: 48); CAB14334 (SEQ ID NO: 49), Z99116 (SEQ ID NO: 50); and CAB14337 (SEQ ID NO: 51), Z99116 (SEQ ID NO: 52)) and Pseudomonas putida (GenBank number: AAA65614 (SEQ ID NO: 53), M57613 (SEQ ID NO: 54); AAA65615 (SEQ ID NO: 55), M57613 ( Column number 56); AAA65617 (SEQ ID NO: 57), M57613 (SEQ ID NO: 58); and AAA65618 (SEQ ID NO: 59), including M57613 (SEQ ID NO: 60)) is available from a wide range of microorganisms, including but not limited to.

  The term “acylated aldehyde dehydrogenase” refers to a polypeptide having an enzymatic activity that catalyzes the conversion of isobutyryl-CoA to isobutyraldehyde, usually using either NADH or NADPH as an electron donor. Examples of acylated aldehyde dehydrogenases are known by EC numbers 1.2.1.10 and 1.2.2.17. Such enzymes include Clostridium beijerinckii (GenBank numbers: AAD31841 (SEQ ID NO: 61), AF157306 (SEQ ID NO: 62)), Clostridium acetobutylicum (Genbank) (Genbank) NP_149325 (SEQ ID NO: 63), NC_001988 (SEQ ID NO: 64); NP_149199 (SEQ ID NO: 65), NC_001988 (SEQ ID NO: 66)), Pseudomonas putida (GenBank) number: AAA89106 (SEQ ID NO: 67) ), U13232 (SEQ ID NO: 68)), and Thermus Mofirusu (Thermus thermophilus) (Genbank (GenBank) Number: YP_145486 (SEQ ID NO: 69), NC_006461 (SEQ ID NO: 70)) including available from multiple sources, including but not limited to.

  The term “transaminase” refers to a polypeptide having an enzymatic activity that catalyzes the conversion of α-ketoisovaleric acid to L-valine using either alanine or glutamate as the amine donor. Examples of transaminases are known by EC numbers 2.6.1.42 and 2.6.1.66. Such enzymes are available from several sources. Examples of sources of alanine-dependent enzymes include, but are not limited to, Escherichia coli (GenBank numbers: YP — 026231 (SEQ ID NO: 71), NC — 000913 (SEQ ID NO: 72)) and Bacillus licheniformis. (Bacillus licheniformis) (GenBank number: YP_093743 (SEQ ID NO: 73), NC_006322 (SEQ ID NO: 74)). Examples of sources of glutamate-dependent enzymes include, but are not limited to, Escherichia coli (GenBank numbers: YP — 026247 (SEQ ID NO: 75), NC — 000913 (SEQ ID NO: 76)), Saccharomyces Saccharomyces cerevisiae (GenBank number: NP_012682 (SEQ ID NO: 77), NC_001142 (SEQ ID NO: 78)) and Methanobacterum thermoautotrophicum (N) SEQ ID NO: 79), NC_000916 (SEQ ID NO: 80)).

  The term “valine dehydrogenase” is a polypeptide having an enzymatic activity that catalyzes the conversion of α-ketoisovaleric acid to L-valine, usually using NAD (P) H as an electron donor and ammonia as an amine donor. Point to. Examples of valine dehydrogenases are known by EC numbers 1.4.1.8 and 1.4.1.9, such enzymes are described in Streptomyces coelicolor (GenBank). Including: NP_628270 (SEQ ID NO: 81), NC_003888 (SEQ ID NO: 82)) and Bacillus subtilis (GenBank numbers: CAB14339 (SEQ ID NO: 83), Z99116 (SEQ ID NO: 84)). Available from several non-limiting sources.

The term “valine decarboxylase” refers to a polypeptide having an enzymatic activity that catalyzes the conversion of L-valine to isobutylamine and CO ₂ . An example of valine decarboxylase is known by EC number 4.1.1.14. Such enzymes include, for example, Streptomyces viridifaciens (GenBank numbers: AAN10242 (SEQ ID NO: 85), AY116644 (SEQ ID NO: 86)) and other Streptomyces genera (Streptomyces). ).

  The term “omega transaminase” refers to a polypeptide having enzymatic activity that catalyzes the conversion of isobutylamine to isobutyraldehyde using an amino acid suitable as an amine donor. An example of an omega transaminase is known by EC number 2.6.1.18, Alcaligenes denitrificans (AAP92672 (SEQ ID NO: 87), AY330220 (SEQ ID NO: 88)), Ralstonia eutropha (Ralstonia eutropha) (GenBank number: YP — 294474 (SEQ ID NO: 89), NC — 007347 (SEQ ID NO: 90)), Shewanella oneidensis (GenBank) number: NP — 719046 (SEQ ID NO: 91) (SEQ ID NO: 92)), and Pseudomonas putida GenBank (GenBank) Number: AAN66223 (SEQ ID NO: 93), which is available from several sources, including, AE016776 (SEQ ID NO: 94)) without limitation.

  The term “acetyl-CoA acetyltransferase” refers to a polypeptide having an enzymatic activity that catalyzes the conversion of two molecules, acetyl-CoA to acetoacetyl-CoA and coenzyme A (CoA). An example of acetyl-CoA acetyltransferase is acetyl-CoA acetyltransferase with substrate selectivity (forward reaction) for short chain acyl-CoA and acetyl-CoA. C. 2.3.1.9 [Enzyme Nomenclature 1992, Academic Press, San Diego], but enzymes with a wider substrate range (EC 2.3.1.16) function similarly. Acetyl-CoA acetyltransferase has several sources, such as Escherichia coli (GenBank numbers: NP_416728, NC_000913; NCBI (National Center for Biotechnology Information) amino acid sequence, NCBI nucleotide sequence), Clostridium acetobutylicum (GenBank number: NP_3494746.1, NC_003030; NP_149242, NC_001988, Bacillus subtilis_N3B) 97, NC_000964), and Saccharomyces cerevisiae (Saccharomyces cerevisiae) (GenBank (GenBank) number: NP_015297, is available from NC_001148).

  The term “3-hydroxybutyryl-CoA dehydrogenase” refers to a polypeptide having an enzymatic activity that catalyzes the conversion of acetoacetyl-CoA to 3-hydroxybutyryl-CoA. Examples of 3-hydroxybutyryl-CoA dehydrogenase may be NADH-dependent and have substrate selectivity for (S) -3-hydroxybutyryl-CoA or (R) -3-hydroxybutyryl-CoA . Examples are shown in E.E. C. 1.1.1.15 and E.I. C. It may be classified as 1.1.1.30. Furthermore, 3-hydroxybutyryl-CoA dehydrogenase may be NADPH-dependent and has substrate selectivity for (S) -3-hydroxybutyryl-CoA or (R) -3-hydroxybutyryl-CoA. And E.E. C. 1.1.1.157 and E.I. C. Classified as 1.1.1.36. 3-Hydroxybutyryl-CoA dehydrogenase is available from several sources, such as Clostridium acetobutylicum (GenBank numbers: NP_349314, NC_003030), Bacillus subtilis (Genbank) Number: AAB09614, U29084), Ralstonia eutropha (GenBank number: YP_2944481, NC_007347), and Alcaligenes eutrophus (obtained from GenBank (Abank 98: GenBank7A984)) It is.

The term “crotonase” refers to a polypeptide having enzymatic activity that catalyzes the conversion of 3-hydroxybutyryl-CoA to crotonyl-CoA and H ₂ O. Examples of crotonases may have substrate selectivity for (S) -3-hydroxybutyryl-CoA or (R) -3-hydroxybutyryl-CoA, respectively. C. 4.2.1.17 and E.I. C. It may be classified as 4.2.1.55. Crotonase can be obtained from several sources, for example, Escherichia coli (GenBank numbers: NP_415911, NC_000913), Clostridium acetobutylicum (GenBank numbers: NP_00303, NP_00303, Available from Bacillus subtilis (GenBank numbers: CAB13705, Z99113), and Aeromonas caviae (GenBank numbers: BAA21816, D88825).

  The term “butyryl-CoA dehydrogenase” refers to a polypeptide having an enzymatic activity that catalyzes the conversion of crotonyl-CoA to butyryl-CoA. Examples of butyryl-CoA dehydrogenase may be NADH-dependent, NADPH-dependent, or flavin-dependent, respectively. C. 1.3.1.44, E.I. C. 1.3.1.38, and E.I. C. It may be classified as 1.3.99.2. Butyryl-CoA dehydrogenase is available from several sources, for example, Clostridium acetobutylicum (GenBank numbers: NP_347102, NC_003030), Euglena gracilis (GenQ5: 82) ), Streptomyces colilinus (GenBank number: AAA92890, U37135), and Streptomyces coelicolor (GenBank number: CAA22721 available from CAA22721) A.

  The term “butyraldehyde dehydrogenase” refers to a polypeptide having enzymatic activity that catalyzes the conversion of butyryl-CoA to butyraldehyde using NADH or NADPH as a cofactor. Butyraldehyde dehydrogenase with selectivity for NADH is E. coli. C. Known as 1.2.1.57, for example, Clostridium beijerinckii (GenBank numbers: AAD31841, AF157306) and Clostridium acetobutylicum (Gen: B NP.sub .-- 149325, NC.sub .-- 001988).

The term “isobutyryl-CoA mutase” refers to a polypeptide having an enzymatic activity that catalyzes the conversion of butyryl-CoA to isobutyryl-CoA. This enzyme uses the coenzyme B ₁₂ as a cofactor. An example of isobutyryl-CoA mutase is known by EC number 5.4.99.13. These enzymes are Streptomyces cinnamonensis (GenBank numbers: AAC08713 (SEQ ID NO: 95), U67612 (SEQ ID NO: 96); CAB59633 (SEQ ID NO: 97), AJ246005 (SEQ ID NO: 98)) Streptomyces coelicolor (GenBank number: CAB70645 (SEQ ID NO: 99), AL939123 (SEQ ID NO: 100); CAB92663 (SEQ ID NO: 101), AL939121 (SEQ ID NO: 102)), and Streptomyces Streptomyces avermitilis (GenBank number) : NP_824008 (SEQ ID NO: 103), NC_003155 (SEQ ID NO: 104); NP_824637 (SEQ ID NO: 105), seen in NC_003155 (SEQ ID NO: 106)) Several Streptomyces genus including, but not limited to a (Streptomyces).

  The term “acetolactic decarboxylase” refers to a polypeptide having an enzymatic activity that catalyzes the conversion of α-acetolactic acid to acetoin. An example of an acetolactate decarboxylase is known as EC 4.1.1.5, for example Bacillus subtilis (GenBank numbers: AAA22223, L04470), Klebsiella terrigena (GenBank numbers: AAA25054, L04507) and Klebsiella pneumoniae (GenBank numbers: AAU43774, AY722056).

  The term “acetoin aminase” or “acetoin transaminase” refers to a polypeptide having an enzymatic activity that catalyzes the conversion of acetoin to 3-amino-2-butanol. Acetoin aminase may use the cofactor pyridoxal 5'-phosphate or NADH or NADPH. The resulting product may have (R) or (S) stereochemistry at the 3-position. Pyridoxal phosphate dependent enzymes may use amino acids such as alanine or glutamate as the amino donor. NADH- and NADPH-dependent enzymes may use ammonia as the second substrate. A suitable example of NADH-dependent acetoin aminase, also known as amino alcohol dehydrogenase, has been described by Ito et al. (US Pat. No. 6,432,688). An example of a pyridoxal-dependent acetoin aminase is the amine: pyruvate aminotransferase (also called amine: pyruvate transaminase) described by Shin and Kim (J. Org. Chem. 67: 2848-2853, 2002). .

  The term “acetoin kinase” refers to a polypeptide having an enzymatic activity that catalyzes the conversion of acetoin to phosphoacetoin. Acetoin kinase may use ATP (adenosine triphosphate) or phosphoenolpyruvate as a phosphate donor in the reaction. An enzyme that catalyzes a similar reaction in a similar substrate, dihydroxyacetone, is known, for example, as EC 2.7.1.29 (Garcia-Alles, et al., Biochemistry 43: 13037-13046, 2004). Enzymes.

  The term “acetoin phosphate aminase” refers to a polypeptide having an enzymatic activity that catalyzes the conversion of phosphoacetoin to 3-amino-2-butanol O-phosphate. Acetoin phosphate aminase may use the cofactor pyridoxal 5'-phosphate, NADH, or NADPH. The resulting product may have (R) or (S) stereochemistry at the 3-position. The pyridoxal phosphate-dependent enzyme may use amino acids such as alanine or glutamate. NADH-dependent and NADPH-dependent enzymes can use ammonia as the second substrate. There are no reports of enzymes that catalyze this reaction in phosphoacetoin, but there is a pyridoxal phosphate-dependent enzyme that is proposed to perform a similar reaction in a similar substrate, serinol phosphate (Yasuta, et al. al., Appl. Environ. Microbial. 67: 4999-5209, 2001).

  The term “aminobutanol phosphate phospholyase”, also referred to as “aminoalcohol O-phosphate lyase”, refers to a polypeptide having an enzymatic activity that catalyzes the conversion of 3-amino-2-butanol O-phosphate to 2-butanone. Point to. The aminobutanol phosphate phospho-lyase can use the cofactor pyridoxal 5'-phosphate. There have been reports of enzymes that catalyze similar reactions in a similar substrate, 1-amino-2-propanol phosphate (Jones, et al., Biochem J. 134: 167-182, 1973). US Patent Application Publication No. 2007/0259410 describes aminobutanol phosphate phospho-lyases derived from the organism Erwinia carotovora.

  The term “aminobutanol kinase” refers to a polypeptide having an enzymatic activity that catalyzes the conversion of 3-amino-2-butanol to 3-amino-2-butanol O-phosphate. Aminobutanol kinase can use ATP as a phosphate donor. There are no reports of enzymes that catalyze this reaction in 3-amino-2-butanol, but there are reports of enzymes that catalyze similar reactions in the similar substrates ethanolamine and 1-amino-2-propanol (Jones, et al., supra). US Patent Application Publication No. 2009/0155870 describes, in Example 14, an amino alcohol kinase of Erwinia carotovora subsp. Atroseptica.

  The term “butanediol dehydrogenase”, also known as “acetoin reductase”, refers to a polypeptide having an enzymatic activity that catalyzes the conversion of acetoin to 2,3-butanediol. Butanediol dehydrogenase is a subset of a broad family of alcohol dehydrogenases. The butanediol dehydrogenase enzyme may have specificity for the production of (R)-or (S) -stereochemistry in the alcohol product. A butanediol dehydrogenase specific for (S) is known as EC 1.1.1.76 and is available, for example, from Klebsiella pneumoniae (GenBank number: BBA13085, D86412) is there. A butanediol dehydrogenase specific for (R) is known as EC 1.1.1.4, for example Bacillus cereus (GenBank numbers NP 830481, NC_004722; AAP07682, AE017000). , And Lactococcus lactis (GenBank numbers AAK04995, AE006323).

  The term “butanediol dehydratase”, also known as “diol dehydratase” or “propanediol dehydratase”, refers to a polypeptide having an enzymatic activity that catalyzes the conversion of 2,3-butanediol to 2-butanone. Butanediol dehydratase may use the cofactor adenosylcobalamin (also known as coenzyme Bw or vitamin B12; vitamin B12 may refer to other forms of cobalamin that are not coenzyme B12). An adenosylcobalamin-dependent enzyme is known as EC 4.2.1.28, for example, Klebsiella oxytoca (GenBank number: AA08099 (α subunit), D45071; BAA08100 ( β subunit), D45071; and BBA08101 (γ subunit), D45071 (note that all three subunits are required for activity), and Klebsiella pneumonia (GenBank) Number: AAC98384 (α subunit), AF102064; GenBank Number: AAC98385 (β subunit), AF102064, GenBank k) Number: AAC 98386 (γ subunit), AF102064) Other suitable diol dehydratases include, but are not limited to, Salmonella typhimurium (GenBank number: AAB84102). (Large subunit), AF026270; GenBank number: AAB84103 (medium subunit), AF026270; GenBank number: AAB84104 (small subunit), AF026270; and Lactobacillus collinoides (GenBank number: CAC82541 (large subunit), AJ2977 3; GenBank number: CAC82542 (medium subunit); AJ297723; GenBank number: CAD01091 (small subunit), AJ297723); B12-dependent diol dehydratase available from Lactobacillus brevis ( Lactobacillus brevis) (especially the strains CNRZ 734 and CNRZ 735, Speranza, et al., J. Agric. Food Chem. 45: 3476-3480, 1997) and nucleotide sequences encoding the corresponding enzymes. Methods for isolating diol dehydratase genes are well known in the art (eg, US Pat. No. 5,686,276).

  The term “pyruvate decarboxylase” refers to a polypeptide having an enzymatic activity that catalyzes the decarboxylation of pyruvate to acetaldehyde and carbon dioxide. Pyruvate dehydrogenase is known by EC number 4.1.1.1. These enzymes are found in several yeasts including Saccharomyces cerevisiae (GenBank number: CAA97575 (SEQ ID NO: 107), CAA97705 (SEQ ID NO: 109), CAA97091 (SEQ ID NO: 111)). .

  It will be appreciated that the microorganism comprising the isobutanol biosynthetic pathway provided herein may further comprise one or more additional modifications. US 2009/0305363 (incorporated by reference) modifies yeast for cytosolic localized expression of acetolactate synthase and substantial loss of pyruvate decarboxylase activity. An increased conversion of pyruvate to acetolactate is thus disclosed. In certain embodiments, the microorganism is modified to reduce glycerol-3-phosphate dehydrogenase activity and / or disruption of at least one gene encoding a polypeptide having pyruvate decarboxylase activity or US Patent Application Publication No. 2009/2009. Disruption of at least one gene encoding a regulatory element that controls pyruvate decarboxylase gene expression as described in US Pat. No. 3,030,363 (incorporated herein by reference) and / or published US patent applications Modifications that provide increased carbon flux or reducing equivalent equilibration via the Entner-Doudoroff pathway as described in 2010/01201005 (incorporated herein by reference) may be included. Other modifications include the incorporation of at least one polynucleotide encoding a polypeptide that catalyzes a step in a biosynthetic pathway that utilizes pyruvate. Other modifications include at least one deletion, mutation, and / or substitution in the endogenous polynucleotide encoding a polypeptide having acetolactate reductase activity. In certain embodiments, the polypeptide having acetolactate reductase activity is Saccharomyces cerevisiae YMR226C (SEQ ID NO: 127, 128) or a homologue thereof. Further modifications include deletions, mutations, and / or substitutions in the endogenous polynucleotide encoding a polypeptide having aldehyde dehydrogenase and / or aldehyde oxidase activity. In certain embodiments, the polypeptide having aldehyde dehydrogenase activity is ALD6 from Saccharomyces cerevisiae or a homologue thereof. A genetic modification with the effect of reducing glucose repression in which the yeast production host cell is pdc- is described in US 2011/0124060, which is incorporated herein by reference. In certain embodiments, the pyruvate decarboxylase that is deleted or down-regulated is selected from the group consisting of PDC1, PDC5, PDC6, and combinations thereof. In certain embodiments, the pyruvate decarboxylase is selected from the enzymes in Table 1. In certain embodiments, the microorganism may comprise a deletion or downregulation of a polynucleotide encoding a polypeptide that catalyzes the conversion of glyceraldehyde-3-phosphate to glycerate 1,3, bisphosphate. In certain embodiments, the enzyme that catalyzes this reaction is glyceraldehyde-3-phosphate dehydrogenase.

  In certain embodiments, any particular nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98 with a nucleotide sequence or polypeptide sequence described herein. %, Or 99% identical. The term “percent identity” as known in the art is a relationship determined by comparing two or more polypeptide sequences or between two or more polynucleotide sequences. In the art, “identity” also means the degree of sequence relatedness determined by a match between strings of polypeptide or polynucleotide sequences, and possibly strings of such sequences. Identity and similarity are calculated using the Computational Molecular Biology (Lesk, AM, Ed.) Oxford University: NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D.W. 1993); Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, Eds.) Humania: NJ (1994); Sequence Analysis in Hole in ul. .) Academic (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., Eds.) Stockton: NY (1991) can be readily calculated by known methods, including but not limited to those disclosed.

  The method for determining identity can be designed to provide the best match between the sequences tested. The method for determining identity and similarity is organized with publicly available computer programs. Sequence alignment and percent identity calculations can be performed using the MEGAlign ™ program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of sequences is disclosed by an alignment method named Clustal V (Higgins and Sharp, CABIOS. 5: 151-153, 1989; Higgins, et al., Comput. Appl. Biosci. 8: 189-191, 1992). Can be performed using a Clustal alignment method that includes several algorithms, including the Clustal V alignment method found in the MEGAlign ™ program of the LASERGENE Bioinformatics Computing Suite (DNASTAR Inc.) . For multiple alignments, the default values correspond to a gap penalty (GAP PENALTY) = 10 and a gap length penalty (GAP LENGTH PENALTY) = 10. The default parameters for pairwise alignment of protein sequences using the Clustal method and calculation of percent identity are: K-tuple (KTUPLE) = 1, gap penalty (GAP PENALTY) = 3, window (WINDOW) = 5 and diagonal saved ( DIAGONALS SAVED) = 5. These parameters for the nucleic acids are K Tuple = 2, GAP PENALTY = 5, WINDOW = 4 and Diagonal Saved = 4. After sequence alignment using the Clustal V program, it is possible to obtain percent identity by looking at the sequence distance table of the same program. In addition, a Clustal W alignment method is available, and an alignment method named Clustal W (Higgins and Sharp, CABIOS. 5: 151-153, 1989; Higgins, et al., Compute. Appl. Biosci. 8: 189-). 191, 1992) and can be found in the MERSAlign ™ v6.1 program of the LASERGENE Bioinformatics Computing Suite (DNASTAR Inc.). Default parameters for multiple alignment (GAP PENALTY = 10, gap length penalty (GAP LENGTH PENALTY) = 0.2, delay divergent sequences (%) = 30, DNA transition weights (DNA) (Transition Weight) = 0.5, Protein Weight Matrix = Gonnet series, DNA Weight Matrix = IUB). After sequence alignment using the Clustal W program, it is possible to obtain percent identity by looking at the sequence distance table of the same program.

  Standard recombinant DNA and molecular cloning techniques are well known in the art and are described in Sambrook et al. (Sambrook, J., Fritsch, EF and Maniatis, T. (Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Lab; Press, Cold Spring Harbor, 1989, referred to herein as Maniatis) and Ausubel et al. (Ausubel, et al., Current Protocols in Molecular Biology, public. Pub. By Greene Publishing Incorporated. Examples of methods for constructing microorganisms that contain a butanol biosynthetic pathway are described, for example, in US Patent No. 7,851,188, and US Patent Application Publication No. 2007/0092957. No. 2007/0259410; No. 2007/0292927; No. 2008/0182308; No. 2008/0274525; No. 2009/0155870; No. 2009 / No. No. 0305363; and 2009/0205370, the entire contents of each of which are incorporated herein by reference.

  Moreover, while various embodiments of the invention have been described herein, it should be understood that they have been presented by way of example only and not limitation. It will be apparent to those skilled in the relevant art that various modifications can be made in the form and details of the invention without departing from the spirit and scope of the invention. Accordingly, the breadth and scope of the present invention is not limited by any of the above-described exemplary embodiments, but is defined only in accordance with the following claims and their equivalents.

  All publications, patents and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains, and each individual publication, patent or patent application is incorporated by reference. To the same extent as specifically and individually indicated herein by reference.

  The following non-limiting examples further illustrate the present invention. While the following examples include corn as a feedstock, it should be understood that other biomass sources can be used for the feedstock without departing from the invention.

As used herein, the abbreviations used have the following meanings: “g” means gram, “kg” means kilogram, “lbm” means pound mass , “Gpm” means gallons / minute, “gal” means gallons, “MMGPY” means 1 million gallons / year, “L” means liters, “mL” means milliliters “ΜL” means microliter, “mL / L” means milliliter / liter, “mL / minute” means milliliter / minute, “min” means minute, “hr” Means time, “DI” means deionized, “uM” means micrometer, “nm” means nanometer, “w / v” means weight / volume. Meaning "% by weight" means percent by weight And, "OD" means optical density, "OD _600" means optical density at a wavelength of 600 nM, "dcw" means dry cell weight, "rpm" means revolutions per minute , “° C.” means degrees Celsius, “° C./min” means degrees Celsius / min, “slpm” means standard liters / min, “ppm” means parts per million, “cP” means centipoise, “ID” means inner diameter, and “GC” means gas chromatograph.

Example 1
Effect of undissolved solids on the rate of mass transfer To mimic the average composition of the liquid phase for SSF batches, an almost intermediate stage of simultaneous saccharification and fermentation (SSF) fermentation (ie, about 50% conversion of oligosaccharides) The following experiment was conducted to determine the effect of undissolved solids on the rate of mass transfer of i-BuOH from the aqueous phase simulating the composition of the fermentation broth obtained from corn mash.

Approximately 100 kg of liquefied corn mash was prepared in three equal batches using a 30 L glass coated resin kettle. The kettle was equipped with mechanical agitation, temperature control, and pH control. The protocol used for the three batches was as follows: (a) ground corn was mixed with tap water (30 wt% corn on a dry basis) and (b) the slurry was stirred with 55 (C) the pH of the slurry is adjusted to 5.8 using either NaOH or H ₂ SO ₄ and (d) α-amylase (0.02 wt% on dry corn basis) (E) heating the slurry to 85 ° C., (f) adjusting the pH to 5.8, (g) holding the slurry at 85 ° C. for 2 hours while maintaining the pH at 5.8, (h) The slurry was cooled to 25 ° C.

Pioneer 3335 hybrid corn, whole yellow corn was used (Pioner Hi-Bred International, Johnston, IA) and it was ground in a hammer mill using a 1 mm sieve. The water content of ground corn was 12% by weight, and the starch content of ground corn was 71.4% by weight on a dry corn basis. The α-amylase enzyme was Liquizyme® SC DS (Novozymes, Franklinton, NC). The total amount of ingredients used in combination of the three batches was: 33.9 kg ground corn (12% moisture), 65.4 kg tap water, and 0.006 kg Liquizyme® SC DS . A total of 0.297 kg NaOH (17 wt%) was added to control the pH. H ₂ SO ₄ was not necessary. The total amount of liquefied corn mash recovered from the three 30 L batches was 99.4 kg.

Solids were removed from the mash by centrifugation in a large floor centrifuge containing six 1 L bottles. The mash (73.4 kg) was centrifuged at 8000 rpm for 20 minutes at 25 ° C., resulting in 44.4 kg of centrifuge and 26.9 kg of wet cake. It was found that the centrifuge contained less than 1% by weight suspended solids and the wet cake contained approximately 18% by weight suspended solids. This suggests that the original liquefied mash contained about 7% by weight suspended solids. This is consistent with the corn filling and the starch content of the corn used, assuming that most of the starch has been liquefied. If all of the starch was liquefied, the 44.4 kg centrifuge recovered directly from the centrifuge would contain approximately 23% by weight dissolved oligosaccharide (liquefied starch). About 0.6 kg i-BuOH was added to 35.4 kg centrifuge and stored. The resulting 36.0 kg centrifuge containing 1.6 wt% i-BuOH was used as the stock solution. This centrifuge liquid mimics the composition of the liquid phase at the start of SSF. Therefore, a portion of it was diluted with an equal amount of H ₂ O on a mass basis to produce a centrifuge that mimics SSF at about 50% conversion. Additional i-BuOH was added to bring the final concentration of i-BuOH in the diluted centrifuge to 3.0 wt% (about 30 g / L).

  A diluted centrifuge solution was prepared as follows: 18 kg centrifuge stock solution containing 1.6 wt% i-BuOH was mixed with 18 kg tap water and 0.82 kg i-BuOH was added. added. The resulting 36.8 kg solution of diluted centrifuge consisted of about 11 wt% oligosaccharides and about 30 g / L i-BuOH. This solution mimics the liquid phase of corn mash fermentation (SSF) and the aqueous titer of 30 g / L i-BuOH at about 50% conversion of oligosaccharides.

After most of the undissolved solids were removed, this solution was used as a water phase to mimic the mass transfer performance in broths obtained from liquefied corn mash and mass transfer tests were performed. The purpose of the mass transfer test is to transfer i-BuOH from a simulated broth obtained from liquefied corn mash to a dispersion of solvent (extractant) droplets rising through the simulated broth. Was to determine the effect of undissolved solids on the total mass transfer coefficient (k _L a). The correlation of k _L a with the main design of the operating parameters can be used to scale up the mass transfer operation. Examples of parameters that should be kept as constant as possible to generate k _L a correlations from smaller scale data that are useful for scaling up are the phase and design parameters that determine droplet size. Both physical properties (eg, nozzle diameter, phase velocity dispersed through the nozzle).

From an aqueous solution of oligosaccharides (obtained from liquefied corn mash) with or without suspended mash solids using a 6 inch diameter, 7 foot high glass coated column The k _L a of i-BuOH transfer to a dispersion of oleyl alcohol (OA) droplets rising through the simulated broth was measured. When i-BuOH was added to the aqueous phase, an initial concentration of i-BuOH of about 30 g / L was obtained. A predetermined amount of aqueous phase (usually about 35 kg) containing about 11% by weight oligosaccharide and about 30 g / L i-BuOH is charged to the column and the column is allowed to flow by flowing warm H ₂ O through the jacket. Heated to 30 ° C. During the test, there was no aqueous phase flow into or out of the column.

  Fresh oleyl alcohol (80/85% grade, Cognis Corporation, Cincinnati, OH) is sprinkled through the single column to the bottom of the column to produce a dispersion of extractant droplets that flow upward through the aqueous phase. did. After reaching the top of the aqueous phase, the extractant droplets formed a separate organic phase that subsequently overflowed from the top of the column and was collected in a receiver. Usually, 3-5 gallons of oleyl alcohol per test flowed through the column.

Aqueous phase samples were collected from the column several times throughout the test, and a composite sample of all “enriched” oleyl alcohol collected from the overflow was collected at the end of the test. All samples were analyzed for i-BuOH using HP-6890 GC (Agilent Technologies, Inc., Santa Clara, Calif.). The concentration profile of i-BuOH in the aqueous phase (ie, i-BuOH concentration over time) was used to calculate k _L a at a given operating condition. The final composite sample of all “enriched” oleyl alcohol collected during the study was used to determine the material balance of i-BuOH.

The nozzle size and nozzle speed (average speed of oleyl alcohol through the feed nozzle) were varied to observe the effect on k _La . A series of tests were performed using an oligosaccharide aqueous solution (diluted centrifuge obtained from liquefied corn mash) from which mash solids had been removed. A similar series of tests were performed using the same aqueous solution of oligosaccharides after mash solids were added back to simulate corn mash (including undissolved solids) liquefied during SSF. Under certain operating conditions (eg, higher oleyl alcohol flow rate), insufficient phase separation is obtained at the top of the column to obtain a typical composite sample of total “enriched” oleyl alcohol collected during the test. It is noted that it becomes difficult. It is also noted that under certain operating conditions, the aqueous phase sample contained a significant amount of organic phase. Special sample processing and preparation techniques were used to obtain an aqueous phase sample that was as typical as possible of the aqueous phase in the column when the sample was collected.

式中、
ｒ_Ｂ＝水相の単位体積当たりの単位時間当たりの水相から溶媒相へと移動されるｉ−ＢｕＯＨの全質量（ｉ−ＢｕＯＨのグラム／水相のリットル／時またはｇ／Ｌ／時）である。
ｋ_Ｌａ＝水相から溶媒相へのｉ−ＢｕＯＨの物質移動を表す全体積物質移動係数（毎時）である。
Ｃ_{Ｂ，ブロス}＝シミュレートされるブロス（水）相中のｉ−ＢｕＯＨの平均濃度（ｉ−ＢｕＯＨのグラム／水相のリットルまたはｇ／Ｌ）である。
Ｃ_Ｂ，溶媒＝試験全体にわたる溶媒相中のｉ−ＢｕＯＨの平均濃度（ｉ−ＢｕＯＨのグラム／溶媒相のリットルまたはｇ／Ｌ）である。
Ｋ_Ｂ＝溶媒と水相との間のｉ−ＢｕＯＨの平均平衡分配係数（（ｉ−ＢｕＯＨのグラム／溶媒相のリットル）／（ｉ−ＢｕＯＨのグラム／水相のリットル））である。 It was found that the aqueous phase in the column was “well mixed” for all practical purposes because the concentration of i-BuOH did not change much along the length of the column at a given time. Assuming that the solvent droplet phase was also well mixed, the total mass transfer of i-BuOH from the aqueous phase to the solvent phase in the column can be estimated by the following equation:

Where
r _B = total mass of i-BuOH transferred from the aqueous phase to the solvent phase per unit time per unit volume of the aqueous phase (grams of i-BuOH / liter of water phase / hour or g / L / hour) It is.
k _L a = total volume mass transfer coefficient (hourly) representing mass transfer of i-BuOH from the aqueous phase to the solvent phase.
_{CB, broth} = average concentration of i-BuOH in the simulated broth (water) phase (grams of i-BuOH / liter of water phase or g / L).
CB _{, solvent} = average concentration of i-BuOH in the solvent phase throughout the test (grams of i-BuOH / liter of solvent phase or g / L).
K _B = average equilibrium partition coefficient of i-BuOH between solvent and aqueous phase ((gram of i-BuOH / liter of solvent phase) / (gram of i-BuOH / liter of water phase)).

The parameters r _B , C _{B, broth} , and C _{B, solvent} were calculated for each test from concentration data obtained from aqueous and solvent phase samples. Aqueous centrifugate from liquefied corn mash was mixed with oleyl alcohol, and i-BuOH, by two liquid phases are mixed vigorously system until equilibrium was measured independently parameter K _B . The concentration of i-BuOH were measured in both phases was determined _{K B.} r _{B, C B, broth, C B,} after _{the solvent,} and _{K B} was determined for a given test, it was possible to calculate the _{k L} a by changing the sequence of formula (1):

  Mass transfer tests were performed using two different sized nozzles at a nozzle speed ranging from 5 ft / sec to 21 ft / sec using a centrifuge (diluted solids) diluted as an aqueous phase. . Three tests were performed using a nozzle having an ID of 0.76 mm, and three tests were performed using a nozzle having an inner diameter of 2.03 mm. All tests were performed at 30 ° C. in the above 6 inch diameter column using oleyl alcohol as solvent. The equilibrium partition coefficient of i-BuOH between oleyl alcohol and diluted centrifuge obtained from corn mash liquefied by removing solids was about 5. Table 2 shows the results of the mass transfer test using the diluted centrifuge (with the solid removed).

An aqueous phase simulating a fermentation broth from a liquefied corn mash (containing undissolved solids) in the middle of the SSF is first obtained by removing the solids from the wet cake (liquefied corn mash. Part) was synthesized by adding to the diluted centrifuge. Some water was also added to dilute the liquid phase retained in the wet cake. This is because this liquid has the same composition as the concentrated centrifuge liquid. Diluted supernatant (17.8 kg), (containing about 18 wt.% Of undissolved mash solids) wet cake 13.0 kg, _H 2 O of 5.0 kg, and the i-BuOH of 0.83kg together To about 6 wt.% Undissolved solids and about 13 wt.% Liquefied starch and about 2.4 wt.% I-BuOH (the remainder being H ₂ O). 36.6 kg of slurry was obtained. This slurry mimics the composition of the fermentation broth in the middle of the corn SSF to i-BuOH at about 30% corn loading. This is because the levels of undissolved solids and oligosaccharides found in these types of broth are about 6-8% and 10-12% by weight, respectively.

  Mass transfer tests were performed using two different sized nozzles at nozzle speeds ranging from 5 feet / second to 22 feet / second using a diluted centrifuge and an undissolved mash solid slurry as the aqueous phase. It was. Three tests were performed using a nozzle having an inner diameter of 0.76 mm, and three tests were performed using a nozzle having an inner diameter of 2.03 mm. All tests were performed at 30 ° C. in the above 6 inch diameter column using oleyl alcohol as solvent. Table 3 shows the results of the mass transfer test using the diluted centrifuge and the slurry of undissolved mash solid.

FIG. 12 shows the i-BuOH transfer from an aqueous solution of liquefied corn starch (ie, oligosaccharides) to a dispersion of oleyl alcohol droplets flowing upward through a bubble column, relative to the total mass transfer coefficient, k _L a The influence of the presence of undissolved corn mash solids is shown. Oleyl alcohol was fed to the column through a nozzle with an inner diameter of 2.03 mm. The ratio of k _{L a} of system solid for k _{L a} of system solids not removed has been removed, it has been discovered that 2 to 5 depending on the nozzle velocity of the nozzle of 2.03 mm.

FIG. 13 shows the total volume mass transfer coefficient, k _L a, of i-BuOH transfer from an aqueous solution of liquefied corn starch (ie, oligosaccharides) to a dispersion of oleyl alcohol droplets flowing upward through a bubble column. The influence of the presence of undissolved corn mash solids is shown. Oleyl alcohol was fed to the column through a 0.76 mm ID nozzle. The ratio of k _{L a} of system solid for k _{L a} of system solids not removed has been removed, it has been discovered that 2 to 4 in accordance with the nozzle velocity of the nozzle of 0.76 mm.

Example 2
Effect of removal of undissolved solids on phase separation between aqueous and solvent phases This example was compared to an aqueous solution of oligosaccharides obtained from liquefied corn mash from which undissolved solids were not removed and the same solvent Figure 2 shows improved phase separation between an aqueous solution of oligosaccharides obtained from liquefied corn mash from which undissolved solids have been removed and the solvent phase. Both systems contained i-BuOH. Sufficient separation of the solvent phase from the aqueous phase is important because liquid-liquid extraction is a viable separation method for performing ISPR.

About 900 g of liquefied corn mash was prepared in a 1 L glass coated resin kettle. The kettle was equipped with mechanical agitation, temperature control, and pH control. The following protocol was used: ground corn was mixed with tap water (26 wt% corn on a dry basis) and the slurry was heated to 55 ° C. with agitation to either NaOH or H ₂ SO _4. Use to adjust the pH to 5.8, add α-amylase (0.02 wt% based on dry corn), continue to heat to 85 ° C, adjust the pH to 5.8, and adjust the pH to 5.8 While maintaining, the temperature was maintained at 85 ° C. for 2 hours and cooled to 25 ° C.

  Pioneer 3335 hybrid corn, whole yellow corn was used (Pioner Hi-Bred International, Johnston, IA) and it was ground in a hammer mill using a 1 mm sieve. The water content of ground corn was 12% by weight, and the starch content of ground corn was 71.4% by weight on a dry corn basis. The α-amylase enzyme was Liquizyme® SC DS made by Novozymes (Franklinton, NC). The total amount of ingredients used was: 265.9 g ground corn (12% moisture), 634.3 g tap water, and 0.056 g Liquizyme® SC DS. The total amount of recovered liquefied corn mash was 883.5 g.

  A portion of the liquefied corn mash was used as it was without removing undissolved solids to prepare an aqueous phase for solid phase separation testing. A portion of the liquefied corn mash was centrifuged to remove most of the undissolved solids and used to prepare a solid phase free aqueous phase for phase separation testing.

  Solids were removed from the mash by centrifugation in a large volume centrifuge. The mash (583.5 g) was centrifuged at 5000 rpm for 20 minutes at 35 ° C. to obtain 394.4 g of centrifuge and 189.0 g of wet cake. It was found that the centrifuge liquid contained about 0.5% by weight suspended solids and the wet cake contained about 20% by weight suspended solids. This suggests that the original liquefied mash contained about 7% by weight suspended solids. This is consistent with the corn filling and the starch content of the corn used, assuming that most of the starch has been liquefied. If all of the starch was liquefied, the centrifuge collected directly from the centrifuge would contain approximately 20% by weight dissolved oligosaccharide (liquefied starch) if it did not contain solids. .

  The purpose of the phase separation test was to measure the effect of undissolved solids on the degree of phase separation between the solvent phase and the aqueous phase simulating broth obtained from liquefied corn mash. In all tests, the aqueous liquid phase contained about 20% oligosaccharides and the organic phase contained oleyl alcohol (OA). In addition, i-BuOH was added to all tests, yielding approximately 25 g / L in the aqueous phase when the phase was in equilibrium. Two shaking tests were performed. The aqueous phase of the first test (including solids) was prepared by mixing 60.0 g of liquefied corn mash with 3.5 g of i-BuOH. The aqueous phase of the second test (with the solids removed) is mixed with 60.0 g of centrifuge obtained from corn mash liquefied by removing the solids with 3.5 g of i-BuOH. Prepared. Oleyl alcohol (15.0 g, 80/85% grade, Cognis Corporation, Cincinnati, OH) was added to each of the shaking test bottles. The oleyl alcohol formed a separate liquid phase on top of the aqueous phase in both bottles, resulting in a phase mass ratio, ie, an aqueous / solvent phase of about 1/4. Both bottles were shaken vigorously for 2 minutes to bring the aqueous and organic phases into close contact so that the i-BuOH reached equilibrium between the two phases. The bottle was set for 1 hour. Photos are taken at various times (0 min, 15 min, 30 min, and 60 min) and contain an aqueous phase obtained from liquefied corn mash, a solvent phase containing oleyl alcohol, and i-BuOH. The effect of undissolved solids on phase separation in the system was observed. Time zero (0) corresponds to the time immediately after 2 minutes when the shaking period is completed.

  Time-dependent organic (solvent) and aqueous phases for systems containing solids (derived from liquefied corn mash) and systems from which solids have been removed (liquid centrifuges from liquefied corn mash) The degree of separation in between appeared to be approximately the same in both systems at any given time. When containing solids, the organic phase was slightly dark and turbid and the interface was a little vague (thicker “rag” layer around the interface). However, in extractive fermentations where the solvent is operated continuously, the composition at the top of the organic phase is involved in downstream processes of extractive fermentation, where the next step is distillation.

  Since microorganisms are thermally inactivated in the distillation column, it can be advantageous to minimize the amount of microorganisms in the upper part of the organic phase. Microorganisms can clog the distillation tower, contaminate the reboiler, cause insufficient phase separation in the solvent / water decanter located at the base of the column, or can be any combination of the above mentioned problems, It may be advantageous to minimize the amount of undissolved solvent at the top of the. It may be advantageous to minimize the amount of phase water at the top of the organic phase. Phase water is water that exists as a separate aqueous phase. The additional amount of aqueous phase increases the load and energy requirements in the distillation column. Ten milliliters (10 mL) of the sample is removed from the top of the organic layer from the “contains solid” and “removed solid” bottles, both samples are centrifuged, and after a settling time of 60 minutes, “ The composition of the organic phase in bottles containing “solids” and “removed solids” was revealed and compared. The results indicate that the “organic phase” at the end of both shaking tests contained some undesirable phase (both organic phases were cloudy). However, the results also show that the top layer from the phase separation test, including the centrifuge, where the solids were removed, was substantially free of undissolved solids. On the other hand, undissolved solids are clearly visible at the bottom of a 10 mL sample collected from the top of the test organic phase containing mash. It was estimated that 3% of the sample collected from the top of the organic layer washed mash solids. If the enriched solvent phase exiting the fermenter of the extractive fermentation process contained 3% undissolved solids, one or more of the following problems may occur: loss of significant amounts of microorganisms, solvent tower reboiler Contamination, solvent tower blockage. The results also show that the upper layer from the phase separation test with the centrifuge liquid contained less phase water. Table 4 shows an estimate of the relative amount of phase dispersed over the upper “organic” layer in both shaking test bottles after a 60 minute settling time.

  This example shows that removal of most of the undissolved solids from liquefied corn mash results in improved phase separation after the liquid, aqueous phase obtained from the mash is contacted with a solvent such as oleyl alcohol. Show. This example shows that if the solids are first removed before contacting the liquid portion of the mash with the organic solvent, the upper phase obtained after phase separation contains significantly less undissolved solids. This shows the benefit of minimizing the undissolved solids content of the mash in the upper (“organic”) layer of the phase separation for extractive fermentation.

  Also, the sample was allowed to stand for several days after completion of sample preparation before repeating the phase separation shaking test described in this example. The sample containing the solid consists of liquefied corn mash, i-BuOH, and oleyl alcohol, and the sample from which the solid has been removed is generated by removing most of the undissolved solid from the liquefied corn mash. Centrifuge, i-BuOH, and oleyl alcohol. The liquefied mash contained about 7% by weight suspended solids and the centrifuge produced from the mash contained about 0.5% by weight suspended solids. If all of the starch in the ground corn is liquefied, the liquid phase in the liquefied mash and the centrifuge produced from the mash will contain about 20% by weight dissolved oligosaccharide ( Liquefied starch). Both samples contained oleyl alcohol in an amount that was about 1/4 of the phase mass ratio, ie solvent / water phase. In addition, i-BuOH was added to all tests, yielding approximately 25 g / L in the aqueous phase when the phase was in equilibrium.

  The purpose of the phase separation test is to measure the effect of undissolved solids on the degree of phase separation after the multiphase mixture has been allowed to stand at room temperature for several days to mimic potential changes in system properties throughout the extraction fermentation Was that. Two shaking tests were performed. Both bottles were shaken vigorously for 2 minutes to bring the aqueous and organic phases into close contact. The bottle was left for 1 hour. Pictures were taken at various times (0 min, 2 min, 5 min, 10 min, 20 min, and 60 min) to observe the effect of undissolved solids on phase separation in these systems aged for several days. Time zero (0) corresponds to the time immediately after placing the bottle on the bench.

  Phase separation began to occur in the sample where the solids were removed after 2 minutes. Based on the organic phase accounting for about 25% of the total volume of the biphasic mixture, it is believed that nearly complete phase separation occurred in the sample if the solid was removed after only 5-10 minutes. If the organic phase accounted for about 20% of the total volume, it can be expected that complete separation can be shown because it corresponds to the initial proportion of the phase. Even after 1 hour, no apparent phase separation occurred in the samples from which no solids were removed.

  The composition of the upper phase of both samples was also compared. The composition of the upper phase has implications for downstream processes of extractive fermentation where the next step is distillation. Since microorganisms are thermally inactivated in the distillation column, it is advantageous to minimize the amount of microorganisms in the upper part of the organic phase. The solids can clog the distillation tower, contaminate the reboiler, cause insufficient phase separation in the solvent / water decanter located at the base of the column, or can be any combination of the above mentioned problems. Another component to be minimized at the top of the is the amount of undissolved solids. Furthermore, another component to be minimized at the top of the organic phase is the amount of phase water, which is water present as a separate aqueous phase, because this additional amount of aqueous phase This is to increase the load and energy requirements in the distillation column.

  Ten milliliters (10 mL) of the sample is removed from the top of the organic layer from the “contains solid” and “removed solid” bottles, both samples are centrifuged, and after a settling time of 60 minutes, “ The composition of the organic phase in bottles containing “solids” and “removed solids” was revealed and compared. The composition of the sample collected from the top of the “contains” sample confirms that virtually no phase separation occurred in the sample within 60 minutes. In particular, the ratio of the solvent phase to the total aqueous phase (aqueous liquid + suspended solid) in the sample collected from the top of the “solids containing” shaking test bottle is about 1/4 w / w, which is The same ratio used to prepare the sample before testing. Also, the amount of undissolved solids in the sample collected from the top of the “solids containing” shaking test bottle is approximately the same as that found in liquefied corn mash, which is within 60 minutes. It shows that substantially no solids settled in the shake test bottle. On the other hand, the upper layer from the phase separation test including the centrifuge from which the solid was removed (“the solid was removed”) contained substantially no undissolved solid. The results also show that the upper layer from the phase separation test with the centrifuge liquid contained less phase water. This is indicated by the ratio of the solvent phase to the aqueous phase in the sample bottle being about 1/1 w / w, which indicates that the organic phase is enriched with solvent (oleyl alcohol) in the test when the solid is removed. It shows that. Table 5 shows an estimate of the relative amount of phase dispersed over the upper “organic” layer in both shaking test bottles after a 60 minute settling time.

  This example removes the undissolved solid from the liquefied corn mash containing i-BuOH, bringing it into contact with the solvent phase, allowing it to stand for a few days, and remixing the phase to remove the undissolved solid. It shows that improved phase separation is obtained compared to the sample that was not removed from the liquefied mash. In fact, this example shows that virtually no phase separation occurs even after 60 minutes in samples that did not remove undissolved solids. This example shows that if the solids are first removed before contacting the liquid portion of the mash with the organic solvent, the upper phase obtained after phase separation contains significantly less undissolved solids. This is important because two of the most important species to be minimized in the upper (“organic”) layer of phase separation for extractive fermentation are the level of microorganisms and the level of undissolved solids from the mash. is there. The previous example showed that removal of solids from liquefied corn mash resulted in improved phase separation immediately after contacting the aqueous phase with the solvent phase. This may allow extractive fermentation to be performed at an earlier point in the fermentation. This example also shows that removal of solids from liquefied corn mash results in improved phase separation in an aged sample containing an aqueous phase (oligosaccharide solution with the solids removed) in contact with the solvent phase. . This may also allow extractive fermentation to be performed at a later point in the fermentation.

Example 3
Effect of removal of undissolved solids on ISPR extraction solvent loss-Disc stack centrifuge This example uses a semi-continuous disc-stack centrifuge to remove undissolved solids from corn mash prior to fermentation This shows the potential to reduce solvent loss via DDGS produced by the extractive fermentation process.

About 216 kg of liquefied corn mash was prepared in a coated stainless steel reactor. The reactor was equipped with mechanical stirring, temperature control, and pH control. The protocol used was as follows: ground corn was mixed with tap water (25 wt% corn on a dry basis) and the slurry was heated to 55 ° C. with stirring at 400 rpm and either NaOH or H Adjust the pH to 5.8 using any of ₂ SO ₄ , add α-amylase (0.02 wt% on dry corn basis), continue to heat to 85 ° C. and adjust the pH to 5.8. While maintaining the pH at 5.8, hold at 85 ° C. for 30 minutes, heat to 121 ° C. using live steam injection, hold at 121 ° C. for 30 minutes to simulate a jet cooker, cool to 85 ° C. , Adjust the pH to 5.8, add a second input of α-amylase (0.02 wt% on dry corn basis), hold at 85 ° C. for 60 minutes while maintaining the pH at 5.8 to liquefy Complete I ended it. The mash was then cooled to 60 ° C. and transferred to the centrifuge supply tank.

Pioneer 3335 hybrid corn, whole yellow corn was used (Pioner Hi-Bred International, Johnston, IA) and it was ground in a hammer mill using a 1 mm sieve. The water content of ground corn was 12% by weight, and the starch content of ground corn was 71.4% by weight on a dry corn basis. The α-amylase enzyme was Liquizyme® SC DS made by Novozymes (Franklinton, NC). The amount of ingredients used is: 61.8 kg ground corn (12% moisture), 147.3 kg tap water, 0.0109 kg Liquizyme in 1 kg water for the first α-amylase input (registered) A solution of SC DS, another solution of 0.0109 kg Liquizyme® SC DS in 1 kg water for the second α-amylase charge (after cooking stage). About 5 kg of H ₂ O was added to the batch via steam condensate during the cooking stage. NaOH in 0.25kg in total (12.5 wt%) and 0.12kg of _H 2 _SO 4 (12.5 wt%) was added throughout the experiment in order to control the pH. The total amount of liquefied corn mash recovered was 216 kg.

  The composition of the final liquefied corn mash slurry was estimated to be about 7 wt% undissolved solids and 93 wt% liquid. The liquid phase contained about 19% by weight (190 g / L) of liquefied starch (soluble oligosaccharide). The mash rheology is important with respect to the ability to separate the slurry into its components. The liquid phase in the mash was found to be a Newtonian fluid with a viscosity of about 5.5 cP at 30 ° C. The mash slurry was found to be a shear-thinning fluid having a volume viscosity of about 10-70 cP at 85 ° C., depending on the shear rate.

  The liquefied mash (209 kg, 190 L) was centrifuged using a disc-stack separation bowl centrifuge (Alfa Laval Inc., Richmond, VA). The centrifuge was operated in semi-batch mode including continuous feed, continuous centrifuge discharge, and wet cake batch discharge. The liquefied corn mash was continuously fed at a rate of 1 L / min, the clarified centrifuge liquid was continuously removed, and the wet cake was discharged periodically every 4 minutes. In order to determine the proper discharge interval for solids from the disk stack, the mash sample fed to the disk stack was centrifuged in a high speed laboratory centrifuge. Mash (48.5 g) was spun at 11,000 rpm (about 21,000 g) at room temperature for about 10 minutes. A clarified centrifuge (36.1 g) and 12.4 g pellet (wet cake) were recovered. The clarified centrifuge was found to contain about 0.3 wt.% Undissolved solids and the pellet (wet cake) contained about 27 wt.% Undissolved solids. Based on this data, a 4 minute discharge interval was selected for operation of the disk stack centrifuge.

  The disk stack centrifuge was operated at 9000 rpm (6100 g) at a liquefied corn mash feed rate of 1 L / min and about 60 ° C. Mash (209 kg) was separated into 155 kg of purified centrifuge and 55 kg of wet cake. The split defined by (amount of centrifuge) / (amount of mash fed) obtained by a semi-continuous disk stack was similar to the split obtained in a batch centrifuge. The split of the disc stack semi-batch centrifuge operated at 6100 g, 1 L / min feed rate, and 4 min discharge interval is (155 kg / 209 kg) = 74% and is run at 21,000 g for 10 min. The split of the experimental batch centrifuge was (36.1 g / 48.5 g) = 74%.

  A 45 mL sample of the purified centrifuge collected from the disk stack centrifuge was spun at 21,000 g for 10 minutes in a laboratory centrifuge to estimate the level of suspended solids in the centrifuge. About 0.15-0.3 g of undissolved solid was recovered from 45 mL of centrifuge. This corresponds to 0.3-0.7 wt% undissolved solids in the centrifuge, which is about a 10-fold reduction of undissolved solids from the mash fed to the centrifuge. In order to remove suspended solids from corn mash prior to fermentation, the level of undissolved solids present during extractive fermentation is reduced by an order of magnitude using several solid / liquid separation devices or device combinations. In cases, it is reasonable to assume that the ISPR extraction solvent loss via DDGS can be reduced by about an order of magnitude. Minimizing solvent loss via DDGS is an important factor in the economics and DDGS quality of the extractive fermentation process.

Example 4
Effect of Removal of Undissolved Solids on ISPR Extraction Solvent Loss-Bottle Rotation Test This example is generated by an extractive fermentation process by removing undissolved solids from corn mash prior to fermentation using a centrifuge. Shows the potential to reduce solvent loss through DDGS.

  A laboratory scale bottle rotation test was performed using liquefied corn mash. The test simulates the operating conditions of a typical decanter centrifuge used to remove undissolved solids from the total distillation waste in commercial ethanol plants. Decanter centrifuges in commercial ethanol plants are typically operated with a relative centrifugal force (RCF) of about 3000 g and a total distillation waste residence time of about 30 seconds. These centrifuges typically remove about 90% of the suspended solids in the total distillation effluent containing about 5% to 6% suspended solids (after the beer tower) and about 0.5% A dilute distillation waste liquid containing suspended solids is obtained.

  A liquefied corn mash was made according to the protocol described in Example 2. About 10 mL of mash was placed in a centrifuge tube. Samples were centrifuged at approximately 3000 g RCF (4400 rpm rotor speed) for a total of 1 minute. Samples were allowed to elapse for about 30-40 seconds at 3000 g and at a speed of less than 3000 g for a total of 20-30 seconds by centrifuge acceleration and deceleration. The sample temperature was about 60 ° C.

  Mash (10 mL) containing about 7 wt% suspended solids was separated into about 6.25 mL of clarified centrifuge and 3.75 mL wet cake (pellet at the bottom of the centrifuge tube). . The split defined by (amount of centrifuge) / (amount of original mash charged) obtained by the bottle rotation test was about 62%. The clarified centrifuge contains about 0.5% by weight suspended solids, which is a reduction of more than 10 times in suspended solids compared to the level of suspended solids in the original mash. I found out. It was also found that the cleaned pellets contained about 18% by weight suspended solids.

  Table 6 summarizes the mass balance of suspended (non-dissolved) solids in a bottle rotation test under typical conditions of operation of a decanter centrifuge that converts total distillation waste to dilute distillation waste in a commercial ethanol process. It is a thing. All values shown in Table 6 are approximate values.

  It was also found that the centrifuge solution contained about 190 g / L of dissolved oligosaccharide (liquefied starch). This is the starch content in the corn used to produce the corn filling used (about 26% by weight on a dry corn basis) and the liquefied mash (about 71.4% by weight starch on a dry corn basis). Is consistent with the assumption that the majority of starch in ground corn was liquefied in the liquefaction process (ie, hydrolyzed to soluble oligosaccharides). Hydrolysis of most of the starch in ground corn at a dry corn loading of 26% results in a suspension of about 7% by weight in the liquefied corn mash that is fed into the centrifuge used for the bottle rotation test. A (non-dissolved) solid is obtained.

  The purified centrifuge liquid contained only about 0.5% by weight of undissolved solids because the conditions used for the bottle rotation test exceeded 10 times the undissolved solids from the input mash. Indicates that it has resulted in a decrease that exceeds. If this same solid removal performance is obtained by a continuous decanter centrifuge before fermentation, it is reasonable to assume that the ISPR extraction solvent loss in DDGS can be reduced by about an order of magnitude. Minimizing solvent loss via DDGS is an important factor in the economics and DDGS quality of the extractive fermentation process.

Example 5
Removal of corn oil by removing undissolved solids This example demonstrates the possibility of removing and recovering corn oil from corn mash by removing undissolved solids prior to fermentation. The effectiveness of the extraction solvent can be improved when corn oil is removed by removal of undissolved solids. Furthermore, removal of corn oil by removal of undissolved solids can minimize the reduction in solvent partition coefficient and may result in an improved extractive fermentation process.

About 1000 g of liquefied corn mash was prepared in a 1 L glass coated resin kettle. The kettle was equipped with mechanical agitation, temperature control, and pH control. The following protocol was used: ground corn was mixed with tap water (26 wt% corn on a dry basis) and the slurry was heated to 55 ° C. with agitation to either NaOH or H ₂ SO _4. Use to adjust the pH to 5.8, add α-amylase (0.02 wt% based on dry corn), continue to heat to 85 ° C, adjust the pH to 5.8, and adjust the pH to 5.8 While maintaining, the temperature was maintained at 85 ° C. for 2 hours and cooled to 25 ° C.

  Pioneer 3335 hybrid corn, whole yellow corn was used (Pioner Hi-Bred International, Johnston, IA) and it was ground in a hammer mill using a 1 mm sieve. The moisture content of ground corn was about 11.7% by weight, and the starch content of ground corn was about 71.4% by weight on a dry corn basis. The α-amylase enzyme was Liquizyme® SC DS made by Novozymes (Franklinton, NC). The total amount of ingredients used was: 294.5 g ground corn (11.7% moisture), 705.5 g tap water, and 0.059 g Liquizyme® SC DS. Water (4.3 g) was added to dilute the enzyme and a total of 2.3 g of 20% NaOH solution was added to control the pH. About 952 g of mash was recovered.

  The liquefied corn mash was centrifuged at 5000 rpm (7260 g) for 30 minutes at 40 ° C. to remove undissolved solids from the oligosaccharide aqueous solution. Removing solids by centrifugation also removes free corn oil as a separate organic liquid layer on top of the aqueous phase. About 1.5 g of corn oil was recovered from the organic layer suspended above the aqueous phase. It was found by hexane extraction that the ground corn used to produce the liquefied mash contained about 3.5 wt% corn oil on a dry corn basis. This corresponds to about 9 g of corn oil fed to the liquefaction process, including ground corn.

  After the corn oil was recovered from the liquefied mash, the oligosaccharide aqueous solution was decanted and removed from the wet cake. When about 617 g of liquefied starch solution was recovered, about 334 g of wet cake remained. The wet cake contained most of the undissolved solid that was in the liquefied mash. The liquefied starch solution contained about 0.2% by weight of undissolved solids. The wet cake contained about 21% by weight undissolved solids. The wet cake was washed with 1000 g of tap water to remove the oligosaccharide remaining in the cake. This was done by mixing the cake with water to form a slurry. The slurry was then centrifuged under the same conditions used to centrifuge the original mash to recover the washed solid. Removal of the washed solids by centrifuging the wash slurry also removed some additional free corn oil that would have remained with the original wet cake produced from the liquefied mash. This additional corn oil was observed as a separate thin organic liquid layer at the top of the aqueous phase of the centrifuged wash mixture. Approximately 1 g of additional corn oil was recovered from the washing process.

  The wet solid was washed twice more with 1000 g of tap water each time to remove substantially all of the liquefied starch. No further visible corn oil was removed from the second and third water wash of the mash solid. The final washed solid was dried in a vacuum oven overnight at 80 ° C. and approximately 20 inches of mercury vacuum. The amount of corn oil remaining in the dry solid (possibly remaining in the germ) was measured by hexane extraction. A 3.60 g sample of a relatively dry solid (3.60 g, about 2 wt% moisture) contained 0.22 g of corn oil. This result corresponds to 0.0624 g corn oil per g dry solid. This was the case for the washed solid, which meant no residual oligosaccharide in the wet solid. After the liquefied corn mash was centrifuged to separate the free corn oil layer and the aqueous solution of oligosaccharides from the wet cake, there remained about 334 g of wet cake containing about 21 wt% undissolved solids. I understood. This corresponds to a wet cake containing about 70.1 g of undissolved solids. At 0.0624 grams of corn oil per gram of dry solids, the solids in the wet cake should contain about 4.4 grams of corn oil.

  In summary, about 1.5 g of free corn oil was recovered by centrifuging the liquefied mash. An additional 1 g of free corn oil was recovered by centrifuging the first (water) wash slurry produced to wash the original wet cake produced from the mash. Finally, it was found that the washed solid still contained about 4.4 g of corn oil. It was also found that the corn charged to liquefaction contained about 9 g of corn oil. Thus, a total of 6.9 g of corn oil was recovered from the following process steps: liquefaction, removal of solids from the liquefied mash, washing of the solids from the mash, and final washed solids. As a result, approximately 76% of the total corn oil in the corn fed to liquefaction was recovered during the liquefaction and solids removal process described herein.

Example 6
Extraction Fermentation Using Mash and Centrifugation as Sugar Source This example illustrates extraction fermentation performed using corn mash and corn mash centrifuge as a sugar source. A corn mash centrifuge was produced by removing undissolved solids from the corn mash prior to fermentation. Four extractive fermentations were performed in parallel, two using liquefied corn mash as a sugar source (with the solids removed) and two from the liquefied corn mash with the majority of the undissolved solids A liquefied mash centrifuge (oligosaccharide aqueous solution) obtained by removal was used. Oleyl alcohol (OA) was added to two of the fermentations (one containing solids and one with the solids removed) and the product (i-BuOH) was extracted from the broth as the product was formed . Add a mixture of corn oil fatty acids (COFA) to the other two of the fermentations (one containing solids and one removing the solids) and extracting the product from the broth as the product is formed did. COFA was made by hydrolyzing corn oil. The purpose of these fermentations is to test the effect of solids removal on the phase separation between the solvent and broth and the residue trapped in undissolved solids recovered from the fermentation broth with or without the removal of solids. It was to measure the amount of solvent.

Preparation of liquefied corn mash Approximately 31 kg of liquefied corn mash was prepared in a 30 L coated glass resin kettle. The reactor was equipped with mechanical stirring, temperature control, and pH control. The protocol used was as follows: ground corn was mixed with tap water (40 wt% corn on a dry basis) and the slurry was heated to 55 ° C. with stirring at 250 rpm and either NaOH or H Adjust the pH to 5.8 using ₂ SO ₄ , add a dilute α-amylase solution (0.16 wt% based on dry corn), hold at 55 ° C for 60 minutes, and heat to 95 ° C Then, the pH was adjusted to 5.8, and kept at 95 ° C. for 120 minutes while maintaining the pH at 5.8 to complete the liquefaction. The mash was transferred to a sterile centrifuge bottle to prevent contamination.

  The corn used was whole yellow corn (Pioner Hi-Bred International, Johnston, IA), which was ground on a pilot scale hammer mill using a 1 mm sieve. The water content of ground corn was about 12% by weight, and the starch content of ground corn was about 71.4% by weight on a dry corn basis. The α-amylase enzyme used was Spezyme® Fred-L (Genencor®, Palo Alto, Calif.). The amount of ingredients used is: α consisting of 14.1 kg ground corn (12% moisture), 16.9 kg tap water, 19.5 g Spezyme® Fred-L in 2.0 kg water -It was a solution of amylase. α-Amylase was sterile filtered. A total of 0.21 kg NaOH (17 wt%) was added throughout the experiment to control the pH.

  The liquefied corn mash is about 28 wt% (about 280 g / L) of liquefaction, based on the corn filling used and the starch content of the corn, assuming that all starch has been hydrolyzed during liquefaction. It was estimated that it contained starch. Mash was prepared with a higher concentration of oligosaccharides required for fermentation to allow dilution when nutrients, inoculum, glucoamylase, and base were added to the initial fermentation broth. After dilution by addition of nutrients, inoculum, glucoamylase, and base, the expected total initial soluble sugar in the mash (which did not remove the solids) was about 250 g / L.

  About 13.9 kg of liquefied mash was centrifuged using a bottle centrifuge containing six 1 L bottles. The centrifuge was operated at 5000 rpm (7260 RCF) for 20 minutes at room temperature. The mash was separated into about 5.5 kg of clarified centrifuge and about 8.4 kg of wet cake (pellet at the bottom of the centrifuge bottle). The split, defined as (amount of centrifuge) / (amount of mash supplied) was about (5.5 kg / 13.9 kg) = 40%.

  Solids were not removed from the mash charged to the 2010Y034 and 2010Y036 fermentations described below. Centrifugation into the fermentations 2010Y033 and 2010Y035 (also described below) was generated by removing most of the suspended solids from the mash by centrifugation according to the protocol described above.

General Methods for Fermentation Seed Flask Growth
Saccharomyces cerevisiae strains modified to produce isobutanol from a carbohydrate source (deficient in pdc1, pdc5, and pdc6) were grown in seed flasks from frozen media at 0.55-1. Grown to 1 g / L dcw (OD ₆₀₀ 1.3-2.6-Thermo Helios α Thermo Fisher Scientific Inc., Waltham, Massachusetts). Saccharomyces cerevisiae strains are described in US Patent Application Publication No. 2012/0164302, which is incorporated herein by reference. The medium was grown at 26 ° C. in an incubator rotating at 300 rpm. The frozen medium was pre-stored at -80 ° C. The composition of the first seed flask medium was as follows:
3.0 g / L dextrose 3.0 g / L absolute ethanol 3.7 g / L ForMedium ™ Synthetic Complete Amino Acid (Kaiser) Drop-Out: no HIS, no URA (reference number DSCK162CK)
6.7 g / L Difco Yeast Nitrogen Base (No. 291920) without amino acids.

12 ml (12 mL) from the first seed flask medium was transferred to a 2 L flask and grown at 30 ° C. in an incubator rotating at 300 rpm. The second seed flask has 220 mL of the following medium:
30.0 g / L dextrose 5.0 g / L absolute ethanol 3.7 g / L ForMedium ™ Synthetic Complete Amino Acid (Kaiser) Drop-Out: no HIS, no URA (reference number DSCK162CK)
6.7 g / L Difco Yeast Nitrogen Base without amino acid (No. 291920)
0.2 M MES buffer titrated to pH 5.5-6.0.

The medium was grown at 0.55-1.1 g / L dcw (OD ₆₀₀ 1.3-2.6). An additional 30 mL solution containing 200 g / L peptone and 100 g / L yeast extract was added to the cell concentrate. Next, an additional 300 mL of 0.2 uM filter sterilized, 90-95% oleyl alcohol (Cognis Corporation, Cincinnati, OH) was added to the flask. Medium was continued to grow to> 4 g / L dcw (OD ₆₀₀ > 10) before being harvested and added to the fermentation.

Fermentation device preparation at the initial stage of fermentation preparation A liquefied mash (centrifugation solution) containing solids or not containing solids was charged into a 2 L fermentation device (Sartorius AG, Goettingen, Germany) coated with glass. A pH probe (Hamilton Easyferm Plus K8, part number: 238627, Hamilton Bondadz AG, Bondaduz, Switzerland) was calibrated by the Sartorius DCU-3 Control Tower Calibration menu. Zero calibration was performed at pH = 7. Span calibration was performed at pH = 4. The probe was then placed into the fermentor through a stainless steel head plate. A dissolved oxygen probe (pO ₂ probe) was also placed in the fermentor through the head plate. Tubes used to deliver nutrients, seed medium, extraction solvent, and base were attached to the head plate and foiled at the ends. The entire fermentation apparatus was placed in an autoclave (Steris Corporation, Mentor, Ohio) and sterilized in a liquid cycle for 30 minutes.

  The fermenter was removed from the autoclave and placed on the load cell. The jacket water supply and return lines were connected to household water and drain drain, respectively. A condenser for cooling the incoming and outgoing water lines was connected to a 6 L recirculation temperature bath operating at 7 ° C. A vent line for transferring gas from the fermenter was connected to a transfer line connected to a Thermo mass spectrometer (Prima dB, Thermo Fisher Scientific Inc., Waltham, Massachusetts). The sparger line was connected to the gas supply line. The tubes for adding nutrients, extraction solvent, seed medium, and base were plumbed through the pump and secured firmly. The autoclave material, 0.9% w / v NaCl, was drained before adding the liquefied mash.

Lipase treatment after liquefaction After the liquefaction cycle was completed, the fermenter temperature was set to 55 ° C instead of 30 ° C. The pH was manually controlled to pH = 5.8 by adding an acid or base bolus as needed. The lipase enzyme stock solution was added to the fermenter so that the final lipase concentration was 10 ppm. The fermenter was held at 55 ° C., 300 rpm, and 0.3 slpm N ₂ overlay for over 6 hours. After the lipase treatment was completed, the fermenter temperature was set to 30 ° C.

Nutritional addition before inoculation 7.0 mL / L (volume after inoculation) of ethanol (200 proof, anhydrous) was added immediately before inoculation. Thiamine was added to a final concentration of 20 mg / L immediately before inoculation. Immediately before inoculation, 100 mg / L nicotinic acid was added.

Fermenter inoculation The fermenter pO ₂ probe was calibrated to zero while N ₂ was added to the fermentor. The fermenter pO ₂ probe was calibrated to its span using a sterile air sparge at 300 rpm. After the second seed flask was over 4 g / L dcw, the fermenter was inoculated. The shake flask was removed from the incubator / shaker for 5 minutes to allow phase separation between the oleyl alcohol phase and the aqueous phase. The aqueous phase (55 mL) was transferred to a sterile inoculation bottle. The inoculum was injected into the fermentor through a peristaltic pump.

Addition of oleyl alcohol or corn oil fatty acid after inoculation Immediately after inoculation, 1 L / L (volume after inoculation) of oleyl alcohol or corn oil fatty acid was added.

Fermenter operating conditions The fermenter was operated at 30 ° C for the entire growth and production stage. The pH was lowered from a pH of 5.7-5.9 to a control set point of 5.2 without any acid added. For the remainder of the growth and production phase, ammonium hydroxide was used to control the pH to pH = 5.2. For the remainder of the growth and production phase, sterile air was added to the fermentor at 0.3 slpm through a sparger. The Sartorius DCU-3 Control Box PID control loop uses only agitation control to set the agitator to a minimum of 300 rpm and a maximum of 2000 rpm to control the pO ₂ to 3.0%. Set. Glucose was supplied by simultaneous saccharification and fermentation of liquefied corn mash by adding α-amylase (glucoamylase). As long as starch was available for saccharification, the glucose was kept in excess (1-50 g / L).

Sample A
The experimental identifier 2010Y033 included: seed flask growth method, initial fermentation device preparation method using corn mash excluding solids, lipase treatment after liquefaction, nutrient addition prior to inoculation method, fermentation device Inoculation method, operating condition method of fermentation apparatus, and analysis method. Corn oil fatty acids were added to a single batch for 0.1-1.0 hours after inoculation.

Sample B
The experimental identifier 2010Y034 included: seed flask growth method, initial fermentation device preparation method using corn mash containing solids, lipase treatment after liquefaction, nutrient addition prior to inoculation method, fermentation device All of the inoculation method, the fermentation apparatus operating condition method, and the analysis method. Corn oil fatty acids were added to a single batch for 0.1-1.0 hours after inoculation.

Sample C
Experiment identifier 2010Y035 included: seed flask growth method, initial fermentation device preparation method using corn mash excluding solids, nutrient addition prior to inoculation method, fermenter inoculation method, fermentation device All of the operating condition methods and analysis methods. Oleyl alcohol was added to a single batch for 0.1-1.0 hours after inoculation.

Sample D
The experimental identifier 2010Y036 included the following: seed flask growth method, initial fermentation device preparation method using solid corn mash, nutrient addition prior to inoculation method, fermenter inoculation method, fermentation device All operating condition methods and analysis methods. Oleyl alcohol was added to a single batch for 0.1-1.0 hours after inoculation. The results of samples A to D are shown in Table 7.

Example 7
Effect of removing undissolved solids from fermenter feedstock on fermenter volumetric efficiency This example shows the effect of removing undissolved solids from mash prior to fermentation on fermenter volumetric efficiency. Undissolved solids in corn mash occupy at least 5% of the mash volume, depending on corn loading and starch content. By removing solids prior to fermentation, at least 5% more sugar can be charged to the fermenter, thus increasing batch productivity.

  The liquefied corn mash produced in Example 5 assumes that all starch was hydrolyzed to soluble oligosaccharides during liquefaction, the corn loading used (40% on a dry corn basis), It was estimated that it contained about 28% by weight (280 g / L) of liquefied starch, based on the starch content (71.4% on a dry corn basis). Mash was prepared with a higher concentration of oligosaccharides required for fermentation to allow dilution when nutrients, inoculum, glucoamylase, and base were added to the initial fermentation broth. After adding these ingredients, the mash was diluted by about 10%. Therefore, the expected concentration of liquefied starch in the mash (including solids) at the start of fermentation 2010Y034 and 2010Y036 was about 250 g / L. The actual glucose equivalents input to the 2010Y034 and 2010Y036 fermentations were 239 g / kg and 241 g / kg, respectively. In comparison, the glucose equivalents input to the 2010Y033 and 2010Y035 fermentations were 257 g / kg and 263 g / kg, respectively. Note that the raw material for these fermentations was centrifuge (mash with most of the solids removed). About 1.2 L of sugar source (mash or centrifuge) was added to each fermentation. Therefore, based on this data, about 8.3% more sugar was loaded into the fermentor using the centrifuge (2010Y033 and 2010Y035) compared to the mash (2010Y034 and 2010Y036). These results show that removal of undissolved solids from corn mash prior to fermentation can significantly increase the sugar input per unit volume. This suggests that if a solid is present, it occupies valuable fermenter volume. If the solid is removed from the feed, more sugar can be added to the fermenter (“suitable”) because there is no undissolved solid. This example shows that fermenter volumetric efficiency can be significantly improved by removing undissolved solids from the mash prior to fermentation.

Example 8
Effect of Removal of Undissolved Solids on Phase Separation between Extraction Solvent and Broth—Extracted Fermentation This example illustrates the use of undissolved solids from corn mash prior to fermentation during and after the extractive fermentation process. Shows improved separation between phase and broth phase. Two extractive fermentations are performed in parallel, one using liquefied corn mash as a sugar source (with the solids removed) and one from the liquefied corn mash with most of the undissolved solids The centrifuge liquid (oligosaccharide aqueous solution) produced by the removal was used. Oleyl alcohol (OA) was added to both fermentations and the product (i-BuOH) was extracted from the broth as the product was formed. The fermentation broth referred to in this example when the solid was not removed from the feed (using corn mash) was 2010Y036 as described in Example 6. The fermentation broth referred to in this example when the solid was removed from the feed (using a centrifuge generated from corn mash) was 2010Y035 as described in Example 6. Oleyl alcohol was the extraction solvent used for both fermentations. The rate and extent of phase separation was measured and compared throughout the fermentation, not just the final fermentation broth. Sufficient phase separation during the extractive fermentation process can lead to minimal loss of microorganisms and minimal solvent loss and lower capital and operating costs of downstream processing.

Phase separation between solvent phase and broth phase during fermentation About 10 mL of sample was withdrawn from each fermentor at 5.3, 29.3, 53.3 and 70.3 hours The phase separation was compared for samples from the fermentation (2010Y035) when the solids were removed from and from the fermentation (2010Y036) when the solids were not removed. Samples were allowed to stand for about 2 hours and phase separation was observed and compared for all samples from all run times by following the position of the liquid-liquid interface. Virtually no phase separation was observed for any of the samples drawn from the fermentation when the solids were not removed. Phase separation was observed for all samples from fermentation when solids were removed from liquefied corn mash before fermentation. Separation began to occur within about 10-15 minutes of pulling the sample from the experiment when the solids were removed for all fermentation times and continued to improve over a period of 2 hours. After about 7 minutes settling time, phase separation began to occur in the sample drawn from the centrifuge fermentation (with the solids removed) at the time of the 5.3 hour fermentation run. After a sedimentation time of about 17 minutes, phase separation began to occur in the sample drawn at 53.3 hours from the centrifuge fermentation (with the solids removed).

  FIG. 13 is a plot of the position of the liquid-liquid interface in the fermentation sample tube as a function of (gravity) settling time. Data are for samples drawn from extractive fermentation when centrifuge was supplied as a sugar source (solids removed from corn mash) and oleyl alcohol was extracted from ISPR extraction solvent (Example 6). Experiment 2010Y035). The phase separation data in this plot is for samples drawn from fermentation 2010Y035 at run times of 5.3 hours, 29.3 hours, 53.3 hours, and 70.3 hours. The interface position is reported as a percentage of the total broth height in the sample tube. For example, the interfacial position of the sample drawn from the 2010Y035 fermentation (centrifugate / oleyl alcohol) at the 5.3 hour run time is 3 from the bottom of the sample tube (no separation) after a 120 minute settling time. Increased to 5 mL. There was about 10 mL of total broth in that particular sample tube. Therefore, the interface position of the sample was reported as 35% in FIG. Similarly, the interfacial position of the sample drawn from the 2010Y035 fermentation (centrifugate / oleyl alcohol) at the 53.3 hour run time is from the bottom of the sample tube (no separation) after 125 minutes settling time. Increased to about 3.9 mL. There was about 10 mL of total broth in that particular sample tube. Therefore, the interface position of the sample was reported as 39% in FIG.

Phase separation between solvent phase and broth phase after completing the fermentation After a run time of 70 hours, the fermentation was stopped and the two broths from the oleyl alcohol extraction fermentation were placed in separate 2 L glass graduated cylinders. Moved. The separation between the solvent phase and the broth phase was observed and compared. For the broth (experiment 2010Y036) where the solid was not removed prior to fermentation, little phase separation was observed after about 3 hours. Phase separation was observed for broth (experiment 2010Y035) when solids were removed from corn mash liquefied prior to fermentation. After about 15 minutes settling time, separation began to occur and continued to improve over a 3 hour period. After 15 minutes, a liquid-liquid interface was established at a level of about 10% of the total height of the two-phase mixture. This indicates that the aqueous phase was first split from the dispersion and began to accumulate at the bottom of the mixture. As time passed, more aqueous phase accumulated at the bottom of the mixture, raising the position of the interface. After a settling time of about 3 hours, the interface increased to a level of about 40% of the total height of the two-phase mixture. This is based on the amount of centrifuge and oleyl alcohol initially charged to the fermentation, with almost complete phase separation of about 3 hours (gravity) for the final two-phase broth when the solids are removed. Indicates what happened after the settling time. Approximately equal volumes of initial centrifuge and solvent were charged to both fermentations. About 1.2 L of liquefied corn mash and about 1.1 L of oleyl alcohol were charged to fermentation 2010Y036. About 1.2 L of centrifuge, produced from the same batch of mash, and about 1.1 L of oleyl alcohol were charged to fermentation 2010Y035. After knowing that about 100 g / kg of the initial sugar in the aqueous phase was consumed and about 75% of the i-BuOH produced was in the solvent phase, It can be expected that the relative volume of the typical aqueous and organic phases will be about 1: 1. FIG. 14 is a plot of the liquid-liquid interface position as a function of (gravity) settling time for the final two-phase broth from extractive fermentation (2010Y035) when the solids are removed. The interface position is reported as a percentage of the total broth height in the 2 L graduated cylinder used to observe the final broth phase separation. The final broth interface position from the 2010Y035 fermentation increased from the bottom of the graduated cylinder (no separation) to a level of about 40% of the total height of the two-phase mixture after a 175 minute settling time. Thus, almost complete separation of the two phases in the final broth occurred after a settling time of 3 hours. About 50% of the interface position may correspond to complete separation.

  A 10 mL sample was withdrawn from the top of the organic phase of the final broth (settled for about 3 hours) from the fermentation when the solids were removed. The sample was rotated in a high speed laboratory centrifuge to measure the amount of aqueous phase present in the organic phase after allowing the broth to settle for 3 hours. The results showed that about 90% of the final broth upper layer was the solvent phase. About 10% of the top layer of the final broth, containing a relatively small amount of undissolved solids, was the aqueous phase. Some solids were located at the bottom of the aqueous phase (darker than the aqueous phase) and a small amount of solids had accumulated at the liquid-liquid interface.

  A 10 mL sample was also drawn from the bottom phase of the final broth (settled for about 3 hours) from the fermentation when the solids were removed. The sample was rotated in a high speed laboratory centrifuge to measure the amount of organic phase present in the aqueous phase after allowing the broth to settle for 3 hours. After allowing the broth to settle for 3 hours, it was found that there was virtually no organic phase in the bottom (water) phase of the final broth from the fermentation that removed the solids. These results confirm that almost complete phase separation occurred for the final broth from the fermentation when the solids were removed. Little phase separation was seen for the final broth from the fermentation when no solids were removed. This data shows that removal of solids from liquefied corn mash prior to extractive fermentation can allow for significant improvements in phase separation during and after fermentation, including microorganisms, undissolved solids, and downstream processing. It suggests that water loss can be reduced.

  A 10 mL sample was withdrawn from the top of the final broth from the fermentation that did not remove solids after allowing the broth to settle for about 3 hours. Samples were rotated in a high speed laboratory centrifuge to measure the relative amounts of solvent and water phases at the top of the final broth. This broth contained all the solids from the liquefied corn mash solids. About half of the sample was the aqueous phase and about half was the organic phase. The aqueous phase contained significantly more undissolved solids (derived from the liquefied mash) compared to the upper sample from the broth when the solids were removed. The amount of aqueous and solvent phase in this sample is about the same, which means that the phase separation is substantially in the final broth when no solids are removed (even after a settling time of 3 hours). Indicates that it did not happen. This data suggests that if solids are not removed from corn mash liquefied prior to extractive fermentation, little or no phase separation is likely to occur during and after fermentation. This can result in considerable loss of microorganisms, undissolved solids, and water to downstream processing.

Example 9
Effect of Removal of Undissolved Solids on ISPR Extraction Solvent Loss-Extraction Fermentation This example demonstrates the solvent loss for DDGS that exits the final stage of the extractive fermentation process by removing undissolved solids from corn mash prior to fermentation. Indicates the possibility of reduction. Example 6 illustrates two extractive fermentations performed in parallel, one using liquefied corn mash (one that does not remove solids 2010Y036-) as the sugar source, one of the undissolved solid A liquefied mash centrifuge (2010Y035-oligosaccharide aqueous solution) obtained by removing most from the liquefied corn mash was used. Oleyl alcohol (OA) was added to both fermentations and the product isobutanol (i-BuOH) was extracted from the broth as the product was formed. The amount of residual solvent entrapped in the undissolved solid recovered from the final fermentation broth was measured and compared.

After the fermentations 2010Y035 and 2010Y036 described in Example 6 were complete, the broth was collected and used to conduct a phase separation test. Next, undissolved solids (fine particles from corn mash that were not removed prior to fermentation) were recovered from each broth and analyzed for total extractable oil. The oil recovered from each lot of solids was analyzed for oleyl alcohol and corn oil. The following protocol was followed for both broths:
-The broth was centrifuged to separate it into an organic phase, an aqueous phase and a solid phase.
The organic and aqueous phases were decanted from the solid, leaving a wet cake at the bottom of the centrifuge bottle.
The wet cake was washed thoroughly with water to remove substantially all of the dissolved solids retained in the cake, such as residual oligosaccharides, glucose, salts, enzymes, etc.
The washed wet cake was dried overnight in a vacuum oven (80 ° C. household vacuum) to remove substantially all of the water in the cake.
A portion of the dry solid was contacted well with hexane in a Soxhlet extractor to remove the oil from the solid.
The oil recovered from the solid was analyzed by GC to determine the relative amount of oleyl alcohol and corn oil present in the oil recovered from the solid.
• The particle size distribution (PSD) for the solids recovered from both fermentation broths was measured.

  Data for the recovery of undissolved solids from both fermentation broths and hexane extraction are shown in Table 8. The data shows that approximately the same amount of oil (per unit mass of solid) was absorbed by the solid in both fermentations.

Example 10
Recovery of soluble starch from wet cake produced from the removal of solids from liquefied corn mash by washing the wet cake with water-a two-step process This example involves washing the cake twice with water Shows the recovery of soluble starch from the wet cake, where the cake was produced by centrifuging the liquefied mash. The liquefied corn mash was fed to a continuous decanter centrifuge to produce a centrifuge liquid stream (C-1) and a wet cake (WC-1). The centrifuge liquid is an aqueous solution of soluble starch that does not contain a solid relatively, and the wet cake was concentrated to a solid as compared with the raw material mash. A portion of the wet cake was mixed with hot water to form a slurry (S-1). The slurry was again injected into the decanter centrifuge to produce a wash water centrifuge (C-2) and a washed wet cake (WC-2). C-2 was a dilute aqueous solution of soluble starch that was relatively free of solids. The concentration of soluble starch in C-2 was lower than the concentration of soluble starch in the centrifuge produced from the mash separation. The liquid phase retained in WC-2 was less starchy than the liquid in the wet cake produced from the mash separation. The washed wet cake (WC-2) was mixed with hot water to form a slurry (S-2). The ratio of input water to the amount of soluble starch in the input wet cake was the same in both washing steps. The second wash slurry was re-injected into the decanter centrifuge to produce a second wash water centrifuge (C-3) and wet cake (WC-3) that was washed twice. C-3 was a dilute aqueous solution of soluble starch that was relatively free of solids. The concentration of soluble starch in C-3 is lower than the concentration of soluble starch in the centrifuge produced in the first washing stage (C-2) and thus WC-3 (second washed wet The liquid phase retained in the cake) was thinner than the starch in WC-2 (first washed wet cake). The total soluble starch in the two wash centrifuges (C-2 and C-3) is a starch that can be recovered and reused for liquefaction. The soluble starch in the liquid retained in the final washed wet cake is much less than in the wet cake produced in the original separation of the mash.

Production of Liquefied Corn Mash Approximately 1000 gallons of liquefied corn mash was produced in a continuous dry grinding liquefaction system consisting of a hammer mill, a slurry mixer, a slurry tank, and a liquefaction tank. Ground corn, water, and α-amylase were fed continuously. The reactor was equipped with mechanical stirring, temperature control, and pH control using either ammonia or sulfuric acid. The reaction conditions were as follows:
Hammer mill sieve size: 7/64 inch Feed rate to slurry mixer-ground corn: 560 mass pounds per hour (14.1 wt% moisture)
-Treated water: 16.6 lbs / min (200F)
-Α-amylase: 61 g / hr (Genecor: Spezyme®)
・ Slurry tank conditions:
-Temperature: 185 ° F (85 ° C)
-PH: 5.8
-Residence time: 0.5 hours-Dry corn filling: 31 wt%
-Enzyme filling amount: 0.028 wt% (based on dry corn)
・ Liquefaction tank conditions:
-Temperature: 185 ° F (85 ° C)
-PH: 5.8
-Residence time: about 3 hours-No further enzyme addition.

  The production rate of liquefied corn mash was about 3 gallons / minute (gpm). The starch content of ground corn was about 70% by weight on a dry corn basis. The total solids (TS) of the liquefied mash was about 31% by weight and the total suspended solids (TSS) was about 7% by weight. The liquid phase contained about 23-24% by weight liquefied starch (soluble oligosaccharide) as measured by HPLC.

The liquefied mash was centrifuged in a continuous decanter centrifuge under the following conditions:
-Bowl speed: 5000 rpm (about 3600 g's)
・ Differential speed: 15rpm
・ Weir diameter: 185 mm (when weir plate is removed)
Feed rate: Fluctuates at 5-20 gallons / minute.

  About 850 gallons of centrifuge and about 1400 mass pounds of wet cake were produced by centrifuging the mash. The total solid content in the wet cake was about 46.3% (suspended solid + dissolved solid) as measured by moisture balance. It was found that the liquid phase contained about 23% by weight soluble starch and it was estimated that the total suspended solids in the wet cake was about 28% by weight. It was estimated that the wet cake contained approximately 12% soluble starch present in the mash liquefied prior to centrifuge operation.

Recovery of soluble starch from wet cake by washing solid with water-first wash Approximately 707 pounds of wet cake recovered from the separation of the liquefied mash was placed in a 300 gallon stainless steel container at 165 Mixed with hot gallon (91 ° C.) water. The resulting slurry was mixed for about 30 minutes. The slurry was continuously fed to a decanter centrifuge to remove the washed solid from the slurry. The centrifuge used to separate the wash slurry is the same as that used to remove solids from the above liquefied mash, and before feeding the slurry, it is washed with fresh water. Rinse. The centrifuge was operated under the following conditions to remove solids from the wash slurry:
-Bowl speed: 5000 rpm (about 3600 g's)
・ Differential speed: 5rpm
・ Weir diameter: 185 mm (when weir plate is removed)
Feed rate: 5 gal / min.

  Approximately 600 pounds of washed wet cake was produced by the centrifuge, but only 400 pounds were recovered due to material loss. The total solid content in the wet cake was about 36.7% (suspended solid + dissolved solid) as measured by moisture balance. Total soluble starch (sum of glucose, DP2, DP3, and DP4 +) in the liquid phase of the slurry and in the wash water centrifuge (obtained from the slurry) was measured by HPLC to be about 6.7% by weight and 6%, respectively. 0.9% by weight. DP2 refers to a dextrose polymer containing two glucose units (glucose dimer). DP3 refers to a dextrose polymer containing three glucose units (glucose trimer). DP4 + refers to a dextrose polymer containing 4 or more glucose units (glucose tetramer and higher order multimer). This confirmed that a well-mixed dilution wash step was performed. Therefore, the concentration of soluble starch in the liquid phase retained in the washed wet cake should have been about 6.8% by weight (by mass balance) for this diluted wash. Based on total solids and dissolved oligosaccharide data, it was estimated that the total suspended solids in the washed wet cake was about 32% by weight. If all 600 mass pounds of cake produced by the centrifuge can be washed, the washed wet cake contains about 2.6% soluble starch present in the original liquefied mash. It was estimated that it was. This represents an approximately 78% reduction in soluble starch in the washed wet cake as compared to the mash wet cake before washing. If the wet cake produced from the separation of the liquefied mash is not washed, about 12% of the total starch in the mash will be lost as soluble (liquefied) starch. If the wet cake produced from the mash separation is washed with water under the conditions shown in this example, 2.6% of the total starch from the mash will be lost as soluble (liquefied) starch.

Approximately 400 pounds of washed wet cake recovered from the first reslurry wash of the liquefied mash wet cake was mixed with 110 gallons of hot (90 ° C.) water in a 300 gallon stainless steel container. The resulting slurry was mixed for about 30 minutes. The slurry was continuously fed to a decanter centrifuge to remove the washed solid from the slurry. The centrifuge used to separate the second wash slurry is the same as that used for the first wash above, and before supplying the second wash slurry, it is washed with fresh water. Rinse. The centrifuge was operated under the following conditions to remove solids from the wash slurry:
-Bowl speed: 5000 rpm (about 3600 g's)
・ Differential speed: 5rpm
・ Weir diameter: 185 mm (when weir plate is removed)
Feed rate: 4 gal / min.

  Approximately 322 pounds of washed wet cake was produced by a centrifuge. The total solid content in the wet cake from the second wash was about 37.4% (suspended solid + dissolved solid) as measured by moisture balance. Total soluble starch (total of glucose, DP2, DP3, and DP4 +) in the liquid phase of the slurry and in the wash water centrifuge (obtained from the slurry) was measured by HPLC to be about 1.6 wt% and 1 respectively 0.6% by weight. This confirmed that a well-mixed dilution wash step was performed in the second wash. Therefore, the concentration of soluble starch in the liquid phase retained in the washed wet cake should have been about 1.6% by weight (by mass balance) for this diluted wash. Based on total solids and dissolved oligosaccharide data, it was estimated that the total suspended solids in the washed wet cake was about 36% by weight. If all 600 mass pounds of cake produced in the first washing stage could be washed, the washed wet cake will have about 0.5% soluble starch present in the original liquefied mash. It was estimated that it contained. This represents a total reduction in soluble starch in the wet cake washed twice compared to the mash wet cake before about 96% washing. If the wet cake produced from the separation of the liquefied mash is not removed, about 12% of the total starch in the mash will be lost as soluble (liquefied) starch. If the wet cake produced from the mash separation is washed twice with water under the conditions shown in this example, 0.5% of the total starch from the mash will be lost as soluble (liquefied) starch. Let's go.

Example 11
The effect of the high temperature stage during liquefaction on the conversion of starch in corn solids to soluble (liquefied) starch. Figure 5 shows that starch in corn solids can lead to higher conversion to soluble (liquefied) starch. The “cooking” stage shown in this example consists of raising the liquefaction temperature at some point after the start of liquefaction, holding it for a higher period, and then lowering the temperature back to its original value. And completing the liquefaction.

A. Procedure for measuring non-hydrolyzed starch remaining in the solid after liquefaction Liquefied corn mash was prepared in one run according to the protocol of Example 1 (no intermediate hot stage). A liquefied corn mash was prepared in a separate run under the same conditions as the first run except for the addition of an intermediate high temperature stage. The mash from both runs was worked up according to the following steps. It was centrifuged to separate the liquefied starch aqueous solution from the undissolved solids. The aqueous solution of liquefied starch was decanted off and the wet cake was collected. The wet cake contained most of the undissolved solid from the mash, but the solid was still wet with the liquefied starch solution. The wet cake was washed thoroughly with water and the resulting slurry was centrifuged to separate the aqueous layer from the undissolved solid. The cake was washed with enough water for a total of 5 times to remove almost all of the soluble starch retained in the original wet cake recovered from liquefaction. As a result, the liquid phase retained in the final washed wet cake consisted of water substantially free of soluble starch.

  The final washed wet cake was reslurried in water and both very excess α-amylase and glucoamylase were added. While controlling the temperature and pH, the slurry is mixed for at least 24 hours to allow α-amylase to convert substantially all unhydrolyzed starch remaining in the undissolved solids to soluble oligosaccharides. did. Subsequently, soluble oligosaccharides produced from residual starch (which was not hydrolyzed during liquefaction under the conditions of interest) were converted to glucose by the existing glucoamylase. The glucose concentration was followed over time by HPLC to confirm that all oligosaccharides produced from the residual starch were converted to glucose and that the glucose concentration no longer increased with time.

B. Production of liquefied corn mash Two batches of liquefied corn mash were prepared using Liquizyme® SC DS (alpha-amylase from Novozymes (Franklinton, NC)) at 85 ° C. (approximately 1 L each). Prepared. Both batches were run for more than 2 hours at 85 ° C. However, a “cooking” period was added in the middle of the second batch (“Batch 2”). The temperature distribution of batch 2 was 85 ° C. for about 30 minutes, the temperature was increased from 85 ° C. to 101 ° C., held at 101 ° C. for about 30 minutes, cooled to 85 ° C. and continued to liquefy for an additional 120 minutes. The ground corn used in both batches was the same as in Example 1. A corn loading of 26% by weight (dry corn basis) was used for both batches. The total amount of enzyme used in both runs corresponded to 0.08 wt% (dry corn basis). The pH was controlled at 5.8 during both liquefaction runs. Liquefaction was performed in a glass-coated resin kettle. The kettle was equipped with mechanical agitation, temperature control, and pH control.

Batch 1 liquefied corn mash was prepared according to the following protocol:
Α-Amylase was diluted with tap water (0.418 g enzyme in 20.802 g water).
-704.5 g of tap water was added to the kettle.
-The stirrer was switched on.
A first charge of ground corn (198 g) was made.
Heated to 55 ° C. with stirring.
• The pH was adjusted to 5.8 using H ₂ SO ₄ or NaOH.
A first charge of α-amylase solution (7.111 g) was made.
• Heated to 85 ° C • Hold at 85 ° C for 30 minutes.
A second charge of α-amylase solution (3.501 g) was made.
A second charge of ground corn (97.5 g) was made.
• Continued operation at 85 ° C for an additional 100 minutes.
-It cooled to 60 degreeC after liquefaction was completed.
-The reactor was dumped and 998.5 g of liquefied mash was recovered.

Batch 2 liquefied corn mash was prepared according to the following protocol:
Α-Amylase was diluted with tap water (0.3366 g enzyme in 16.642 g water).
-562.6 g of tap water was added to the kettle.
-The stirrer was switched on.
-Ground corn (237.5 g) was added.
Heated to 55 ° C. with stirring.
Adjust the pH to 5.8 using dilute H ₂ SO ₄ or NaOH.
A first charge of α-amylase solution (4.25 g) was made.
-Heated to 85 ° C.
-Hold at 85 ° C for 30 minutes.
-Heated to 101 ° C.
-Hold at 101 ° C for 30 minutes.
-The mash temperature was again reduced to 85 ° C.
Adjust the pH to 5.8 using dilute H ₂ SO ₄ or NaOH.
A second charge of α-amylase solution (4.2439 g) was made.
• Continued operation at 85 ° C for an additional 120 minutes.
-It cooled to 60 degreeC after liquefaction was completed.

C. Removal of undissolved solids from the liquefied mash and washing of the wet cake with water to remove soluble starch. Most of the solids were removed from both batches. Centrifugation of 500 g mash from batch 1 gave 334.1 g of centrifuge and 165.9 g of wet cake. Centrifugation of 872 g of mash from batch 2 yielded 654.7 g of centrifuge and 217 g of wet cake. The wet cake recovered from each batch of liquefied mash was washed 5 times with tap water to remove substantially all of the soluble starch retained in the cake. Washing was done in the same bottle used to centrifuge the original mash to prevent the cake from moving between the containers. In each wash step, the cake was mixed with water and the resulting wash slurry was centrifuged at room temperature (5000 rpm for 20 minutes). This was done for all five washing steps performed on wet cakes recovered from both batches of mash. Approximately 165 g of water was used for each of the 5 washes of the wet cake from Batch 1, which totaled 828.7 g of water used to wash the wet cake from Batch 1. About 500 g of water was used for each of the 5 washes of the wet cake from Batch 2, which resulted in a total of 2500 g of water used to wash the wet cake from Batch 2. The total wash centrifuge recovered from all 5 water washes of the wet cake from Batch 1 was 893.1 g. The total wash centrifuge recovered from all 5 water washes of the wet cake from batch 2 was 2566.3 g. The final washed wet cake recovered from batch 1 was 101.5 g and the final washed wet cake recovered from batch 2 was 151.0 g. The final washed wet cake obtained from each batch was substantially free of soluble starch; therefore, the liquid retained in each cake was primarily water. The total solids (TS) of the wet cake was measured using a moisture balance. The total solids of the wet cake from batch 1 was 21.63 wt% and the TS of the wet cake from batch 2 was 23.66 wt%.

D. Liquefaction / saccharification of the washed wet cake to measure the level of non-hydrolyzed starch remaining in the non-dissolved solid after liquefaction Non-hydrate remaining in the solid present in both washed wet cakes The level of degraded starch was measured by reslurrying the cake in water and adding excess α-amylase and excess glucoamylase. α-Amylase converts residual non-hydrolyzed starch in solids into soluble oligosaccharides that dissolve in the aqueous phase of the slurry. Thereafter, glucoamylase converts soluble oligosaccharides produced by α-amylase into glucose. Run the reaction at 55 ° C (recommended maximum temperature for glucoamylase) for at least 24 hours to convert all residual starch in the solid to soluble oligosaccharides and all soluble oligosaccharides to glucose. I was sure. Residual non-hydrolyzed starch in solids that is not hydrolyzed during liquefaction can be calculated from the amount of glucose produced by this procedure.

  The α-amylase and glucoamylase enzymes used in the following protocols were Liquizyme® SC DS and Spirizyme® Fuel (Novozymes, Franklinton, NC), respectively. The container used to process the washed wet cake was a 250 mL coated glass resin kettle with mechanical stirring, temperature control, and pH control. The amount of Liquizyme® used corresponded to an enzyme loading of 0.08% by weight on a “dry corn basis”. The amount of Spirizyme® used corresponded to an enzyme loading of 0.2% by weight on a “dry corn basis”. This criterion is defined as the amount of ground corn required to obtain the amount of undissolved solids retained in the washed cake, assuming that all starch is hydrolyzed to soluble oligosaccharides. Is done. Most of the undissolved solids retained in the washed cake are considered to be non-fermentable parts of corn starch. These enzyme loadings are at least four times higher than required to obtain complete liquefaction and saccharification at 26% corn loading. Enzymes were used in great excess to ensure complete hydrolysis of the residual starch in the solid and complete conversion of the oligosaccharide to glucose.

The level of non-hydrolyzed starch in the solid present in the washed wet cake from the batch 1 mash was measured according to the following protocol:
Α-Amylase was diluted with tap water (0.1297 g enzyme in 10.3607 g water).
Glucoamylase was diluted with tap water (0.3212 g enzyme in 15.6054 g water).
-132 g of tap water was added to the kettle.
-The stirrer was switched on.
-Charged 68 g of washed wet cake (TS = 21.63% wt) produced from liquefaction batch 1.
Heated to 55 ° C. with stirring.
Adjust the pH to 5.5 using dilute H ₂ SO ₄ or NaOH.
-An α-amylase solution (3.4992 g) was added.
-A glucoamylase solution (5.319 g) was added.
-It was operated at 55 ° C for 24 hours while controlling the pH to 5.5, and a slurry for glucose was collected periodically.

The level of non-hydrolyzed starch in the solid present in the washed wet cake from batch 2 was measured according to the following protocol.
Α-Amylase was diluted with tap water (0.2384 g enzyme in 11.709 g water).
Glucoamylase was diluted with tap water (0.3509 g enzyme in 17.5538 g water).
-154.3 g of tap water was added to the kettle.
-The stirrer was switched on.
-70.7 g of washed wet cake produced from liquefaction batch 1 (TS = 23.66 wt%) was charged.
Heated to 55 ° C. with stirring.
Adjust the pH to 5.5 using dilute H ₂ SO ₄ or NaOH.
-An α-amylase solution (2.393 g) was added.
-A glucoamylase solution (5.9701 g) was added.
-It was operated at 55 ° C for 24 hours while controlling the pH to 5.5, and a slurry for glucose was collected periodically.

Comparison of the results of liquefaction / saccharification of the washed wet cake As described above, the washed wet cake from batches 1 and 2 is reslurried in water to hydrolyze any remaining starch in the solid. In order to convert it to glucose, both very excess α-amylase and glucoamylase were added to the slurry. FIG. 15 shows the concentration of glucose in the aqueous phase of the slurry as a function of time.

  The glucose concentration increased with time and stabilized at a maximum at about 24 hours for both reactions. The slight decrease in glucose between 24 hours and 48 hours may be due to microbial contamination; therefore, the residual unhydrolyzed starch in the washed wet cake solids using the maximum level of glucose reached in each system. The level of was calculated. The maximum level of glucose reached by reacting the washed wet cake obtained from liquefaction of Batch 1 (in the presence of α-amylase and glucoamylase) was 4.48 g / L. In comparison, the maximum level of glucose reached by reacting the washed wet cake obtained from batch 2 liquefaction (in the presence of α-amylase and glucoamylase) was 2.39 g / L.

Glucose data obtained from washed wet cakes obtained from corresponding batches of mash, levels of residual non-hydrolyzed starch (not hydrolyzed during liquefaction) in non-dissolved solids in liquefied mash Calculated based on
Liquefaction batch 1: Residual non-hydrolyzed starch in solids represents 2.1% of the total starch in corn fed to liquefaction. This suggests that 2.1% of the starch in the corn was not hydrolyzed under the batch 1 liquefaction conditions. During liquefaction batch 1, no intermediate high temperature (“cooking”) step was performed.
Liquefaction batch 2: Residual non-hydrolyzed starch in solids represents 1.1% of total starch in corn fed to liquefaction. This suggests that 1.1% of the starch in the corn was not hydrolyzed under the batch 2 liquefaction conditions. During liquefaction batch 2, a high temperature (“cooking”) stage was performed.

  This example shows that the addition of a high temperature “cooking” stage at some point during liquefaction can result in higher starch conversion. This results in less residual non-hydrolyzed starch remaining in the undissolved solids in the liquefied corn mash, and less starch loss in the process when the undissolved solids are removed from the mash prior to liquefaction. .

Example 12
Effect of high temperature stage during liquefaction on the conversion of starch in corn solids to soluble (liquefied) starch. It was prepared at 85 ° C using Novozymes (α-amylase manufactured by Franklinton, NC). Both batches were run for more than 2 hours at 85 ° C. However, a “cooking” period was added in the middle of batch 4. The temperature distribution in batch 4 was 85 ° C. for about 30 minutes, the temperature was raised from 85 ° C. to 121 ° C., held at 121 ° C. for about 30 minutes, cooled to 85 ° C. and continued to liquefy for an additional 90 minutes. The ground corn used in both batches was the same as in Example 1. A corn loading of 26% by weight (dry corn basis) was used for both batches. The total amount of enzyme used in both runs corresponded to 0.04 wt% (dry corn basis). The pH was controlled at 5.8 during both liquefaction runs. Batch 3 liquefaction was performed in a 1 L glass coated resin kettle and Batch 4 liquefaction was performed in a 200 L stainless steel fermenter. Both reactors were equipped with mechanical stirring, temperature control, and pH control.

  The experimental conditions for this example were similar to those described for Example 9 with the following differences:

  For production of batch 3 liquefied corn mash: 0.211 g α-amylase was diluted with 10.403 g tap water. The first charge of α-amylase solution was 3.556 g. The second charge of α-amylase solution was 1.755 g and the reaction continued to run at 85 ° C. for an additional 90 minutes.

  In the production of batch 4 liquefied corn mash: 22 g of α-amylase was diluted with 2 kg of tap water, 147.9 kg of tap water was charged to the fermentor, and 61.8 kg of ground corn was charged. . The first charge of α-amylase solution is 1.0 kg, the reaction is heated to 85 ° C. and held at 85 ° C. for 30 minutes, then the reaction is heated to 121 ° C. and at 121 ° C. for 30 minutes. Retained. The second charge of α-amylase solution was 1 kg and the reaction continued to run at 85 ° C. for an additional 90 minutes.

Removal of undissolved solids from liquefied mash and washing of wet cake with water to remove soluble starch Liquefied mash by centrifuging the batch in a high-capacity centrifuge at 5000 rpm for 15 minutes at room temperature Most of the solids were removed from both batches. Centrifugation of 500.1 g mash from Batch 3 gave 337.2 g of centrifuge and 162.9 g of wet cake. Centrifugation of 509.7 g mash from batch 4 yielded 346.3 g of centrifuge and 163.4 g of wet cake. The wet cake recovered from each batch of liquefied mash was washed 5 times with tap water to remove substantially all of the soluble starch retained in the cake. Washing was done in the same bottle used to centrifuge the original mash to prevent the cake from moving between the containers. In each washing step, the cake was mixed with water and the resulting washing slurry was centrifuged at room temperature (5000 rpm for 15 minutes). This was done for all five washing steps performed on wet cakes recovered from both batches of mash. Approximately 164 g of water was used for each of the 5 washes of the wet cake from Batch 3, which totaled 819.8 g of water used to wash the wet cake from Batch 3. Approximately 400 g of water was used for each of the 5 washes of the wet cake from Batch 4, which resulted in a total of 2000 g of water used to wash the wet cake from Batch 4. The total wash centrifuge recovered from all 5 water washes of the wet cake from Batch 3 was 879.5 g. The total wash centrifuge recovered from all 5 water washes of the wet cake from Batch 4 was 2048.8 g. The final washed wet cake recovered from batch 3 was 103.2 g and the final washed wet cake recovered from batch 4 was 114.6 g. The final washed wet cake obtained from each batch was substantially free of soluble starch; therefore, the liquid retained in each cake was primarily water. The total solids (TS) of the wet cake was measured using a moisture balance. The total solids of the wet cake from batch 3 was 21.88 wt% and the TS of the wet cake from batch 4 was 18.1 wt%.

  The experimental conditions for this example were similar to those described for Example 9 with the following differences:

  For batch 3 liquefaction / saccharification of the washed wet cake to measure the level of unhydrolyzed starch remaining in the undissolved solids after liquefaction: 68 g of washed from the liquefaction of batch 3 Wet cake was added (TS = 21.88% by weight). 3.4984 g of α-amylase solution and 5.3042 g of glucoamylase were added. The reaction was carried out at 55 ° C. for 47 hours while controlling the pH at 5.5, and a slurry for glucose was collected periodically.

  For batch 4 liquefaction / saccharification of the washed wet cake to measure the level of unhydrolyzed starch remaining in the undissolved solids after liquefaction: 0.1663 g α-amylase And 0.213 g of glucoamylase was diluted with 20.8002 g of tap water. 117.8 g of tap water was charged into the kettle. 82.24 g of washed wet cake produced from liquefaction of batch 4 was charged (TS = 18.1 wt%). 3.4952 g of α-amylase solution and 10.510 g of glucoamylase were added. The reaction was carried out at 55 ° C. for 50 hours while controlling the pH at 5.5, and a slurry for glucose was collected periodically.

Comparison of the results of liquefaction / saccharification of the washed wet cake As described above, the washed wet cake from batches 3 and 4 was reslurried in water to hydrolyze any remaining starch in the solid. In order to convert it to glucose, both very excess α-amylase and glucoamylase were added to the slurry. FIG. 16 shows the concentration of glucose in the aqueous phase of the slurry as a function of time.

  The glucose concentration increased with time and stabilized at a maximum at about 26 hours for the washed wet cake from Batch 3. In batch 4 washed wet cake, the glucose concentration continued to increase slightly from 24 to 47 hours. It is assumed that the glucose concentration measured at 47 hours for the batch 4 wet cake is approximately equal to the maximum value. The maximum level of glucose reached by reacting the washed wet cake obtained from batch 3 liquefaction (in the presence of α-amylase and glucoamylase) was 8.33 g / L. In comparison, the maximum level of glucose reached by reacting the washed wet cake obtained from the liquefaction of batch 4 (in the presence of α-amylase and glucoamylase) was 4.92 g / L.

The level of residual non-hydrolyzed starch (which was not hydrolyzed during liquefaction) in non-dissolved solids in the liquefied mash was adjusted to the corresponding batch of mash (in the presence of excess α-amylase and glucoamylase). Was calculated based on glucose data obtained by “hydrolyzing” the washed wet cake obtained from
Liquefaction batch 3: Residual non-hydrolyzed starch in solids represents 3.8% of total starch in corn fed to liquefaction. This suggests that 3.8% of the starch in the corn was not hydrolyzed under the batch 3 liquefaction conditions. During liquefaction batch 3, no intermediate high temperature (“cooking”) step was performed.
Liquefaction batch 4: Residual non-hydrolyzed starch in solids represents 2.2% of total starch in corn fed to liquefaction. This suggests that 2.2% of the starch in the corn was not hydrolyzed under the batch 4 liquefaction conditions. During liquefaction batch 4, a high temperature ("cooking") stage was performed.

  This example shows that the addition of a high temperature “cooking” stage at some point during liquefaction can result in higher starch conversion. This results in less residual non-hydrolyzed starch remaining in the undissolved solids in the liquefied corn mash, and less starch loss in the process when the undissolved solids are removed from the mash prior to liquefaction. .

Overview and Comparison of Examples 11 and 12 Liquefaction conditions can affect the conversion of starch in corn solids to soluble (liquefied) starch. Possible liquefaction conditions that may affect the conversion of starch in ground corn to soluble starch are temperature, enzyme (α-amylase) loading, and +/- high temperature (“cooking”) stages being liquefied. Whether it is done at some point. Examples 11 and 12 showed that implementation of the high temperature (“cooking”) stage at some point during liquefaction resulted in a higher conversion of starch in corn solids to soluble (liquefied) starch. The high temperature stage during liquefaction described in Examples 11 and 12 consists of increasing the liquefaction temperature at some point after liquefaction has started, maintaining it at a higher temperature for a period of time, and then adjusting the temperature to And reducing to the original value again to complete the liquefaction.

  The liquefaction reaction compared in Example 11 was performed with a different enzyme loading than the reaction compared in Example 12. These examples show the effect of two main liquefaction conditions on starch conversion: (1) enzyme loading and (2) +/− whether a high temperature step is applied at some point during liquefaction. .

  The conditions used to prepare the four batches of liquefied corn mash described in Examples 11 and 12 are summarized below and in Table 9.

Conditions common to all batches:
・ Liquefaction temperature -85 ℃
-Total time at liquefaction temperature-about 2 hours-Screen size used to grind corn-1mm
・ PH-5.8
・ Dry corn filling -26%
Α-Amylase-Liquizyme® SC DS (Novozymes, Franklinton, NC).

  The temperature distribution for batches 2 and 4 (all values are approximate) were: 85 ° C. for 30 minutes, 30 minute high temperature phase, 85 ° C. for 90 minutes. Half of the enzyme was added before the first 85 ° C period and half after the high temperature phase over the last 85 ° C period.

  FIG. 17 shows the effect of enzyme loading and whether the +/− hot stage was applied at some point during liquefaction on starch conversion. The level of residual non-hydrolyzed starch in the solid is starch that was not hydrolyzed at the intended liquefaction conditions. FIG. 17 shows that the level of non-hydrolyzed starch in the solid was reduced by almost half by the application of a high temperature (“cooking”) stage at some point during liquefaction. This was shown with two different enzyme loadings. The data in FIG. 17 shows that doubling the enzyme loading, whether or not the high temperature step was applied during liquefaction, almost halved the level of unhydrolyzed starch remaining in the solid. Show. These examples show that the implementation of liquefaction with higher enzyme (α-amylase) loading and / or the addition of a high temperature (“cooking”) stage at some point during the reaction is present in the liquefied corn mash. It shows that residual unhydrolyzed starch in dissolved solids can be significantly reduced and that starch loss in the process can be reduced when undissolved solids are removed from the mash before liquefaction. Any residual starch in the solid after liquefaction has no opportunity to hydrolyze during fermentation in a process where the solid is removed prior to fermentation.

Example 13
Screening Separation of Starch and Insoluble After Enzymatic Digestion at 85 ° C. Mash (301 grams) prepared for each method described in Example 1 is used with drops of NaOH solution if adjustment is required. The pH was maintained at 5.8 and was treated with a manufacturer-specified dose of approximately 0.064 grams of Liquizyme® α-amylase enzyme (Novozyme, Franklinton, NC) and held at 85 ° C. for 5 hours. The product was refrigerated.

  The refrigerated product was warmed to about 50 ° C. and 48 g was poured into a filter assembly containing a 100 mesh screen and connected to a household vacuum source at -15 inches of mercury (in Hg) to -20 inches of mercury. The sieve pan had an exposed sieve surface area of 44 cm 2 and was sealed in a plastic filter housing provided by Nalgene® (Thermo Fisher Scientific, Rochester, NY). When the slurry was filtered, a wet cake was formed on the sieve, and 40.4 g of a yellow turbid filtrate was formed in the receiver bottle. The wet cake was immediately washed with water in place and then interrupted while the vacuum source continued to draw free moisture through the final washed cake. Filtration was stopped when dripping from the bottom of the filter stopped. An additional 28.5 g of wash filtrate was collected over three stages, where the final stage filtrate showed the least color and turbidity. The final wet cake mass of 7.6 g was air dried to 2.1 g over 24 hours at room temperature. 2.1 g was found to contain 7.73% water after drying with a heat lamp. The vacuum filtration of this experiment produced a wet cake containing a total of 25% dry solids.

  A sample of the filtrate was mixed vigorously with oleyl alcohol at room temperature and allowed to settle. The interface was allowed to recover for about 15 minutes, but a cloudy rag layer remained.

  1 g of> 99.99% (based on trace metals) iodine, 2 g of ReagentPlus® grade (> 99%) potassium iodide (both from Sigma-Aldrich (St. Louis, MO)), and 1 A drop volume of 17 g of Lugol's solution (starch indicator) consisting of domestic deionized water was added to a sample of the filtrate, a re-slurried, dried cake solid and water control sample. The filtrate turned dark blue or purple, the solid slurry turned very dark blue, and the water color became light amber. Darker than amber indicates the presence of oligosaccharides longer than 12 units long.

  This experiment showed that most of the suspended solids could be separated from the starch solution prepared as described above at a moderate rate in a 100 mesh screen and that the starch remained with the filter cake solids. . This indicates incomplete washing of the cake leaving a portion of the hydrolyzed starch.

  The experiment was repeated with 156 grams of mash on a 63 mesh diameter 100 mesh screen. The maximum temperature was 102 ° C., the enzyme was Spezyme®, and the slurry was held above 85 ° C. for 3 hours. The sieving speed was measured and determined to be less than 0.004 gallons per minute per square foot of sieving area.

Example 14
Screening Separation of Starch and Insoluble After Enzymatic Digestion at 115 ° C. Household deionized water (200 g) is charged into an empty Parr Model 4635 1 liter pressure vessel (Moline, IL) at a temperature of 85 ° C. Until heated. Water was stirred using a magnetic stir bar. Dry ground corn (90 g) prepared as described in Example 1 was added with a spoon. The pH was raised from 5.2 to about 6.0 using a stock solution of aqueous ammonia. About 0.064 grams of Liquizyme® solution was added with a small calibrated pipette. The pressure vessel lid was closed and the vessel was pressurized to 50 psig with household nitrogen. The stirred mixture was heated to 110 ° C. within 6 minutes and held at 106-116 ° C. for a total of 20 minutes. Heating was suppressed, pressure was reduced, and the container was opened. An additional 0.064 g of Liquizyme® was added and the temperature was held at 63-75 ° C. for an additional 142 minutes.

  A small amount of slurry was removed from the Parr vessel and screened by gravity through a stack of 100, 140, and 170 mesh screens. The solid remained only on the 100 mesh screen.

  A portion of the slurry, approximately 40%, was transferred hot to the top of the 75 and 100 mesh dish double sieve assembly of 75 millimeters in diameter. Some gravity filtration was performed. A vacuum of −15 to −20 inches of mercury was drawn into the filtrate receiver to set a constant filtration. The filtrate was yellow and cloudy, but the dispersion was stable. The cake surface was exposed for less than 5 minutes. The cake was washed with a spray of deionized water for 2-3 minutes and repeated with different receptacles until the turbidity of the filtrate was constant (5 sprays total). Examination of the sieve resulted in the conclusion that all solids were on a 100 mesh screen and no solids were on 200 mesh. The wet cake was 5 mm thick. When the mass of the wet cake was measured, it was 18.9 g, and the total mass of the filtrate was 192 g.

  The remaining mass of the slurry was transferred to a filter assembly equipped with a 100 mesh screen in place at 65 ° C. and filtered for 5-10 minutes. The cake was washed with a spray of deionized water for 3-4 minutes and repeated with different receptacles until the filtrate was turbid (8 sprays total). The vacuum was continued until no drops were observed from the bottom of the filter. The wet cake had a thickness of 8 mm, a diameter of 75 mm, and a mass of 36.6 g. The total weight of the filtrate was 261 g.

  Three containers were tested for starch for each of the above methods. One container contained water and the other two containers contained a sample of wet cake slurried in deionized water. The color of all containers changed to yellow amber. This is taken to mean that the filter cake was washed to remove starch oligosaccharides. These solids were accurately analyzed using long term liquefaction and subsequent saccharification to determine that on a glucose basis, the wet cake contained less than 0.2% of the total starch in the original corn. confirmed.

  A sample of the filtrate was combined with oleyl alcohol in a container and mixed vigorously and allowed to settle. A clear oil layer was obtained quickly, with almost no lag layer at the interface and was well defined. This example shows that in a process where corn mash is heated to hot water conditions of about 110 ° C. for 20 minutes cooking and further liquefied for more than 2 hours at 85 ° C. before being filtered and washed, To contain substantially all of the starch supplied to Further, less interference between oleyl alcohol and impurities contained in the filtrate is not observed.

  This experiment was repeated with 247 grams of mash in an 80 mesh screen with a diameter of 75 mm. The maximum cooking temperature was 115 ° C., the enzyme was Liquizyme®, and the slurry was held at 85 ° C. or higher for 3 hours. The sieving speed was measured and determined to be greater than 0.1 gallon per minute per square foot of sieving area.

Example 15
This example showed the removal of solids from the distillation waste and extraction with a desolvator to recover fatty acids, esters, and triglycerides from the solids. During the fermentation, the solid is separated from the total distillation effluent and fed to the desolventizer where the solid is contacted with 1.1 ton / hour of steam. The total distillation waste liquid wet cake (extractor raw material), the solvent, the extractor miscella, and the flow rate of the discharged solids of the extractor are as shown in Table 10. The unit of the values in the table is short ton / hour.

  The solid leaving the desolvator is fed to the dryer. The vapor exiting the desolvator contains 0.55 ton / hour hexane and 1.102 ton / hour water. This stream is condensed and fed to the decanter. The water-enriched phase exiting the decanter contains about 360 ppm of hexane. This stream is fed to a distillation column where hexane is removed from the water-enriched stream. The hexane enriched stream exiting the top of the distillation column is condensed and fed to the decanter. The organic enriched stream leaving the decanter is fed to a distillation column. Steam (11.02 tons / hour) is fed to the bottom of the distillation column. Table 11 shows the composition of the top and bottom products of this tower. The unit of the values in the table is ton / hour.

Example 16
Byproduct Recovery This example illustrates the recovery of byproducts from mash. The conditions described in Example 6 except that a three-phase centrifuge (Flottweg Tricanter® Z23-4 bowl diameter, 230 mm, length to diameter ratio 4: 1) was used under these conditions. Under separate corn oil from mash:
・ Bowl speed: 5000rpm
・ Differential speed: 10rpm
・ Supply speed: 3gpm
・ Phase separator disk: 138mm
・ Impeller setting: 144 mm.

  The separated corn oil has 81% triglycerides, 6% free fatty acids, 4% diglycerides, and a total of 5% phospholipids and monoglycerides as measured by gas chromatography and thin layer chromatography. (See, eg, US Patent Application Publication No. 2012/0164302).

  The solid separated from the mash under the above conditions has a moisture content of 58% as measured by weight loss on drying, 1.2% triglyceride and 0.27 as measured by gas chromatography. % Free fatty acids (see, for example, US 2012/0164302).

  The composition of the solids separated from the total distillation effluent in Table 14, the oil extracted during the evaporator stage, the by-product extractant and the condensed distillation solubles (CDS) is shown in Table 12 The calculation was made on the assumption of Table 13 (separation in a three-phase centrifuge). The values in Table 11 were obtained from an Aspen Plus® model (Aspen Technology, Inc., Burlington, Mass.). This model assumes that no corn oil has been extracted from the mash. It is estimated that the protein content on a dry basis of cells, lysed solids and suspended solids is about 50%, 22% and 35.5%, respectively. The composition of the byproduct extractant is estimated to be 70.7% fatty acid and 29.3% fatty acid isobutyl ester on a dry basis.

Example 17
Removal of corn oil from liquefied corn mash This example illustrates the use of a three-phase centrifuge to remove corn oil from liquefied corn mash. Whole grain corn typically contains about 3-6% by weight corn oil, most of which is present in the germ. Corn oil is released from the germ during dry grinding and liquefaction. As a result, corn mash contains free corn oil.

  For example, a liquefied corn mash was produced using a standard continuous liquefaction process used in the dry ground corn-ethanol process. The ground corn contained 4.16 wt% corn oil (dry corn basis) and had a moisture content of 14.7 wt%. Ground corn and water were fed to the slurry tank at 10.2 lb / min and 17.0 lb / min, respectively, resulting in a dry corn charge of 32 wt%. α-Amylase was fed to the slurry tank at a rate corresponding to an enzyme loading of about 0.025% by weight on a dry corn basis. Both the slurry tank and the liquefaction tank were operated at 85 ° C. and a pH of 5.8. The total residence time at 85 ° C. was about 2 hours. Mash was produced at a rate of about 3 gpm, which contained about 1.3 wt% corn oil on a wet basis. Some of this oil was present as free oil and some was present as undissolved solids. This corresponds to a total corn oil content of mash that is approximately 2.0 pounds of corn oil per bushel of corn. Total solids (TS) in the mash was 32% by weight and total suspended solids (TSS) was 7.7% by weight.

The liquefied corn mash was fed to a three-phase centrifuge (Model Z23-4 / 441, Flottweg Tricanter®, Flottweg AG, Vilsibiburg, Germany) at a rate of about 3 gpm. The feed temperature was about 80 ° C. The mash was separated into three streams: (1) corn oil, (2) an oligosaccharide (liquefied starch) aqueous solution, and (3) a wet cake. The operating conditions of Tricanter® were as follows:
・ Bowl speed: 5000rpm
・ Gravity: about 4000g's
・ Differential speed: 10rpm
・ Impeller setting: about 145
-Phase separator disk: about 138 mm.

  Table 15 summarizes the data (flow rate, density, solids content, and corn oil content) measured for the feed stream and the three outlet streams from Tricanter®.

  Corn oil removed from the mash by Tricanter® accounted for 39% of the total corn oil in the mash feed. Corn oil removal rate was equal to about 0.8 pounds per corn bushel. The corn oil separated and recovered from the liquefied corn mash contained about 85% by weight glycerides.

In a process where about 0.8 pounds of corn oil is removed per corn bushel, the mash flow rate will be reduced by 3.9 gallons / minute:

  In a production plant where the total mash flow to fermentation is about 700 gpm, the oil removed will account for about 0.55% of the total mash flow. Assuming that the production plant increases the throughput proportionally to take advantage of the additional capacity, the increase in annual production by 0.55% means that the 56MMGPY plant has an additional 310,000 gallons of ethanol. Means that it will generate

Example 18
Corn oil removal from liquefied corn mash-feed rate adjustment In this example, liquefied corn mash was fed to a three-phase centrifuge at a feed rate of 1 gpm. For example, a liquefied corn mash was produced using a standard continuous liquefaction process used in the dry ground corn-ethanol process. The ground corn contained 4.16 wt% corn oil (dry corn basis) and had a moisture content of 14.7 wt%. Ground corn and water were fed to the slurry tank at 8.2 lb / min and 19.0 lb / min, respectively, resulting in a dry corn fill of about 26 wt%. α-Amylase was fed to the slurry tank at a rate of 50 g / hr corresponding to an enzyme loading of about 0.026 wt% on a dry corn basis. Both the slurry tank and the liquefaction tank were operated at 85 ° C. and a pH of 5.8. A jet cooker was not used. The total residence time at 85 ° C. was about 2 hours. Mash was produced at a rate of about 3 gpm and was inventoried in a 1500 gallon tank for centrifuge testing. The mash contained about 1.1 wt% corn oil on a wet basis. Some of this oil was present as free oil and some was present as undissolved solids. This corresponds to a total corn oil content of mash that is approximately 2.0 pounds of corn oil per bushel of corn. TS in the mash was 25.6% by weight and TSS was 5.3% by weight.

Liquefied corn mash was fed at a rate of about 1 gpm from a feed tank to a three-phase centrifuge (Model Z23-3, Flottweg Tricanter®, Flottweg AG, Vilsibiburg, Germany). The feed temperature was about 80 ° C. The mash was separated into three streams: (1) corn oil, (2) an oligosaccharide (liquefied starch) aqueous solution, and (3) a wet cake. The operating conditions of Tricanter® were as follows:
・ Bowl speed: 5000rpm
・ Gravity: about 4000g's
・ Differential speed: 12rpm
・ Impeller setting: about 156
-Phase separator disk: about 140 mm.

  Table 16 summarizes the data (flow rate, density, solids content, and corn oil content) measured for the feed stream and the three outlet streams from Tricanter®. The mass balance quality of corn oil was 102% and the mass balance quality of the total solids was 105%.

  Corn oil removed from the mash by Tricanter® accounted for 17% of the total corn oil in the mash feed. The corn oil removal rate was equal to about 0.34 pounds per corn bushel. The corn oil separated and recovered from the liquefied corn mash contained about 81.4 wt% glycerides and 8.3 wt% free fatty acids.

Example 19
Corn oil removal from liquefied corn mash-feed rate adjustment In this example, liquefied corn mash was fed to a three-phase centrifuge at a feed rate of 10.1 gpm. For example, a liquefied corn mash was produced using a standard continuous liquefaction process used in the dry ground corn-ethanol process. The ground corn contained 4.16 wt% corn oil (dry corn basis) and had a moisture content of 14.7 wt%. Ground corn and water were fed to the slurry tank at 8.2 lb / min and 19.0 lb / min, respectively, resulting in a dry corn fill of about 26 wt%. α-Amylase was fed to the slurry tank at a rate of 50 g / hr corresponding to an enzyme loading of about 0.026 wt% on a dry corn basis. Both the slurry tank and the liquefaction tank were operated at 85 ° C. and a pH of 5.8. A jet cooker was not used. The total residence time at 85 ° C. was about 2 hours. Mash was produced at a rate of about 3 gpm and was stocked in a 1500 gallon tank for centrifuge testing. The mash contained about 1.1 wt% corn oil on a wet basis. Some of this oil was present as free oil and some was present as undissolved solids. This corresponds to a total corn oil content of mash that is approximately 2.0 pounds of corn oil per bushel of corn. TS in the mash was 26.2% by weight and TSS was 6.7% by weight.

Liquefied corn mash was fed at a rate of approximately 10.1 gpm from a feed tank to a three-phase centrifuge (Model Z23-4 / 441, Flottweg Tricanter®, Flottweg AG, Vilsibiburg, Germany). The feed temperature was about 80 ° C. The mash was separated into three streams: (1) corn oil, (2) an oligosaccharide (liquefied starch) aqueous solution, and (3) a wet cake. The operating conditions of Tricanter® were as follows:
・ Bowl speed: 5000rpm
・ Gravity: about 4000g's
・ Differential speed: 20rpm
・ Impeller setting: about 148
-Phase separator disk: about 138 mm.

  Table 17 summarizes the data (flow rate, density, solids content, and corn oil content) measured for the feed stream and the three outlet streams from Tricanter®. The mass balance quality of corn oil was 95%.

  Corn oil removed from the mash by Tricanter® accounted for 18% of the total corn oil in the mash feed. The corn oil removal rate was equal to about 0.36 pounds per corn bushel. The corn oil separated and recovered from the liquefied corn mash contained about 81.4 wt% glycerides and 8.3 wt% free fatty acids.

Example 20
Effect of liquefied corn mash pH on corn oil recovery from mash Liquefied corn mash was produced using a standard continuous liquefaction process commonly used in dry ground corn-ethanol processes. The ground corn contained 4.6 wt% corn oil (based on dry corn) and had a moisture content of 12.5 wt%. Ground corn and water were fed into the slurry tank at a rate to produce 3 gpm corn mash with a dry corn charge of 25.9 wt%. The slurry tank was operated at 85 ° C. with a residence time of 30 minutes. The slurry was then heated to 105 ° C. with live steam in a jet cooker and held at that temperature for about 30 minutes. After exiting the holding tube, the slurry was fed into a liquefaction tank operating at 85 ° C. with a residence time of 90 minutes. α-Amylase (Spezyme® ALPHA, Genencor®, Palo Alto, Calif.) was continuously added to the process at a rate corresponding to a total enzyme loading of 0.04 wt% enzyme on dry corn basis. Supplied. Forty percent (40%) of the total enzyme was added to the slurry tank and 60% was added to the liquefaction tank. Both the slurry tank and the liquefaction tank were operated at a pH of 5.8. Mash was produced at a rate of about 3 gpm and was stocked in a 1500 gallon tank for centrifuge testing. The liquefied corn mash contained about 1.12% by weight corn oil on a wet basis. This corresponds to a total corn oil content of mash of approximately 2.2 pounds of corn oil per bushel of corn. Some of this oil was present as free oil; some was still in undissolved solids. The ratio of glycerides to free fatty acids in corn oil in mash was about 7.6-1. Total solids (TS) in the mash was 25.9% by weight and total suspended solids (TSS) was 4.7% by weight. The final mash DE (dextrose equivalent) and pH were 15.9 and 5.75, respectively. The density of the mash was 1.08 g / mL.

  Three different centrifuges (Model Z23-4 / 441, Flottweg Tricanter®, Flottweg AG, Vilbiburg) at three different feed flow rates: 1.24 gal / min, 5.1 gal / min and 10 gal / min , Germany) was used to separate the liquefied mash. The feed temperature was about 80 ° C. The mash was separated into three streams: (1) corn oil, (2) an oligosaccharide (liquefied starch) aqueous solution, and (3) a wet cake. The bowl speed was held constant at about 5000 rpm (about 4000 g's). Table 18 compares corn oil recovery as a function of mash feed rate to Tricanter® at a mash pH of 5.8. The data shown in Table 18 shows that there is an impact of the feed rate to Tricanter® on the recovery of corn oil at pH = 5.8.

  Corn oil recovery is based on the total oil contained in the mash (both free oil and oil in solids). The mash fed to Tricanter® contained 1.1-1.2% by weight corn oil (including free oil and oil in solids).

  The data in Table 18 shows that there is an impact of mash feed rate on corn oil recovery (in the conditions tested). Table 19 summarizes the amount of oil in the aqueous centrifuge, the aqueous phase in the oil centrifuge, and the solids in the oil centrifuge for the three conditions tested.

  The data in Table 19 shows that the recovered corn oil was fairly clean because it contained very little aqueous phase and very little solids. The corn oil separated and recovered from the liquefied corn mash contained about 85.1% by weight glycerides and 8.0% by weight free fatty acids. The rest were solids, aqueous phases, and other extractables (eg, phospholipids, sterols, etc.).

  The pH of the liquefied corn mash remaining in the Tricanter® feed tank was lowered to about 3. Using two-phase centrifuges (Model Z23-4 / 441, Flottweg Tricanter®, Flottweg AG, Vilsibiburg, Germany) at two different feed flow rates: 1.3 gal / min and 5 gal / min, The acidic mash was isolated. The feed temperature was about 80 ° C. The mash was separated into three streams: (1) corn oil, (2) an oligosaccharide (liquefied starch) aqueous solution, and (3) a wet cake. The bowl speed was held constant at about 5000 rpm (about 4000 g's). Table 20 compares corn oil recovery as a function of mash feed rate to Tricanter® at a mash pH of 3.0.

  Mash was produced at pH = 5.8 and then the final mash pH was lowered to 3 before feeding it to the centrifuge. Corn oil recovery is based on the total oil contained in the mash (both free oil and oil in solids). The mash fed to Tricanter® contained about 0.9% by weight corn oil (including free oil and oil in solids). The total solids of the mash fed to Tricanter® was 27.1% by weight and the total suspended solids of the mash was 5.5% by weight.

  Table 21 summarizes the amount of oil phase in the aqueous centrifuge, aqueous phase in the oil centrifuge, and solids in the oil centrifuge for the two conditions tested. The data in Table 21 shows that the recovered corn oil was fairly clean because it contained very little aqueous phase and very little solids.

  A comparison of the results of Test A (Table 18) to Test D (Table 20) and a comparison of the results of Test B (Table 19) to Test E (Table 21) was performed on a mash against corn oil recovery using Tricanter®. The effect of pH is shown. The data suggests that lowering the mash pH prior to separating the mash using Tricanter® results in higher corn oil recovery. These comparisons are shown in Table 22.

  The percentages shown in Table 22 are the recoveries of corn oil based on the total oil contained in the mash (both free oil and oil in solids). The composition of corn oil in mash fed to Tricanter® was 0.9% to 1.2% by weight of corn oil (in free oil and solids) for all tests described in this example. Of oil). The Tricanter® was operated at 5000 rpm (about 4000 G's), and the differential speed and impeller settings were 5-10 rpm and 145 mm, respectively.

Example 21
Recovery of corn oil and solids from corn mash A liquefied corn mash was produced using a standard continuous liquefaction process used in the dry ground corn-ethanol process at 30-31% by weight on a dry corn basis. . Recycled water consisting of cooking water and backflow with total solids (TS) increased to about 33% by weight was used. α-Amylase (Spezyme® RSL, Genencor®, Palo Alto, Calif.) was added to a slurry tank (85 ° C.) at a rate corresponding to an enzyme charge on a dry corn basis of about 0.02 wt%. PH about 5.8, residence time of 30 minutes). Using a jet cooker, the temperature was increased from 105 to 110 ° C. with a cooking time of 18 minutes. The liquefaction tank was operated at 85 ° C. at a pH of about 5.8. Spezyme® RSL (Genencor®, Palo Alto, Calif.) Is also added to the liquefaction tank at a rate corresponding to an enzyme charge on a dry corn basis of about 0.005 wt%. The residence time was about 90 minutes. The mash side stream was collected from the liquefaction tank and transferred to a small dilution tank, where process condensate was added to obtain the desired dilution. The original mash contained about 1.55 wt% corn oil on a wet basis. Some of this oil was present as free oil and some was present as undissolved solids. This corresponds to the total corn oil content of the original mash, which is approximately 3.0 pounds of corn oil per corn bushel. TS in the original mash was 33.2 wt% and total suspended solids (TSS) was 6.5 wt%. Dilution with process condensate reduced TS to about 27 wt%, TSS to about 5.5 wt%, and oil content to about 1.3 wt% (wet basis).

Liquefied corn mash was fed at a rate of 9-11 gpm from a feed tank to a three-phase centrifuge (Model Z23-4 / 441, Flottweg Tricanter®, Flottweg AG, Vilsibiburg, Germany). The feed temperature was about 85 ° C. The mash was separated into three streams: (1) corn oil, (2) an oligosaccharide (liquefied starch) aqueous solution, and (3) a wet cake. The operating conditions of the three-phase centrifuge were as follows:
・ Bowl speed: 5000rpm
・ Gravity: about 3200g
・ Differential speed: 25rpm
・ Impeller setting: See table ・ Phase separator disk: 138mm

  Table 23 summarizes the conditions and characteristics of the three-phase centrifuge after separation. The stream at both 33 wt% and 26 wt% corn loading was separated into a very clean corn oil stream and wet cake at 38-41 wt% total solids. The suspended solids concentration in the heavy phase was strongly influenced by corn loading. 33 wt% sample produced about 3.5-4 wt% centrifuge TSS, while 26 wt% TS produced about 1.7-2 wt% lower TSS centrifuge To do.

  The results are also shown in FIGS. FIG. 19A shows that the centrifuge TSS is about 3.3% at a low flow rate of about 4 gpm, and the centrifuge TSS is increased from about 4.2 to 4.7% at a flow rate of 11.5 gpm. Indicates that

  FIG. 19B shows the recovery rate of suspended solids as a function of flow rate. About 60% suspended solids were collected in the wet cake at a low flow rate of about 4 gpm. By increasing the flow rate to about 11.5 gpm, the recovery was reduced to about 40-50%.

  FIG. 19C shows the wet cake total solids as a function of flow rate. At a low flow rate of about 4 gpm, the wet cake total solids was about 41%. By increasing the flow rate to about 11.5 gpm, the wet cake total solids was reduced to about 39%.

FIG. 19D shows the effect of differential speed rpm on the total wet cake solids. The wet cake solids decreased at the increased differential rpm.

  FIG. 19E shows the effect of feed rate on corn oil recovery. At low flow rates, the oil recovery was about 48%. When the flow rate is increased to about 11.5 gpm, the oil recovery decreases to about 35%. It appears that less oil is separated from the feed stream at higher flow rates.

Example 22
Rheological properties of corn mash The viscosity of corn mash is measured using an AR-G2 rotary rheometer (TA Instruments, New Castle, DE) configured in a blade shape. A ground corn and water slurry is prepared, mixed and heated to 55 ° C. in a resin kettle. The pH is adjusted to 5.8 and an enzyme (eg, α-amylase and / or glucoamylase) is added to the slurry. The slurry is heated to 65 ° C. at a rate of 2 ° C./min. The sample is removed and transferred to a rheometer equipped with a narrow gap concentric cylinder geometry preheated to 65 ° C. Next, the temperature is increased to increase the temperature from 65 ° C. to 85 ° C. at a rate of 2 ° C./min. The temperature rise is performed at a fixed shear rate (for example, 75 to 200 s ⁻¹ ). Viscosity is measured as a function of time.

  While various embodiments of the invention have been described above, it should be understood that they have been presented by way of example only and not limitation. It will be apparent to those skilled in the relevant art that various modifications can be made in the form and details of the invention without departing from the spirit and scope of the invention. Accordingly, the breadth and scope of the present invention is not limited by any of the above-described exemplary embodiments, but is defined only in accordance with the following claims and their equivalents.

  All publications, patents and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains, and each individual publication, patent or patent application is incorporated by reference. To the same extent as specifically and individually indicated herein by reference.

Claims (47)

A method for producing a fermentation product comprising:
Providing a raw slurry comprising a fermentable carbon source, an undissolved solid, and oil;
Separating a portion of the undissolved solid and oil from the raw slurry, thereby forming an aqueous solution containing a fermentable carbon source, a wet cake containing a solid, and an oil stream;
Adding the aqueous solution to a fermentation broth containing microorganisms, thereby producing the fermentation product.   The method of claim 1, further comprising recovering the oil stream.   The method of claim 1, further comprising washing the wet cake to obtain a water stream containing carbohydrates.   4. The method of claim 3, further comprising adding the water stream of claim 3 to the fermentation broth.   The method of claim 1, wherein the aqueous solution contains no more than about 5 wt% undissolved solids.   The method of claim 1, wherein the oil is corn oil and comprises one or more of triglycerides, fatty acids, diglycerides, monoglycerides, and phospholipids.   The method of claim 1, further comprising combining a portion of the wet cake and a portion of oil to produce a wet cake comprising triglycerides, free fatty acids, diglycerides, monoglycerides, and phospholipids.   The method of claim 1, further comprising combining the aqueous solution with a portion of the wet cake to form a mixture of the aqueous solution and wet cake, and adding the mixture to the fermentation broth.   The method of claim 1, wherein the step of separating the raw slurry is a single step process.   The undissolved solid and oil are decanter bowl centrifuge, three-phase centrifuge, disc stack centrifuge, filtration centrifuge, decanter centrifuge, filtration, vacuum filtration, belt filter, membrane filtration, crossflow filtration, pressure filtration, Claims separated from the raw slurry by sieving filtration, sieving separation, grid, porous grid, flotation, hydroclone, filter press, screw press, gravity settler, vortex separator, or combinations thereof The method according to 1.   The method of claim 10, wherein one or more control parameters of the separation device are adjusted to improve separation of the raw slurry.   The method of claim 11, wherein the one or more control parameters are selected from differential speed, bowl speed, flow rate, impeller position, cough position, scroll pitch, dwell time, and discharge volume.   The method of claim 1, wherein the fermentation product is a product alcohol selected from ethanol, propanol, butanol, pentanol, hexanol, and fusel alcohol.   The method of claim 1, wherein the microorganism comprises a butanol biosynthetic pathway.   15. The method of claim 14, wherein the butanol biosynthesis pathway is a 1-butanol biosynthesis pathway, a 2-butanol biosynthesis pathway, or an isobutanol biosynthesis pathway.   The method of claim 1, wherein real time measurements are used to monitor the separation of the raw slurry.   Separation is monitored by Fourier transform infrared spectroscopy, near infrared spectroscopy, Raman spectroscopy, high pressure liquid chromatography, viscometer, density meter, tension meter, droplet size analyzer, particle analyzer, or combinations thereof The method of claim 16, wherein: a) providing a raw slurry comprising a fermentable carbon source and an undissolved solid;
b) separating at least a portion of the undissolved solid from the raw slurry, thereby producing an aqueous solution containing a fermentable carbon source and a wet cake containing the solid;
c) contacting the wet cake with a liquid to form a wet cake mixture;
d) separating at least a portion of the undissolved solid from the wet cake mixture, thereby producing a second aqueous solution comprising a fermentable carbon source and a second wet cake comprising a solid.   The method of claim 18, wherein the liquid is selected from fresh water, reflux, cooking water, process water, distilled residual water, evaporating water, or combinations thereof.   19. A method according to claim 18, wherein steps c) and d) are repeated. Providing a raw slurry comprising a fermentable carbon source, an undissolved solid, and oil;
Separating at least a portion of the oil and undissolved solids from the raw slurry, thereby producing an aqueous solution containing a fermentable carbon source, an oil stream; and a wet cake containing solids;
c) contacting the wet cake with a liquid to form a wet cake mixture;
d) separating at least a portion of the undissolved solids and oil from the wet cake mixture, whereby a second aqueous solution comprising a fermentable carbon source, a second oil stream; and a second wet cake comprising solids Generated.   The method of claim 21, wherein the step of separating the raw slurry is a single step process.   The undissolved solid and oil are decanter bowl centrifuge, three-phase centrifuge, disc stack centrifuge, filtration centrifuge, decanter centrifuge, filtration, vacuum filtration, belt filter, membrane filtration, crossflow filtration, pressure filtration, Claims separated from the raw slurry by sieving filtration, sieving separation, grid, porous grid, flotation, hydroclone, filter press, screw press, gravity settler, vortex separator, or combinations thereof The method according to 21.   24. The method of claim 23, wherein one or more control parameters of the separation device are adjusted to improve separation of the raw slurry.   25. The method of claim 24, wherein the one or more control parameters are selected from differential speed, bowl speed, flow rate, impeller position, cough position, scroll pitch, dwell time, and discharge volume.   The method of claim 21, wherein real-time measurements are used to monitor the separation of the raw slurry.   Separation is monitored by Fourier transform infrared spectroscopy, near infrared spectroscopy, Raman spectroscopy, high pressure liquid chromatography, viscometer, density meter, tension meter, droplet size analyzer, particle analyzer, or combinations thereof 27. The method of claim 26, wherein: Providing a raw slurry comprising a fermentable carbon source, an undissolved solid, and oil;
Separating said raw slurry, thereby (i) a first aqueous solution comprising a fermentable carbon source; (ii) a first wet cake comprising a solid; and (iii) an oil, solid and fermentable carbon source. Forming a stream comprising a water stream comprising;
Adding the first aqueous solution to a fermentation broth containing microorganisms, thereby producing a fermentation product. Separating said stream comprising an oil, a solid and a water stream comprising a fermentable carbon source, thereby (i) a second aqueous solution comprising a fermentable carbon source, (ii) a second wet cake comprising a solid, and 30. The method of claim 28, further comprising (iii) forming an oil stream.   30. The method of claim 29, wherein the first and second aqueous solutions are combined prior to the addition to the fermentation broth.   30. The method of claim 29, wherein the second aqueous solution further comprises an oil.   32. The method of claim 31, wherein the oil or portion thereof of the second aqueous solution is treated to produce an extractant.   35. The method of claim 32, wherein the oil is treated chemically or enzymatically.   Separation is decanter bowl centrifugation, three-phase centrifugation, disk stack centrifugation, filtration centrifugation, decanter centrifugation, filtration, vacuum filtration, belt filter, pressure filtration, sieve filtration, sieving separation, lattice, porous 30. The method of claim 28 or 29, wherein the method is performed by a mass grid, flotation method, hydroclone, filter press, screw press, gravity settler, vortex separator, or combinations thereof. Providing a raw slurry comprising a fermentable carbon source, an undissolved solid, and oil;
The raw slurry is separated, thereby forming (i) a first aqueous solution containing a fermentable carbon source and solids, (ii) a first wet cake containing solids, and (iii) a first oil stream. And a process of
Adding oil to said first aqueous solution, thereby forming an oil layer comprising a solid and a second aqueous solution comprising a fermentable carbon source.   The oil layer containing solids is separated to form a third aqueous solution containing (i) a second oil stream, (ii) a second wet cake containing solids, and (iii) a fermentable carbon source. 36. The method of claim 35.   37. The method of claim 36, wherein the second aqueous solution and the third aqueous solution are added to a fermentation broth containing microorganisms, thereby producing a fermentation product.   36. The method of claim 35, wherein the second aqueous solution and the third aqueous solution further comprise oil.   39. The second aqueous solution and the third aqueous solution are combined, and the oil or part thereof of the second aqueous solution and the third aqueous solution is treated to produce an extractant. The method described in 1.   40. The method of claim 39, wherein the oil is treated chemically or enzymatically.   Separation is decanter bowl centrifugation, three-phase centrifugation, disk stack centrifugation, filtration centrifugation, decanter centrifugation, filtration, vacuum filtration, belt filter, pressure filtration, sieve filtration, sieving separation, lattice, porous 37. The method of claim 35 or 36, wherein the method is performed by a mass grid, flotation method, hydroclone, filter press, screw press, gravity settling device, vortex separator, or combinations thereof. One or more liquefaction units configured to liquefy the raw material to produce a raw slurry,
An inlet for receiving the raw material; and an outlet for discharging the raw slurry, wherein the raw slurry includes a fermentable carbon source, oil, and undissolved solids;
One or more configured to remove the oil and undissolved solids from the raw slurry to produce an aqueous solution comprising the fermentable carbon source, an oil stream, and a wet cake comprising a portion of the undissolved solids; A separation unit, wherein the centrifuge is
An inlet for receiving the raw slurry;
A first outlet for discharging the aqueous solution;
A second outlet for discharging the wet cake; and a separation unit including a third outlet for discharging the oil. One or more cleaning systems configured to recover the fermentable carbon source from the wet cake, the cleaning system comprising:
43. The system of claim 42, comprising one or more mixing units; and one or more separation units.   The separation unit uses decanter bowl centrifuge, three-phase centrifuge, disc stack centrifuge, filtration centrifuge, decanter centrifuge, filtration, vacuum filtration, belt filter, membrane filtration, crossflow filtration, pressure filtration, sieve 45. The system of claim 43, wherein the system is selected from: filtration, sieving separation, grid, porous grid, flotation method, hydroclone, filter press, screw press, gravity settler, vortex separator, and combinations thereof. One or more fermenters configured to ferment the aqueous solution to produce product alcohol, the fermenter comprising:
44. The system of claim 43, comprising an inlet for receiving the aqueous solution and / or wet cake; and an outlet for discharging fermentation broth containing product alcohol.   43. The system of claim 42, further comprising an online measurement device.   The online measuring device is a particle size analyzer, Fourier transform infrared spectrometer, near infrared spectrometer, Raman spectrometer, high pressure liquid chromatography, viscometer, density meter, tension meter, droplet size analyzer, pH meter, dissolved 48. The system of claim 46, wherein the system is selected from: an oxygen probe, or a combination thereof.

Images