JP6653576B2 - Methods and systems for the production of fermentation products - Google Patents

Description

  This application is based on US Provisional Patent Application No. 61 / 699,976 filed on Sep. 12, 2012; US Provisional Patent Application No. 61 / 712,385 filed on Oct. 11, 2012. US Patent Application No. 13 / 828,353 filed March 14, 2013; and US Patent Application No. 13 / 836,115 filed March 15, 2013. The entire contents of each of which are incorporated herein by reference.

  The sequence listing associated with this application is submitted in electronic form via EFS-Web and is incorporated herein by reference in its entirety.

  The present invention relates to the production of fermentation products such as alcohols, including ethanol and butanol, and methods using in situ product removal methods.

  Many chemicals and consumer products can be produced using fermentation as a manufacturing process. For example, alcohols such as ethanol and butanol have various industrial and scientific uses such as fuels, reagents, and solvents. 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 perfumery industries. The production of butanol or butanol isomers from materials such as plant-derived materials can minimize the use of petrochemicals and will make progress in the art. In addition, the production of chemicals and fuels using plant-derived materials or other biomass sources will provide an environmentally friendly and sustainable alternative to petrochemical processes.

  Techniques such as genetic engineering and metabolic engineering can be used to modify microorganisms to produce specific products from plant-derived materials or other biomass sources. However, some microorganisms that produce butanol in high yields, for example, when producing butanol, also have a low butanol toxicity threshold. Removing butanol from the fermentation as it is produced is a means of addressing such low butanol toxicity thresholds. Therefore, despite the low butanol toxicity threshold of butanologenic microorganisms, there is a continuing need to develop efficient methods and systems for producing butanol in high yields.

  In situ product removal (ISPR) (also called extractive fermentation) can be used to remove butanol or other fermentation products from the fermentation as butanol or other fermentation products are produced, whereby microorganisms can be used. Butanol can be produced in high yield. One ISPR method for removing fermented alcohol described in the art is liquid-liquid extraction (see, for example, US Pat. Generally, for butanol fermentation, the fermentation broth containing the microorganism is contacted with the extractant at a time before the butanol concentration reaches, for example, toxic levels. Butanol is distributed in the extractant to reduce the concentration of butanol in the fermentation broth containing the microorganism, thereby reducing exposure of the microorganism to inhibitory butanol.

  To be technically and economically viable, liquid-liquid extraction involves contacting the extractant with the fermentation broth for efficient mass transfer of the alcohol into the extractant; the phase of the extractant from the fermentation broth Separation (during and / or after fermentation); efficient recovery and reuse of the extractant; and requiring minimal reduction in the extractant partition coefficient over extended periods of operation. The extractant can become contaminated over time each time it is reused, for example, due to the accumulation of lipids present in the biomass used as a raw material for fermentation, and this contamination can be accompanied by a decrease in the extractant's partition coefficient .

  Further, the presence of undissolved solids during the extractive fermentation can adversely affect the efficiency of alcohol production. For example, the presence of undissolved solids may reduce the mass transfer coefficient, hinder phase separation, cause oil to accumulate from the undissolved solids in the extractant, and reduce extraction efficiency over time, It may slow the detachment of the extractant droplets from the fermentation broth, reduce the fermenter volumetric efficiency, and cause the extractant to become trapped in solids and be dissolved as a soluble matter-containing dry grain distillation cake (DDGS). Removal may increase the loss of extractant.

US Patent Application Publication No. 2009/0305370

  Therefore, there is a continuing need for alternative extractive fermentation processes that can reduce the toxic effects of fermented alcohols such as butanol on microorganisms and also reduce the decrease in the partition coefficient of the extractant. The present invention fulfills the needs described herein and provides methods, processes, and systems for the fermentative production of alcohols such as ethanol and butanol.

The present invention provides a fermentation broth comprising: providing a fermentation broth comprising a microorganism that produces a fermentation product in a fermentation apparatus; contacting the fermentation broth with at least one extractant; and collecting the fermentation product. For recovering fermentation products from a fermentation product. In certain embodiments, the step of contacting the fermentation broth with at least one extractant is performed in a fermentation device, an external unit, or both. In one embodiment, the external unit is an extractor. In some embodiments, the extractor is a siphon, decanter, centrifuge, gravity settler, phase splitter, mixer settler, column extractor, centrifugal extractor, stirred extractor, hydrocyclone, spray tower, And combinations thereof. In certain embodiments, the extraction _agent, fatty _alcohols, fatty acids C 7 _{-C 22} of C 7 _{-C 22,} esters of fatty acids of C 7 _{-C 22,} fatty aldehydes of _{C 7 ~C 22, C 7 ~C} 22 Fatty amides, and mixtures thereof. In certain embodiments, the extractant is oleyl alcohol, behenyl alcohol, cetyl alcohol, lauryl alcohol, myristyl alcohol, stearyl alcohol, oleic acid, lauric acid, linoleic acid, linolenic acid, myristic acid, stearic acid, octanoic acid, decanoic acid, Undecanoic acid, methyl myristate, methyl oleate, 1-nonanol, 1-decanol, 2-undecanol, 1-nonanal, 1-undecanol, undecanal, laurinaldehyde, 2-methylundecanal, oleamide, linoleamide, palmitoamide, stearyl It is selected from amides, 2-ethyl-1-hexanol, 2-hexyl-1-decanol, 2-octyl-1-dodecanol, and mixtures thereof. In certain embodiments, a hydrophilic solute is added to the fermentation broth. In certain embodiments, the hydrophilic solute is selected from polyhydroxylated compounds, polycarboxylic acids, polyol compounds, ionic salts, and mixtures thereof. In certain embodiments, contacting the fermentation broth with at least one extractant occurs in two or more external units. In certain embodiments, contacting the fermentation broth with at least one extractant is performed in two or more fermenters. In certain embodiments, a fermentation apparatus includes an interior or device to enhance phase separation. In certain embodiments, the interior or device is selected from a coalescer, baffle, perforated plate, well, lamella separator, cone, and combinations thereof. In certain embodiments, real-time measurements are used to monitor fermentation product extraction. In certain embodiments, extraction of the fermentation product is monitored by real-time measurement of phase separation. In certain embodiments, phase separation is monitored by measuring the rate of phase separation, the droplet size of the extractant, and / or the composition of the fermentation broth. In certain embodiments, phase separation is monitored by conductivity, dielectric constant, viscoelasticity, and / or ultrasonic measurements. In certain embodiments, the step of providing a fermentation broth comprising the microorganism is performed in two or more fermenters. In certain embodiments, the fermentation product may be a product alcohol. 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 biosynthesis pathway is the 1-butanol biosynthesis pathway, the 2-butanol biosynthesis pathway, the isobutanol biosynthesis pathway, or the 2-butanone pathway. In certain embodiments, the microorganism is a recombinant microorganism. In certain embodiments, the method comprises providing a feed slurry comprising a fermentable carbon source, an undissolved solid, an oil, and water; separating the feed slurry to provide three streams: (i) fermentable Forming an aqueous solution containing a carbon source, (ii) a wet cake containing solids, and (iii) an oil; and adding the aqueous solution to the fermentation broth. In certain embodiments, the oil is hydrolyzed to form fatty acids. In certain embodiments, the fermentation broth is contacted with a fatty acid. In certain embodiments, the oil is hydrolyzed by an enzyme. In certain embodiments, the enzyme is one or more lipases or phospholipases. In some embodiments, the raw slurry is produced by hydrolysis of the raw material. In certain embodiments, the ingredients are selected from rye, wheat, corn, sugarcane, barley, cellulosic or lignocellulosic materials, and combinations thereof. In certain embodiments, the raw slurry is decanter bowl centrifugation, three-phase centrifugation, disk stack centrifugation, filtration centrifugation, decanter centrifugation, filtration, membrane filtration, microfiltration, vacuum filtration, belt filtration, pressure filtration, Filtration using a sieve, sieving separation, grating, porous grid, flotation, hydrocyclone, filter press, screw press, gravity settler, vortex separator, or a combination thereof Separated. In certain embodiments, the step of separating the feedstock is a single step process. In some embodiments, the wet cake is combined with an aqueous solution. In certain embodiments, the method further comprises contacting the aqueous solution with a catalyst to convert oils in the aqueous solution to fatty acids. In certain embodiments, the aqueous solution and the fatty acids are added to the fermentation broth. In some embodiments, the catalyst is deactivated.

  The present invention relates to one or more fermenters comprising: an inlet for receiving a raw slurry; and one or more fermenters including an outlet for discharging a fermentation broth containing fermentation products; and one or more extractions. Vessel: a first inlet for receiving fermentation broth; a second inlet for receiving extractant; a first outlet for discharging lean fermentation broth; and for discharging rich extractant. And one or more extractors including a second outlet of the system. In certain embodiments, the system further comprises one or more liquefaction units; one or more separation means; and, optionally, one or more cleaning systems. In one embodiment, the separating means is decanter bowl centrifugation, three-phase centrifugation, disk stack centrifugation, filtration centrifugation, decanter centrifugation, filtration, vacuum filtration, belt filter, pressure filtration, membrane filtration, microfiltration, It is selected from filtration using a sieve, sieving separation, a grid, a porous grid, a flotation method, a hydrocyclone, a filter press, a screw press, a gravity settler, a vortex separator, and combinations thereof. In certain embodiments, the system also includes an online measurement device. In certain embodiments, the on-line measurement device is a particle size analyzer, Fourier transform infrared spectrometer, near infrared spectrometer, Raman spectrometer, high pressure liquid chromatography, viscometer, densitometer, tensiometer, droplet size analyzer, pH analyzer And a dissolved oxygen probe, and combinations thereof.

  BRIEF DESCRIPTION OF THE DRAWINGS 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 provide those skilled in the relevant art with an understanding of the invention. And make it usable.

1 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 method and system of the invention in which ISPR is performed downstream of fermentation. 2 schematically illustrates another exemplary alternative method and system of the present invention where the oil stream is drained. Figure 4 schematically illustrates another exemplary alternative method and system of the present invention, wherein the wet cake is subjected to a wash cycle. 3 schematically illustrates another exemplary alternative method and system of the present invention where the oil stream is drained and the wet cake is subjected to a wash cycle. Another exemplary alternative method of the invention wherein the aqueous solution and the wet cake are combined and sent to the fermentation (FIG. 6A) and the aqueous solution, oil and wet cake are combined and sent to the fermentation (FIG. 6B) and 1 schematically shows a system. Another exemplary alternative method of the invention wherein the aqueous solution and the wet cake are combined and sent to the fermentation (FIG. 6A) and the aqueous solution, oil and wet cake are combined and sent to the fermentation (FIG. 6B) and 1 schematically shows a system. 1 schematically illustrates an exemplary alternative method and system of the invention in which an aqueous solution is subjected to conversion (eg, hydrolysis, transesterification) and / or deactivation. 1 schematically illustrates an exemplary alternative method and system of the invention in which an aqueous solution is subjected to conversion (eg, hydrolysis, transesterification) and / or deactivation. 1 schematically illustrates an exemplary alternative method and system of the invention in which an aqueous solution is subjected to conversion (eg, hydrolysis, transesterification) and / or deactivation. 1 schematically illustrates an exemplary alternative method and system of the invention in which an aqueous solution is subjected to conversion (eg, hydrolysis, transesterification) and / or deactivation. 1 schematically illustrates an exemplary fermentation process of the present invention, including downstream processing. 1 schematically illustrates an exemplary fermentation process of the present invention, including downstream processing. Various systems have been shown that can be used in the processes described herein. Various systems have been shown that can be used in the processes described herein. Various systems have been shown that can be used in the processes described herein. Various systems have been shown that can be used in the processes described herein. Various systems have been shown that can be used in the processes described herein. Various systems have been shown that can be used in the processes described herein. Various systems have been shown that can be used in the processes described herein. Various systems have been shown that can be used in the processes described herein. Various systems have been shown that can be used in the processes described herein. 1 schematically illustrates a multiple extractant flow system. 1 schematically illustrates a multiple extractant flow system. 1 schematically illustrates an exemplary fermentation process of the present invention using online, in-line, at-line, and / or real-time measurements to monitor the fermentation process. A, B schematically illustrate an exemplary method of the present invention for mitigating the formation of a rag layer. 1 schematically illustrates an exemplary method of the invention including a fermentation, extraction, and distillation process. 4 shows the effect of the ratio of fermentation broth to extractant (aqueous / organic phase (aq / org)) on extraction column efficiency. A, B show the effect of ISPR with an external extraction column on isobutanol concentration and glucose profile. Figure 4 shows the effect of ISPR with a mixer settler on isobutanol removal. Figure 4 shows FTIR spectra of starch concentration range using in-line measurement. FIG. 4 shows an FTIR spectrum of the starch concentration of a wet cake during the processing of corn mash. 2 shows the FTIR spectrum of corn oil during the processing of corn mash. Figure 4 shows a real-time measurement of isobutanol in COFA.

  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. 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 incorporated by reference in their entirety for all purposes.

  The following terms and definitions are provided herein to further define the invention.

  As used herein, "comprises", "comprising", "includes", "includes", "has", "having" The term "contains," or "contains," or any other conjugation thereof, encompasses the integer or group of integers described, but any other integer or It will be understood that this does not exclude the group of integers. For example, a composition, mixture, process, method, article, or device comprising a enumeration of elements is not necessarily limited to only those elements, and is not explicitly enumerated or such compositions, It may include mixtures, processes, methods, articles, or other elements specific to the device. Further, unless otherwise indicated, "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 with respect to the number of instances, i.e., 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 the element or component is the plural unless it is obvious that the numerical value is singular. Including.

  The terms "invention" or "invention" as used herein are non-limiting terms, are not intended to refer to any one embodiment of the invention, and are not described in the present application. All possible embodiments.

  As used herein, the term "about", which modifies the amount of a component or reactant of the present invention used, refers to, for example, the typical term used to make concentrates or solutions in the real world. By quantitative measurements and liquid handling procedures; by inadvertent errors in these procedures; by the quantity of materials that can occur due to differences in the manufacture, sources, or purity of the components used to make the compositions or perform the methods. Refers to fluctuations. The term "about" also encompasses different amounts due to different equilibrium conditions for a composition resulting from a particular initial mixture. The claims, whether modified by the term "about", include equivalents to the amounts. In one embodiment, the term "about" means within 10% of the reported number, or within 5% of the reported number.

  "Biomass" as used herein refers to corn, sugarcane, wheat, cellulosic or lignocellulosic materials, and cellulose, hemicellulose, lignin, starch, oligosaccharides, disaccharides, and / or monosaccharides, and Refers to any sugar derived from natural resources such as materials including mixtures thereof and natural products containing fermentable sugars including starch and / or hydrolysable polysaccharides that provide starch. Biomass can also include additional components such as proteins and / or lipids. Biomass may be from one source, or biomass may include a mixture from two or more sources. For example, biomass can include 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, sludge from papermaking, garden litter, wood chips and forestry waste (eg, forest thinnings) Can be Examples of biomass include, but are not limited to, corn, corn cobs, corn husk, corn stover and other crop residues, grass, wheat, rye, wheat straw, spelled wheat, triticale, barley, Barley straw, oats, pasture, rice, rice straw, switchgrass, potatoes, sweet potatoes, cassava, jersey, sugar cane marc, sorghum, sugar cane, sugar beet, feed beet, soybeans, palm, coconut, rape, safflower, Sunflower, foxtail, eucalyptus, kaya, ingredients obtained from grinding cereals, trees (eg, branches, roots, leaves), wood chips, sawdust, shrubs and shrubs, vegetables, fruits, flowers, animal manure, and mixtures thereof Is mentioned. For example, a mash, juice, molasses, or hydrolysate can be formed from biomass by any process known in the art for treating biomass for fermentation, such as grinding and liquefaction. For example, cellulosic and / or lignocellulosic biomass is known to those skilled in the art, such as the low ammonia pretreatment disclosed in US Patent Application Publication No. 2007/0031918, which is incorporated herein by reference. It can be treated to obtain a hydrolyzate containing fermentable sugars by any method. Enzymatic saccharification of cellulosic and / or lignocellulosic biomass typically involves enzymatic mixtures (e.g., to break down cellulose and hemicellulose to produce hydrolysates containing sugars, including glucose, xylose, and arabinose, e.g., Cellulase, xylanase, glucosidase, glucanase, lyase). Saccharifying enzymes suitable for cellulosic and / or lignocellulosic biomass are described in Lynd, et al. (Microbiol. Mol. Biol. Rev. 66: 506-577, 2002).

  “Fermentable carbon source” or “fermentable carbon substrate” as used herein refers to a carbon source that can be metabolized by microorganisms. 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; And mixtures thereof.

  As used herein, "fermentable sugar" refers to one or more saccharides that can be metabolized by the microorganisms disclosed herein for the production of a fermentation product.

  As used herein, `` raw material '' refers to a raw material in a fermentation process, wherein the raw material contains a fermentable carbon source with or without undissolved solids and oils, where applicable, the raw material is: It contains a fermentable carbon source before or after the fermentable carbon source has been removed from the starch or obtained from the degradation of the complex saccharide by further processing, such as by liquefaction, saccharification, or other processes. The feedstock may 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 materials, lignocellulosic materials, or mixtures thereof. It will be understood that when reference is made to "feedstock", the term encompasses oils produced from a given feedstock.

  As used herein, "fermentation broth" refers to a microorganism that produces water, a fermentable carbon source (eg, sugars), dissolved solids, and optionally a fermentation product (eg, product alcohol). Optionally refers to a mixture of a fermentation product (eg, product alcohol) and other ingredients. In certain embodiments, a fermentation broth refers to a material in which a fermentation product (eg, product alcohol) is retained in a fermentation apparatus made by metabolism of a fermentable carbon source by a microorganism. Optionally, the term “fermentation broth” as used herein may be used synonymously with “fermentation medium” or “fermented mixture”. In certain embodiments, a fermentation broth comprising product alcohol may be referred to as a fermentation beer or beer.

  As used herein, a "fermenter" or "fermentor" is a fermentation reaction in which a fermentation product (e.g., a product alcohol such as ethanol or butanol) is produced from a fermentable carbon source. Refers to the unit to be used. The term "fermenter" can be used interchangeably herein with "fermentor."

  “Liquefaction unit” as used herein refers to a unit where liquefaction takes place. Liquefaction is the process by which oligosaccharides are released from raw materials. In certain embodiments where the raw material is corn, the oligosaccharide is released from the corn starch content during liquefaction.

  As used herein, a "saccharification unit" refers to a unit that undergoes saccharification (ie, the degradation of oligosaccharides to monosaccharides). When fermentation and saccharification are performed simultaneously, the saccharification unit and the fermentation apparatus may be the same unit.

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

  As used herein, a "saccharification enzyme" is one or more enzymes capable of hydrolyzing polysaccharides and / or oligosaccharides, such as glycogen or the alpha-1,4-glucosidic bond of starch. Point to. Saccharification enzymes may also include enzymes capable of hydrolyzing cellulosic or lignocellulosic materials.

  "Undissolved solid" as used herein refers to the non-fermentable portion of a raw material that does not dissolve in an aqueous medium, such as germ, fiber, gluten, and any additional components. For example, the non-fermentable portion of the feedstock may include portions that remain as solids in the feedstock and absorb liquid from the fermentation broth.

  “Oil” as used herein refers to lipids obtained from a plant (eg, biomass) or animal. Examples of oils include, but are not limited to, tallow, corn oil, canola oil, capric acid / caprylic triglyceride, castor oil, coconut oil, cottonseed oil, fish oil, jojoba oil, lard, linseed oil, beef leg oil, Deer oil, coconut oil, peanut oil, rapeseed oil, rice oil, safflower oil, soybean oil, sunflower oil, tung oil, jatropha oil, and vegetable oil blends.

"Product alcohol" as used herein refers to any alcohol that can be produced by a microorganism in a fermentation process using biomass as a source of fermentable carbon substrate. 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 understood that C ₁ -C ₈ alkyl alcohols include, but are not limited to, methanol, ethanol, propanol, butanol, pentanol, and hexanol. Similarly, C ₂ -C ₈ alkyl alcohols include, but are not limited to, ethanol, propanol, butanol, pentanol, and hexanol. In certain embodiments, the product alcohol may also include fusel alcohol (or fusel oil) and glycerol. "Alcohol" may also be used herein in reference to product alcohol.

  “Butanol” as used herein refers to the butanol isomer 1-butanol (1-BuOH), 2-butanol (2-BuOH), tert-butanol (t-BuOH), and / or isobutanol ( iBOH, also known as iBuOH, i-BuOH, I-BUOH, 2-methyl-1-propanol, is referred to individually or as a mixture thereof. Optionally, when referring to esters of butanol, the terms "butyl ester" and "butanol ester" may be used interchangeably.

  "Propanol" as used herein refers to the propanol isomer isopropanol or 1-propanol.

  “Pentanol” as used herein refers to the pentanol isomer 1-pentanol, 3-methyl-1-butanol, 2-methyl-1-butanol, 2,2-dimethyl-1-propanol, Refers to 3-pentanol, 2-pentanol, 3-methyl-2-butanol, or 2-methyl-2-butanol.

  As used herein, "in situ product removal (ISPR)" refers to a biological process, such as fermentation, for controlling the product concentration in a biological process as the product is produced. Refers to the selective removal of specific products from the process.

  “Extractant” as used herein refers to the solvent used to extract the fermentation product (eg, product alcohol). Optionally, the term "extractant" as used herein may be used synonymously with "solvent."

  "Water-immiscible" as used herein refers to 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.

  "Carboxylic acid" as used herein refers to a carbon atom bonded to an oxygen atom by a double bond to form a carbonyl group (-C = O) and a hydroxyl group (-OH by a single bond. ) Refers to any organic compound represented by the general formula -COOH. The carboxylic acid may exist in the form of a protonated carboxylic acid, in the form of a salt of the carboxylic acid (eg, an ammonium salt, a sodium salt, or a potassium salt), or as a mixture of a protonated carboxylic acid and a salt of a carboxylic acid. . The term carboxylic acid refers to a single species (eg, oleic acid) or a mixture of carboxylic acids that can be produced, for example, by hydrolysis of fatty acid esters or triglycerides, diglycerides, monoglycerides, and phospholipids from biomass. obtain.

Carboxylic "fatty acid" when used herein, having either a saturated or unsaturated, carbon atoms C ₄ -C ₂₈ (most commonly, carbon atoms C ₁₂ -C ₂₄₎ Refers to acids (eg, aliphatic monocarboxylic acids). Fatty acids can also be branched or unbranched. Fatty acids may be derived from or included in esterified forms, animal or vegetable fats, oils or waxes. Fatty acids may be naturally occurring in the form of glycerides in fats and fatty oils, or obtained by the hydrolysis of fats or by synthesis. The term fatty acid may represent a single species or a mixture of fatty acids. Furthermore, the term fatty acid also includes free fatty acids.

“Fatty alcohol” as used herein refers to an alcohol having an aliphatic chain of C _{4 to} C ₂₂ carbon atoms that is either saturated or unsaturated.

“Fatty aldehyde” as used herein refers to an aldehyde having an aliphatic chain of C _{4 to} C ₂₂ carbon atoms that is either saturated or unsaturated.

“Fatty amide” as used herein refers to an amide having an aliphatic chain of C _{4 to} C ₂₂ carbon atoms that is either saturated or unsaturated.

“Fatty ester” as used herein refers to an ester having an aliphatic chain of C _{4 to} C ₂₂ carbon atoms that is either saturated or unsaturated.

  An “aqueous phase” as used herein includes, for example, an aqueous phase of a two-phase mixture, including a liquid phase and a gas phase, two liquid phases (eg, an organic phase and an aqueous phase) and a gas phase. The aqueous phase of a three-phase mixture, the aqueous phase of either a two-phase or three-phase mixture in which the aqueous phase contains some amount of suspended solids, or a four-phase mixture comprising a gas phase, an organic phase, an aqueous phase and a solid phase Point to. In certain embodiments, a three-phase mixture can include a gas phase, a liquid phase, and a solid phase. In certain embodiments, the aqueous phase is obtained by contacting the fermentation broth with a water-immiscible organic extractant. In one embodiment of the process described herein involving fermentation extraction, the term "fermentation broth" may in this case refer to the aqueous phase in a two-phase fermentation extraction.

  An “organic phase” as used herein refers to a non-mixture of a mixture (eg, a two-phase mixture, a three-phase mixture, a four-phase mixture) obtained by contacting a fermentation broth with a water-immiscible organic extractant. Refers to the aqueous phase. Optionally, the term “organic phase” as used herein may be used synonymously with “extractant phase”.

  “Effective potency” as used herein refers to the total amount of a particular fermentation product (eg, product alcohol) produced by fermentation per liter of fermentation broth.

  A “portion” as used herein in the context of a process stream refers to 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 fraction of the retained stream.

The present invention provides processes and methods for producing fermentation products, such as product alcohols, 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 carbon 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.

  The present invention provides methods and systems for producing product alcohol by a fermentation process and recovering the product alcohol produced by the fermentation process. As an example of one embodiment of the method described herein, fermentation may be initiated by introducing the raw materials directly into the fermentor. In certain embodiments, one or more fermenters can be used in the methods described herein. Suitable raw materials include, but are not limited to, rye, wheat, corn, corn mash, sugar cane, sugar cane mash, barley, cellulosic materials, lignocellulosic materials, or mixtures thereof. These raw materials can be processed using a method such as dry grinding or wet grinding. In certain embodiments, prior to introduction into the fermentor, the feed may be liquefied to produce a feed slurry that may include undissolved solids, a fermentable carbon source (eg, sugar), and oil. Liquefaction of the feedstock may be performed by any known liquefaction process, including but not limited to an acid process, an enzymatic process (eg, α-amylase), an acid-enzyme process, or a combination thereof. In certain embodiments, liquefaction may be performed in one or more liquefaction units.

  If the raw slurry is fed directly to the fermentor, undissolved solids and / or oils may prevent efficient removal and recovery of product alcohol. In particular, when liquid-liquid extraction is used to extract product alcohol from the fermentation broth, the presence of undissolved solids (eg, particulates) may prevent the extractant from contacting the fermentation broth with the extractant. Reducing the mass transfer rate of the product alcohol of the present invention; creating or promoting an emulsion in the fermenter, thereby preventing phase separation of the extractant and fermentation broth; at least a portion of the extractant and product alcohol To reduce the efficiency of recovering and reusing the extractant because it is "trapped" by solids that can be removed as solubles-containing dried grain distillation cake (DDGS); some solids occupy volume in the fermenter The fermenter volumetric efficiency due to the extraction and slower release of the extractant from the fermentation broth; and the life of the extractant due to oil contamination. But it is not limited to including being shorter, can lead to inefficiency of the system. These effects can increase capital and operating costs. In addition, extractants that are "entrapped" by DDGS can deviate from DDGS values and requirements for sale as animal feed. Thus, to avoid and / or minimize these problems, at least some of the undissolved solids can be removed from the raw slurry before adding the raw slurry to the fermentor. The extraction activity and efficiency of product alcohol production can be enhanced if the extraction is performed in a fermentation broth from which undissolved solids have been removed.

  A method and system for processing a feedstock to produce a feedstock slurry, separating the feedstock slurry, and producing an aqueous phase containing a fermentable carbon source and a solid phase (e.g., a wet cake) are illustrated in FIG. Described herein by reference. As shown in FIG. 1, in one embodiment, the system includes a liquefaction 10 configured to liquefy the feedstock to produce a feedstock slurry. For example, feedstock 12 may be introduced to liquefaction 10 (eg, via an inlet of a liquefaction unit). Ingredient 12 comprises a fermentable carbon source such as sugar and / or starch, including, but not limited to, barley, oats, rye, sorghum, wheat, triticale, spelled wheat, millet, sugar cane, corn, or combinations thereof. Can be any suitable biomass material known in the art. Water may also be introduced into liquefaction 10.

  The process of liquefying feedstock 12 includes hydrolyzing the starch in feedstock 12 to water-soluble sugars. Any known liquefaction process used in the art, including but not limited to acid processes, enzymatic processes, or acid-enzyme processes, as well as liquefaction units may be used. Such processes can be used alone or in combination. In certain embodiments, an enzymatic process can be used, wherein a suitable enzyme 14, eg, α-amylase, is introduced into liquefaction 10. Examples of α-amylases that can be used in the systems and methods of the present invention include US Patent No. 7,541,026; US Patent Application Publication No. 2009/02009026; US Patent Application Publication No. 2009/0238923. Specification; U.S. Patent Application Publication No. 2009/0252828; U.S. Patent Application Publication No. 2009/0314286; U.S. Patent Application Publication No. 2010/02278970; U.S. Patent Application Publication No. 2010/0048446 Described in U.S. Patent Application Publication No. 2010/0021587, the entire contents of each of which is incorporated herein by reference.

  In certain embodiments, enzymes for liquefaction and / or saccharification can be produced by microorganisms. Examples of microorganisms that produce such enzymes are U.S. Patent No. 7,498,159; U.S. Patent Application Publication No. 2012/0003701; U.S. Patent Application Publication No. 2012/0129229; PCT International. WO 2010/096562; and PCT International Publication WO 2011/153516, the entire contents of each of which are incorporated herein by reference. In certain embodiments, enzymes for liquefaction and / or saccharification can be expressed by microorganisms that also produce product alcohol. In certain embodiments, enzymes for liquefaction and / or saccharification can be expressed by microorganisms that also express the butanol biosynthetic pathway. In certain embodiments, the butanol biosynthetic pathway can be a 1-butanol biosynthetic pathway, a 2-butanol biosynthetic pathway, an isobutanol biosynthetic pathway, or a 2-butanone biosynthetic pathway.

  The process of liquefying feedstock 12 produces a feedstock slurry 16 (also referred to as a mash or a high viscosity mash) that includes a fermentable carbon source (eg, sugar) and undissolved solids. In certain embodiments, the raw slurry 16 may include a fermentable carbon source (eg, sugar), an oil, and an undissolved solid. The undissolved solid can be a non-fermentable portion of feedstock 12. In certain embodiments, feedstock 12 may be corn, such as dry-milled, unfractionated corn fruit, and feedstock slurry 16 is a corn mash slurry. Raw slurry 16 may be discharged from the outlet of liquefaction 10 and may be sent to separation 20.

  The separation 20 has an inlet for receiving the raw slurry 16 and may be configured to remove undissolved solids from the raw slurry 16. Separation 20 may also be configured to remove oil and / or oil and undissolved solids. Separation 20 may agitate or spin the raw slurry 16 to produce a liquid or aqueous solution 22 and a solid or wet cake 24.

  The aqueous solution 22 can include, for example, sugar in the form of an oligosaccharide, and water. 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. The aqueous solution 22 can be discharged from the separation 20 via an outlet. In certain embodiments, the outlet may be located near the top of separation 20.

  Wet cake 24 may include undissolved solids. Wet cake 24 may be discharged from separation 20 via an outlet. In certain embodiments, the outlet may be located near the bottom of separation 20. The wet cake 24 may also include some of the sugar and water. The wet cake 24 may be washed with additional water in the separation 20 after the aqueous solution 22 has drained from the separation 20. Alternatively, the wet cake 24 may be washed with additional water by an additional separation device. By washing the wet cake 24, sugars (eg, oligosaccharides) present in the wet cake are collected, and the collected sugar and water can be reused for the liquefaction 10. After washing, the wet cake 24 may be further processed by any suitable known method to form soluble matter-containing dry cereal distillers cake (DDGS). Forming DDGS from wet cake 24 formed in separation 20 has several advantages. DDGS is not subjected to fermenter conditions because undissolved solids are not sent to the fermentor. For example, DDGS does not come into contact with microorganisms present in the fermentor or any other material that may be present in the fermenter (eg, extractant and / or product alcohol), so that microorganisms and / or other materials may be present. , DDGS. These effects provide advantages, for example, for subsequent processing and sale of DDGS as animal feed.

  Separation 20 may be performed by any conventional method used in the art, including, for example, a centrifuge such as a decanter bowl centrifuge, a three-phase centrifuge, a disc stack centrifuge, a filtration centrifuge, or a decanter centrifuge. Separation device. In certain embodiments, the removal of undissolved solids from the raw slurry 16 may be filtration, vacuum filtration, belt filtration, pressure filtration, membrane filtration, microfiltration, filtration using a sieve, sieving separation, grate or It can be performed by a grid, a porous grid, a flotation method, a hydrocyclone, a filter press, a screw press, a gravity settler, a vortex separator, or any method or device that can be used to separate solids from liquids. In some embodiments, separation 20 is a single-step process. In one embodiment, undissolved solids are removed from the raw slurry 16 and compared to two product streams, for example, an aqueous solution of oligosaccharide containing a lower concentration of solids compared to the raw slurry 16 and the raw slurry 16. Thus, a wet cake containing a higher concentration of solids can be formed. Further, if, for example, three-phase centrifugation is used to remove solids from the raw slurry 16, a third stream containing oil may be generated. Thus, several product streams may be produced using different separation techniques or combinations thereof.

  Three-phase centrifuges are used for three-phase separation of raw slurry, such as separation of raw slurry, to produce two liquid phases (eg, water and oil streams) and a solid phase (eg, solid or wet cake). (See, eg, Flotteweg Tricanter®, Flotteweg AG, Vilsibiburg, Germany). The two liquid phases may be separated or decanted from the centrifuge bowl, for example, via two discharge systems to prevent cross-contamination, and the solid phase may be removed via separate discharge systems. obtain.

  In certain embodiments using corn as a feedstock, a three-phase centrifuge can be used to simultaneously remove solids and corn oil from liquefied corn mash. The solid can be an undissolved solid that remains after the starch is hydrolyzed to soluble oligosaccharides during liquefaction. Corn oil can be removed from corn 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 may consist of liquefied corn mash produced during liquefaction. The mash may consist of an aqueous solution of an oligosaccharide (eg, liquefied starch); an undissolved solid consisting of insoluble non-starch components derived from corn; and corn oil consisting of glycerides and free fatty acids. The three outlet streams from the three-phase centrifuge are a wet cake containing most of the undissolved solids from the mash; a heavy centrate stream containing most of the liquefied starch from the mash; And a light centrate stream containing the majority of the corn oil from the mash. The heavy centrifuge stream can be fed to the fermentation. The wet cake may be washed with a process recycle water, such as an evaporator condensate and / or a backset as described herein, to recover soluble starch from the wet cake. The light centrifuge stream may be sold as a by-product, converted to another by-product, or used in processes such as converting corn oil to corn oil fatty acid (COFA). In certain embodiments, COFA may be used as an extractant.

  Referring to FIG. 1, a fermentation 30 (or fermenter 30) configured to ferment an aqueous solution 22 to produce a product alcohol has an inlet for receiving the aqueous solution 22. Fermentation 30 can be any suitable fermentation apparatus known in the art. Fermentation 30 may include a fermentation broth. In certain embodiments, a simultaneous saccharification and fermentation (SSF) can be performed inside fermentation 30. Any known saccharification process used in the art, including but not limited to an acid process, an enzymatic process, or an acid-enzyme process, may be used. In certain embodiments, an enzyme 38 (such as, for example, glucoamylase) may be introduced at the inlet of fermentation 30 to hydrolyze oligosaccharides in aqueous solution 22 to form monosaccharides. Examples of glucoamylases that can be used in the systems and methods of the present invention include: 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; and U.S. Patent Application Publication No. 2011/0020899, each of which is hereby incorporated by reference in its entirety. Is hereby incorporated by reference herein. In certain embodiments, the glucoamylase can be expressed by a microorganism. In certain embodiments, glucoamylase can be expressed by a microorganism that also produces product alcohol. In certain embodiments, glucoamylase can be expressed by a microorganism that also expresses the butanol biosynthetic pathway. In certain embodiments, the butanol biosynthetic pathway can be a 1-butanol biosynthetic pathway, a 2-butanol biosynthetic pathway, an isobutanol biosynthetic pathway, or a 2-butanone biosynthetic pathway.

  In certain embodiments, an enzyme such as glucoamylase may be added to the liquefaction. By adding an enzyme such as glucoamylase to the liquefaction, the viscosity of the raw material 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 (eg, viscometer, rheometer).

  Microorganisms 32 may be introduced into fermentation 30. In certain embodiments, microorganisms 32 may be included in the fermentation broth. In certain embodiments, the microorganisms 32 can be propagated in a separate tank or tank (eg, a breeding tank). In certain embodiments, microorganisms from a breeding tank can be used to inoculate one or more fermenters. In certain embodiments, one or more breeding tanks may be used in the methods and systems described herein. In certain embodiments, the breeding tank may be between about 2% and about 5% of the size of the fermentor. In certain embodiments, the breeding tank may include one or more of the following mashes, water, enzymes, nutrients, extractants, and microorganisms. In certain embodiments, product alcohol may be produced in a breeding tank.

  In certain embodiments, microorganism 32 can be a bacterium, a cyanobacterium, a mold, or a yeast. In certain embodiments, microorganisms 32 metabolize sugars in aqueous solution 22 to produce product alcohol. In certain embodiments, microorganism 32 may be a recombinant microorganism. In certain embodiments, microorganisms 32 may be immobilized by adsorption, covalent bonding, crosslinking, entrapment, and encapsulation, and the like. Methods for confining cells are known in the art, for example, as described in US Patent Application Publication No. 2011/03030616, which is incorporated herein by reference.

  In certain embodiments, in situ product removal (ISPR) may be used to remove product alcohol from fermentation 30 as the product alcohol is produced by microorganism 32. In certain embodiments, liquid-liquid extraction may be used for ISPR. In certain embodiments, fermentation 30 may have an inlet for receiving extractant 34. In certain embodiments, extractant 34 may be added to fermentation broth downstream of fermentation 30. An alternative means of adding fermentation 30 or extractant 34 downstream of fermentation 30 is represented by the dotted line. In certain embodiments, ISPR may be performed in a breeding tank. In certain embodiments, ISPR may be performed in fermenters and breeding tanks. In certain embodiments, ISPR may be performed at the beginning of fermentation and / or breeding (eg, at time 0). By starting ISPR at the beginning of fermentation and / or breeding, the concentration of product alcohol in the fermentor and breeding tank is maintained at a low level, thereby minimizing the effect of product alcohol on microorganisms. Alternatively, it may be possible for a microorganism to obtain an increased cell mass. In certain embodiments, the extractant may be added to a breeding tank. In certain embodiments, the extractant may be added prior to inoculation of the breeding tank. In certain embodiments, the extractant may be added after inoculation of the breeding tank. In certain embodiments, the extractant may be added at various times after inoculation of the breeding tank. In certain embodiments, an extractant may be added to the fermentor. In certain embodiments, the extractant may be added prior to inoculation of the fermentor. In certain embodiments, the extractant may be added after inoculation of the fermentor. In certain embodiments, the extractant may be added at various times after inoculation of the fermentor. In certain embodiments, the extractant may be added to the fermentor and breeding tank. Examples of liquid-liquid extraction are described herein. Methods for producing and recovering alcohol from fermentation broth using extractive fermentation are disclosed in US Patent Application Publication No. 2009/0305370; US Patent Application Publication No. 2010/0221802; US Patent Application Publication 2011 / 0097773; US Patent Application Publication No. 2011/0312044; US Patent Application Publication No. 2011/0312043; and PCT International Publication No. WO 2011/159998 pamphlet; Is incorporated herein by reference in its entirety.

  The extractant 34 contacts the fermentation broth to form a stream 36 comprising, for example, a two-phase mixture (e.g., an extractant-enriched phase containing product alcohol and an aqueous phase rich in product alcohol). In certain embodiments, stream 36 can be, for example, a four-phase mixture including a gas phase, an organic phase, an aqueous phase, and a solid phase. The product alcohol, or a portion thereof, in the fermentation broth is transferred to extractant 34. In certain embodiments, stream 36 may be discharged via the outlet of fermentation 30. Product alcohol can be separated from the extractant in stream 36 using conventional techniques.

In certain embodiments, the interior or device of the fermentor may be used to enhance the phase separation between the fermentation broth and the extractant. For example, the interior or device may serve as a coalescer to promote phase separation between the fermentation broth and the extractant, and / or may serve as a physical barrier to enhance phase separation. The interior or device of these fermenters also prevents solids from settling in the extractant phase (or layer), promotes aggregation of water droplets that can be entrained in the extractant layer, and reduces offgas (eg, CO ₂ , Facilitates the removal of (air), thereby minimizing the disturbance of the extractant phase and / or liquid-liquid interface. Examples of internals or devices that can be used in the methods and systems described herein include, but are not limited to, baffles, perforated plates, deep wells, lamella separators, cones, and the like. In some embodiments, the perforated plate can be a flat horizontal perforated plate. In certain embodiments, the cone may be an inverted cone or a concentric cone. In some embodiments, the interior may rotate. In certain embodiments, the interior or device may be located at or near the liquid-liquid interface of the fermentation broth and extractant. In certain embodiments, a coalescing pad may be added and / or the outlet port may be repositioned to improve the coagulation and recovery of the aqueous phase.

In certain embodiments, prior to completion of the ISPR and / or fermentation, stream 35 may be discharged from the outlet of fermentation 30. The discharged stream 35 may include microorganisms 32. Microorganisms 32 may be separated from stream 35, for example, by centrifugation or membrane filtration. In certain embodiments, by removing the microorganisms prior to addition of the extractant to the fermentation broth, the microorganisms are not exposed to the extractant and thus do not have any adverse effects that the extractant may have on the microorganisms. Further, by removing the microorganisms in the upstream of the extraction process, more aggressive extraction process (e.g., heating or cooling of the mixture to facilitate separation, a higher K _D and / or higher selectivity of the extraction agent, Or use of an extractant with lower biocompatibility but with improved properties) can be used to recover the product alcohol. In certain embodiments, microorganisms 32 may be recycled to fermentation 30 which may increase the rate of product alcohol production, thereby increasing the efficiency of product alcohol production.

  Referring to FIG. 2, in one embodiment, the ISPR may be performed downstream of fermentation 30. In certain embodiments, a stream 33 containing product alcohol and microorganisms 32 may be discharged from the outlet of fermentation 30 and sent downstream, for example, to an extraction column for product alcohol recovery. In certain embodiments, stream 33 may be treated by separating microorganisms 32 prior to ISPR. For example, removal of microorganisms 32 from stream 33 includes centrifugation, filtration, vacuum filtration, belt filtration, pressure filtration, membrane filtration, microfiltration, filtration using a sieve, sieving separation, grate or grate, porous grate. , Flotation, hydrocyclone, filter press, screw press, gravity settler, vortex separator, or any method or separation device that can be used to separate solids (eg, microorganisms) from liquids. After removal of microorganisms 32, stream 33 may be sent to an extraction column for recovery of product alcohol.

  Further embodiments of the methods and systems described herein are shown in FIGS. FIGS. 3-6, which include the option of adding an extractant to the fermentor (e.g., producing stream 36) or extracting downstream of the fermenter (e.g., producing stream 33), are shown in FIGS. Since it is the same as described above, it will not be described again in detail.

  Referring to FIG. 3, the system and method of the present invention may include draining oil 26 from the outlet of separation 20. The raw slurry 16 is converted into a first liquid phase or aqueous solution 22 containing fermentable sugars, a solid phase or wet cake 24 containing undissolved solids, and a second liquid phase containing oil 26 that can exit the separation 20. Can be separated. In some embodiments, separation of the raw slurry 16 into a first liquid phase, a second liquid phase, and a solid phase can be performed in a single step. In some embodiments, feedstock 12 is corn and oil 26 is corn oil. In some embodiments, oil 26 may be sent to a storage tank or any unit suitable for storing oil. Any suitable separation device, for example, a three-phase centrifuge, can be used to drain aqueous solution 22, wet cake 24, and oil 26. In some embodiments, if the ingredient is corn, a portion of the oil from ingredient 12, such as corn oil, will remain in wet cake 24. In certain embodiments, when oil 26 is removed from feedstock 12 (eg, corn) via separation 20, the fermentation broth in fermentation 30 includes a reduced amount of corn oil.

  As described herein, in certain embodiments, the oil may be separated from the feed or feed slurry and stored in an oil storage unit. For example, the oil may be separated from the feed or feed slurry using a three-phase centrifuge or any suitable means for separation including mechanical extraction. Surfactants, demulsifiers, or flocculants as well as oil extraction aids such as enzymes may be used to enhance the removal of oil from the feed or feed slurry. Examples of oil extraction aids include, but are not limited to, non-polymeric liquid surfactants; talcum powder; fine talcum powder; salt (NaOH); calcium carbonate; and Pectinex® Ultra SP-L; And enzymes such as Celluclast®, and Viscozyme® L (Sigma-Aldrich, St. Louis, MO), and NZ 33095 (Novozymes, Franklinton, NC).

  If the oil is not drained separately, as shown in FIG. 4, the oil may be removed along with the wet cake 24. When the wet cake 24 is removed via the separation 20, in some embodiments, if the feed is corn, a portion of the oil from the feed 12 such as corn oil will remain in the wet cake 24. The wet cake 24 may be sent to a mix 60 and combined with water or other solvent to form a wet cake mixture 65. In some embodiments, the water is fresh water, reflux, cook water, process water, luter water, evaporative water, or any water source available at the fermentation facility. Or any combination thereof. The wet cake mixture 65 may be sent to a separation 70 to produce a wash centrifuge 75 containing the fermentable sugars recovered from the wet cake 24, and the wet cake 74. The wash centrifuge liquid 75 can be recycled to the liquefaction 10.

  In certain embodiments, the separation 70 is, for example, a decanter bowl centrifuge, a three-phase centrifuge, a disc stack centrifuge, a filtration centrifuge, a decanter centrifuge, a filtration, a vacuum filtration, a belt filter, a pressure filtration, a membrane filtration, a sieve. Separates solids and liquids, including filtration, sieving separations, grids, porous grids, suspension methods, hydrocyclones, filter presses, screw presses, gravity sedimenters, vortex separators, or combinations thereof Any separation device.

  In certain embodiments, the wet cake may be subjected to one or more washing cycles or systems. For example, the wet cake 74 can be further processed by sending the wet cake 74 to a second cleaning system. In some embodiments, the wet cake 74 can be sent to a second mix 60 'to form a wet cake mixture 65'. The wet cake mixture 65 'may be sent to a second separation 70' to produce a wash centrifuge 75 'and a wet cake 74'. The wash centrifuge 75 'can be recycled to liquefaction 10. In some embodiments, the wash centrifuge 75 ′ can be combined with the wash centrifuge 75 and recycled to the liquefaction 10. In certain embodiments, the wet cake 74 'may be combined with the wet cake 74 for further processing as described herein. In certain embodiments, the separation 70 'is, for example, a decanter bowl centrifuge, a three-phase centrifuge, a disc stack centrifuge, a filtration centrifuge, a decanter centrifuge, a filtration, a vacuum filtration, a belt filter, a pressure filtration, a membrane filtration, Separation of solids and liquids, including microfiltration, sieving filtration, sieving separation, grids, porous grids, flotation, hydrocyclones, filter presses, screw presses, gravity sedimenters, vortex separators, or combinations thereof It can be any separation device capable of doing so. In certain embodiments, the wet cake may be subjected to one, two, three, four, five, or more washing cycles or systems.

  The wet cake 74 can be combined with the syrup and then dried to form DDGS through any suitable known process. Forming DDGS from wet cake 74 has several advantages. Since no undissolved solids are added to the fermentor, DDGS has no trapped extractant and / or product alcohol, is not subjected to fermentor conditions, and does not come into contact with microorganisms present in the fermenter. . These advantages make it easier, for example, to process DDGS as animal feed.

  In certain embodiments, a portion of the undissolved solid may be sent to fermentation 30. In certain embodiments, this portion of the undissolved solid can have a smaller particle size (eg, fines). In certain embodiments, this portion of the undissolved solid can form a whole stillage. In certain embodiments, the total distillation effluent can be treated to form a thin stillage and a wet cake. In certain embodiments, the wet cake and wet cake 74 and / or 74 'formed from the total distillation effluent can be combined and further processed to produce DDGS.

  As shown in FIG. 4, the oil is not separately drained from the wet cake, and the oil is included as part of the wet cake and is finally present in the DDGS. When corn is used as a feedstock, corn oil contains triglycerides, diglycerides, monoglycerides, fatty acids, and phospholipids, which provide the animal's metabolizable energy source. The presence of an oil (eg, corn oil) in the wet cake and ultimately in the DDGS provides a desirable animal feed, eg, a high fat content animal feed.

  In certain embodiments, the oil may be separated from the wet cake and DDGS and converted to an ISPR extractant for subsequent use in the same or a different alcohol fermentation process. Methods for obtaining extractants from biomass are described in 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 of which are incorporated herein by reference. The oil may be separated from the wet cake and DDGS using any suitable known process, including, for example, a solvent extraction process. In one embodiment of the present invention, the wet cake or DDGS can be added to the extraction unit 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 butanol, isohexane, ethanol, petroleum ether, or mixtures thereof. After oil extraction, the wet cake or DDGS can be treated to remove any residual solvent. For example, the wet cake or DDGS can be heated to evaporate any residual solvent using any method known in the art. After removing the solvent, the wet cake or DDGS may be subjected to a drying process to remove any residual water. The treated wet cake can be used to produce DDGS. The treated DDGS can be used as a supplemental feed for animals such as dairy and beef cattle, poultry, pigs, livestock, horses, aquaculture, and pets.

  In certain embodiments, the extractant may be used as a means to adjust the color of the wet cake. For example, raw materials such as corn contain pigments (eg, xanthophylls) that can be used as colorants in foods, including animal feeds (eg, poultry feeds). Exposure to the extractant allows these pigments to be adjusted, for example, to obtain a lighter colored wet cake. A lighter colored wet cake may produce a lighter colored DDGS, which may be of a desired quality for some animal feeds.

  In certain embodiments, when corn is used as a raw material, xanthophylls are isolated from corn and / or undissolved solids and supplemented as a pigment component in DDGS or animal feed, or for pharmaceutical and dietary supplement applications. Can be used as Methods for isolating xanthophylls include, but are not limited to, chromatography such as size exclusion chromatography, solvent extraction such as ethanol extraction, and enzymatic treatment such as alcalase hydrolysis (eg, Tsui, et al., J. Food Eng. 83: 590-595, 2007; Li, et al., Food Science 31: 72-77, 2010, each of which is incorporated by reference herein in its entirety. : U.S. Pat. No. 5,648,564; U.S. Pat. No. 6,169,217; U.S. Pat. No. 6,329,557; U.S. Pat. No. 8,236,929. ). In certain embodiments, xanthophylls can be isolated from corn and / or undissolved solids and added to COFA. In certain embodiments, COFA and / or xanthophyll may be used for food, pharmaceutical, and dietary supplement applications.

  After extraction from the wet cake or DDGS, the resulting oil and solvent mixture can be collected for oil and solvent separation. In one embodiment, the oil / solvent mixture may be processed by evaporation, whereby the solvent can be evaporated, collected, and reused. The recovered oil may be converted to an ISPR extractant for subsequent use in the same or a different alcohol fermentation process.

  Removal of the raw oil component is advantageous for product alcohol production because the oil present in the fermentor can be broken down into fatty acids and glycerin. Glycerin can accumulate in water and reduce the amount of water available for reuse throughout the system. Thus, the removal of feedstock oil components can increase the efficiency of product alcohol production by increasing the amount of water that can be recycled through the system.

  Referring to FIG. 5, oil may be removed at various points during the process described herein. The raw slurry 16 can be separated into a first liquid phase or aqueous solution 22, a second liquid phase including oil 26, and a solid phase or wet cake 24, for example, using a three-phase centrifuge. The wet cake 24 may be further processed to recover fermentable sugars and oils. The wet cake 24 may be sent to a mix 60 and combined with water or other solvent to form a wet cake mixture 65. In certain embodiments, the water can be reflux, cooking water, process water, distillation 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 separator 70 (eg, a three-phase centrifuge) to produce a wash centrifuge 75 containing fermentable sugars, an oil stream 76, and a wet cake 74. The wash centrifuge liquid 75 can be recycled to the liquefaction 10.

  As described herein, the wet cake may be subjected to one or more washing cycles or systems. In some embodiments, the wet cake 74 can be sent to a second mix 60 'to form a wet cake mixture 65'. The wet cake mixture 65 'may be sent to a second separation 70' to produce a wash centrifuge 75 ', an oil stream 76', and a wet cake 74 '. The wash centrifuge 75 'can be recycled to liquefaction 10. In some embodiments, the wash centrifuge 75 ′ can be combined with the wash centrifuge 75 and recycled to the liquefaction 10. In some embodiments, the wet cake 74 'may be combined with the wet cake 74 for further processing as described below. In certain embodiments, the oil stream 76 'and the oil 26 can be combined and further processed to produce an extractant that can be used in the fermentation process, or the oil stream 76' and the oil 26 can be combined and consumed. Can be further processed for the manufacture of consumer products.

  The wet cake 74 can be combined with the syrup and then dried to form DDGS using any suitable process. Forming DDGS from wet cake 74 has several advantages. Because no undissolved solids are added to the fermentor, DDGS does not contain extractant and / or product alcohol, is not subjected to fermentor conditions, and does not come into contact with microorganisms present in the fermenter. These advantages make it easier, for example, to process DDGS as animal feed. As described herein, in certain embodiments, the wet cakes 74, 74 ', and the wet cake formed from the total distillation effluent can be combined and further processed to produce DDGS.

  As shown in FIG. 6A, the aqueous solution 22 and the wet cake 24 may 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 including oil 26, and a solid phase or wet cake 24, for example, using a three-phase centrifuge. In some embodiments, oil 26 may be sent to a storage tank or any unit suitable for storing oil. The aqueous solution 22 and wet cake 24 may be sent to a mixer 80 and reslurried to form an aqueous solution / wet cake mixture 82. The mixture 82 may be sent to a cooler 90 to produce a cooled mixture 92, which may be sent to the fermentation 30. In certain embodiments, if oil 26 is removed from feed slurry 16 via separation 20, mixtures 82 and 92 include a reduced amount of oil.

  In another embodiment, as shown in FIG. 6B, the raw slurry 16 is separated using a separation device (eg, a three-phase centrifuge) and includes a first liquid phase or aqueous solution 22, oil 26. A second liquid phase, and a solid phase or wet cake 24 may be produced. Aqueous solution 22, wet cake 24, and oil 26, or a portion thereof, may be sent to fermentation 30. In certain embodiments, the aqueous solution 22, the wet cake 24, and the oil 26, or a portion thereof, may be combined, for example, by mixing, to form an aqueous solution, a wet cake, and an oil mixture, wherein the mixture is fermented. 30. In certain embodiments, aqueous solution 22 and wet cake 24 may be combined to form an aqueous solution and wet cake mixture, and oil 26 is then added to the mixture to form an aqueous solution, wet cake, and oil mixture. May be formed and the mixture may be sent to fermentation 30. In certain embodiments, the aqueous solution 22 and the wet cake 24 may be combined to form an aqueous solution and wet cake mixture, wherein the mixture and the oil 26, or a portion thereof, are sent to the fermentation 30 as separate streams. Can be

  In further embodiments of the methods and systems described herein, saccharification can be performed in a separate saccharification system. In certain embodiments, a saccharification system may be located between the liquefaction 10 and the separation 20 or between the separation 20 and the fermentation 30. In certain embodiments, liquefaction and / or saccharification is performed using a live starch or low-temperature hydrolase such as Stargen ™ (Genencor International, Palo Alto, Calif.) And BPX ™ (Novozymes, Franklinton, NC). Can be done. In certain embodiments, the raw slurry can be subjected to raw starch hydrolysis (also known as low temperature cooking or low temperature hydrolysis).

  In certain embodiments, the systems and methods of the present invention can include a series of two or more separation devices (eg, a centrifuge) for removal of undissolved solids and / or oils. For example, the aqueous solution discharged from the first separation unit can be sent to the inlet of the second separation unit. The first and second separation units can be the same (eg, two three-phase centrifuges) or different (eg, a three-phase centrifuge and a decanter centrifuge). Separation includes decanter bowl centrifugation, three-phase centrifugation, disk stack centrifugation, filtration centrifugation, decanter centrifugation, filtration, vacuum filtration, belt filter, pressure filtration, membrane filtration, filtration using a sieve, sieving separation, It can be performed by any number of means including, but not limited to, a grid, a porous grid, a flotation method, a hydrocyclone, a filter press, a screw press, a gravity settler, a vortex separator, or a combination thereof.

  There are several advantages to having no or minimal undissolved solids in the fermentation broth. For example, the need for an operating unit in downstream processing can be eliminated, thereby increasing the efficiency of product alcohol production. Also, as a result of less undissolved solids in the fermentation broth exiting the fermentor, some or all of the centrifuge used to treat the total distillation effluent can be omitted. Removal of undissolved solids from the raw slurry can improve biomass processing productivity and cost effectiveness. Improved productivity includes improved efficiency of product alcohol production and / or improved extraction activity compared to methods and systems that do not remove undissolved solids prior to fermentation. For a further description of methods and systems for separating undissolved solids from a raw slurry, see, for example, US Patent Application Publication No. 2012/0164302 and PCT International, each of which is incorporated herein by reference in its entirety. See Patent Application No. PCT / US2013 / 51571.

  As described herein, product alcohol can be recovered from the fermentation broth using several methods, including liquid-liquid extraction. In certain embodiments of the methods and systems described herein, an extractant may be used to recover product alcohol from the fermentation broth. The extractant used herein may, for example, have the following properties and / or characteristics: (i) biocompatible with microorganisms, (ii) immiscible with fermentation broth, (iii) production High partition coefficient (Kp) for extraction of alcohol, (iv) low partition coefficient for nutrient and / or water extraction, (v) low viscosity (μ), (vi) High selectivity of product alcohol, (vii) lower density compared to fermentation broth (ρ) or different density compared to fermentation broth density, (viii) boiling point suitable for downstream processing of extractant and product alcohol (Xi) a melting point below ambient temperature, (x) minimal absorption to solids, (xi) a low tendency to form emulsions with fermentation broth, (xii) stability throughout the fermentation process, (xiii) ) Low cost, and (xiv) harmless.

  In certain embodiments, an extractant may be selected based on certain properties and / or characteristics described herein. For example, the viscosity of the extractant can affect the mass transfer properties of the system, ie, the efficiency with which product alcohol can be extracted from the aqueous phase into the extractant phase (ie, the organic phase). The density of the extractant can affect phase separation. In certain embodiments, selectivity refers to the relative amount of product alcohol to water taken up by the extractant. The boiling point can affect the cost and method of product alcohol recovery. For example, if butanol is recovered from the extractant phase by distillation, the boiling point of the extractant can be determined while minimizing the thermal decomposition or side reactions of the extractant, or the need for deep vacuum in the distillation process. Should be low enough to allow for

  The extractant can be biocompatible with the microorganism, ie, harmless to the microorganism or only toxic to the extent that the microorganism is harmed to an acceptable level. 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. The degree of biocompatibility of the extractant can be determined, for example, by the glucose utilization of the microorganism in the presence of the extractant and the product alcohol. In certain embodiments, a non-biocompatible extractant refers to an extractant that interferes with a microorganism's ability to utilize a fermentable carbon source. For example, a non-biocompatible extractant may cause microorganisms to be in excess of about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% of the rate in the absence of the extractant. Glucose is not available at more than% rate.

  One of skill in the art can select an extractant to maximize the desired properties and / or characteristics described herein and optimize product alcohol recovery. One skilled in the art will also understand that it may be advantageous to use a mixture of extractants. For example, the extractant mixture can be used to increase the partition coefficient of the product alcohol. Further, the extractant mixture can be used to adjust and optimize physical properties of the extractant, such as density, boiling point, and viscosity. For example, an extractant having a sufficient partition coefficient and sufficient biocompatibility of the product alcohol to allow the appropriate combination to economically use the extractant to remove product alcohol from the fermentation broth. Can provide.

In certain embodiments, the extractant useful in the methods and systems described herein can be an organic solvent. In certain embodiments, an extractant useful in the methods and systems described herein can be a water-immiscible organic solvent. In certain embodiments, the extraction agent is saturated, monounsaturated, fatty _alcohols, fatty acids C 12 _{-C 22} polyunsaturated _C 12 _{-C 22,} esters of fatty acids of _C 12 ~C _22, C 12 ~C ₂₂ fatty aldehydes, may be C ₁₂ fatty amides -C _22, and an organic extraction agent selected from the group consisting of mixtures thereof. In certain embodiments, the extraction may also contain saturated, monounsaturated, esters of polyunsaturated fatty alcohols _C 4 _{-C 22,} fatty acids C 4 _{-C 28,} fatty acids _C 4 ~C _28, C 4 ~ fatty aldehydes of C _22, may be a C ₄ fatty amide -C _22, and an organic extraction agent selected from the group consisting of mixtures thereof. In some embodiments, the fatty acid _may be a fatty acid of C 4 _{-C 24,} and / or esters _may be esters of fatty acids C 4 _{-C 24.} In certain embodiments, the extraction agent is saturated, monounsaturated, fatty _alcohols, fatty acids C 12 _{-C 18} polyunsaturated _C 12 _{-C 18,} esters of fatty acids of _C 12 ~C _18, C 12 ~C ₁₈ fatty aldehydes, may be C ₁₂ fatty amides -C _18, and an organic extraction agent selected from the group consisting of mixtures thereof. In certain embodiments, the extraction agent is saturated, monounsaturated, fatty alcohols polyunsaturated _C 14 _{-C 18,} fatty acids C 14 _{-C 18,} esters of fatty acids of _C 14 ~C _18, C 14 ~C ₁₈ fatty aldehydes, organic extraction agent selected fatty amides of C ₁₄ -C _18, and from mixtures thereof. In certain embodiments, the extraction agent is saturated, monounsaturated, esters of polyunsaturated fatty alcohols _C 16 _{-C 18,} fatty acids C 16 _{-C 18,} fatty acids _C 16 ~C _18, C 16 ~C It can be an organic extractant selected from the group consisting of ₁₈ fatty aldehydes, C ₁₆ -C ₁₈ fatty amides, and mixtures thereof. In certain embodiments, the extractant may include a carboxylic acid. In certain embodiments, an ester of a fatty acid can be a combination of a fatty acid and an alcohol (eg, a fatty ester). In some embodiments, the alcohol can be a product alcohol. In certain embodiments, the ester can be a methyl, ethyl, propyl, butyl, pentyl, hexyl, or glyceride.

In certain embodiments, the extraction _agent, fatty _alcohols, fatty acids C 12 _{-C 22} of _C 12 _{~C 22,} C 12 esters of fatty acids _{-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 and _C 12 _{-C 22,} fatty acids C 12 _{-C 22,} esters of fatty acids of _C 12 ~C _22, C 12 ~C ₂₂ fatty aldehydes _may include aliphatic amides of C 12 _{-C 22,} and a second extraction agent selected from mixtures thereof. In certain embodiments, the extraction agent _is first extraction agent selected from the C 12 fatty alcohols _{-C 22,} fatty acids C 12 _{-C 22,} esters of fatty acids of C 12 _{-C 22,} and mixtures thereof; and fatty alcohols _C 12 _{-C 22,} fatty acids C 12 _{-C 22,} may include esters of fatty acids of C 12 _{-C 22,} and a second extraction agent selected from mixtures thereof. In certain embodiments, the extraction agent _is first extraction agent selected from C 12 _{-C 18} fatty _alcohols, fatty acids C 12 _{-C 18,} esters of fatty acids of C 12 _{-C 18,} and mixtures thereof; and fatty alcohols _C 12 _{-C 18,} may include fatty acids C 12 _{-C 18,} esters of fatty acids of C 12 _{-C 18,} and a second extraction agent selected from mixtures thereof. In certain embodiments, the extraction agent _is first extractant selected fatty _alcohols, fatty acids C 14 _{-C 18} in C 14 _{-C 18,} esters of fatty acids of C 14 _{-C 18,} and mixtures thereof; and fatty alcohols _C 14 _{-C 18,} fatty acids C 14 _{-C 18,} may include esters of fatty acids of C 14 _{-C 18,} and a second extraction agent selected from mixtures thereof. In certain embodiments, the extraction _agent, fatty _alcohols, fatty acids C 12 _{-C 22} of _C 12 _{~C 22,} C 12 esters of fatty acids _{-C 22,} fatty aldehydes of _{C 12 ~C 22, C 12 ~C} 22 And a first extractant selected from fatty amides, and mixtures thereof; and C ₇ -C ₁₁ fatty alcohols, C ₇ -C ₁₁ fatty acids, esters of C ₇ -C ₁₁ fatty acids, C ₇ -C _Eleven fatty aldehydes, and mixtures thereof.

  In certain embodiments, the extractant is oleyl alcohol, behenyl alcohol, cetyl alcohol, lauryl alcohol (also referred to as 1-dodecanol), myristyl alcohol, stearyl alcohol, oleic acid, lauric acid, linoleic acid, linolenic acid, myristic acid, palmitic acid. , Stearic acid, octanoic acid, decanoic acid, undecanoic acid, methyl myristate, methyl oleate, 1-nonanol, 1-decanol, 2-undecanol, 1-nonanal, 1-undecanol, undecanol, laurinaldehyde, 2-methyl Undecanal, oleamide, linoleamide, palmitoamide, stearylamide, 2-ethyl-1-hexanol, 2-hexyl-1-decanol, 2-octyl-1-dodecanol, and the like It may be an organic extractant such compounds. In certain embodiments, the extractant may include one or more of the following oleic, lauric, linoleic, linolenic, myristic, palmitic, stearic, octanoic, decanoic, and undecanoic acids. . In certain embodiments, the extractant may include one or more of the following oleic, linoleic, linolenic, myristic, palmitic, and stearic acids. In certain embodiments, the extractant may include one or more of the following oleic, linoleic, palmitic, and stearic acids. In certain embodiments, the extractant comprises one or more of the following oleic, lauric, linoleic, linolenic, myristic, palmitic, stearic, octanoic, decanoic, and undecanoic acids, and oleic acid. It may include one or more esters of acids, lauric, linoleic, linolenic, myristic, palmitic, stearic, octanoic, decanoic, and undecanoic acids. In certain embodiments, the extractant comprises one or more of the following oleic, linoleic, linolenic, myristic, palmitic, and stearic acids, and oleic, linoleic, linolenic, myristic, palmitic, Acids and one or more esters of stearic acid may be included. In certain embodiments, the extractant comprises one or more of the following oleic, linoleic, palmitic, and stearic acids, and one or more esters of oleic, linoleic, palmitic, and stearic acids. May be included. In certain embodiments, the extractant may include one or more of the following oleyl alcohol, behenyl alcohol, cetyl alcohol, lauryl alcohol, myristyl alcohol, stearyl alcohol. In certain embodiments, the extractant comprises the following 1-nonanol, 1-decanol, 2-undecanol, 1-nonanal, 1-undecanol, undecanol, 2-ethyl-1-hexanol, 2-hexyl-1-decanol, It may include one or more of 2-octyl-1-dodecanol.

  In certain embodiments, the extractant can be a mixture of a biocompatible extractant and a non-biocompatible 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. Further examples of biocompatible and non-biocompatible extractants are described in U.S. Patent Application Publication No. 2009/0305370 and U.S. Patent Application Publication No. 2011/0097773; the entire contents of each of which are incorporated by reference. Is hereby incorporated by reference herein. In certain embodiments, the biocompatible extractant can have a high atmospheric boiling point. For example, a biocompatible extractant can have an atmospheric boiling point that is higher than the atmospheric boiling point of water.

  In certain embodiments, a hydrophilic solute may be added to a fermentation broth that is contacted with an extractant. The presence of a hydrophilic solute in the aqueous phase can improve phase separation and increase the proportion of product alcohol separated into the organic phase. Examples of hydrophilic solutes include, but are not limited to, polyhydroxylated compounds, polycarboxylic compounds, polyol compounds, and dissociated ionic salts. Saccharides such as glucose, fructose, sucrose, maltose, and oligosaccharides can serve as hydrophilic solutes. Other polyhydroxylated compounds include glycerol, ethylene glycol, propanediol, polyglycerol, and hydroxylated fullerene. Polycarboxylic acid compounds include citric acid, tartaric acid, maleic acid, succinic acid, polyacrylic acid, and their sodium, potassium, or ammonium salts. Ionic salts that can be used as hydrophilic solutes in fermentation broths include cations, including sodium, potassium, ammonium, magnesium, calcium, and zinc; and anions, including sulfate, phosphate, chloride, and nitrate. The amount of hydrophilic solute in the fermentation broth can be changed from the aqueous phase (eg, fermentation broth) to the organic phase (eg, extractant) without adversely affecting the growth and / or productivity of the microorganisms that produce the product alcohol. Can be selected by one skilled in the art to maximize the transfer of the product alcohol. High levels of hydrophilic solutes can impart osmotic stress and / or toxicity to microorganisms. One of skill in the art can use any number of known methods for determining the optimal amount of hydrophilic solute to minimize the effects of osmotic stress and / or toxicity on the microorganism.

  In certain embodiments where the product alcohol is butanol, the extractant can be selected to attract the alkyl portion of butanol and provide little or no affinity for water. For example, extractants that do not provide hydrogen bonds to water selectively absorb alcohol. In certain embodiments, the extractant may include an aromatic compound. In certain embodiments, the extractant is cumene, para-cymene (also known as 1-methyl-4- (1-methylethyl) benzene), meta-cymene (1-methyl-3- (1-methyl). (Also known as ethyl) benzene), meta-diisopropylbenzene, para-diisopropylbenzene, triethylbenzene, ethylbutylbenzene, and alkyl-substituted benzenes, including but not limited to tert-butylstyrene. The advantage of using an alkyl-substituted benzene is the relatively high butanol affinity compared to other hydrocarbons. In addition, isopropyl- or isobutyl-substituted benzenes may provide certain advantages in butanol affinity over other substituted benzenes. Another advantage is lower viscosity, lower surface tension, lower density, higher thermal stability, and higher chemical stability, which aid in phase separability and long-term recycling. In certain embodiments, an extractant that attracts the alkyl portion of butanol may be combined with another extractant that provides affinity, for example, in the form of a hydrogen bond to the hydroxyl portion of butanol, whereby the mixture comprises: Provides an optimal balance of selectivity and partitioning over water. In certain embodiments, the butanol-containing extractant can be a phase that is separated from the fermentation broth and distilled in a column that operates under vacuum. The distillation can operate at reflux to maintain a high purity butanol distillate with little extractant. The bottoms may include a portion of butanol contained in the distillation feed such that the reboil temperature under vacuum is suitable to provide heat indirectly from available steam. The distillation may be performed using a condensator, wherein only the reflux liquid is condensed and substantially the distillate of the butanol composition is decanted from the condensed beer column overhead vapor. Butanol stream can be sent directly to the bottom of the rectification column fed simultaneously. The advantage of this type of distillation is that the heat that captures the vapor generated by removing butanol from the extractant eliminates the need for a reboiler to purify the decanted butanol stream.

  In certain embodiments, the extractant may be produced from a raw material. For example, oils such as corn oil present in the feedstock can be used to produce an extractant for extractive fermentation. The glycerides in the oil are chemically or chemically converted to reaction products such as fatty acids and / or fatty esters (eg, ethyl esters, butyl esters, fusel esters) which can be used as extractants for the recovery of product alcohols. It can be converted enzymatically. Using corn oil as an example, corn oil triglyceride is reacted with a base such as ammonia hydroxide to give fatty amides and glycerol. In certain embodiments, the oil in the feed may be hydrolyzed by a catalyst to produce fatty acids. In certain embodiments, at least a portion of the acylglycerides in the oil can be hydrolyzed to a carboxylic acid by contacting the oil with a catalyst. In certain embodiments, the resulting acid / oil composition comprises monoglycerides and / or diglycerides from partial hydrolysis of the acylglycerides in the oil. In certain embodiments, the resulting acid / oil composition comprises glycerol, a by-product of acylglyceride hydrolysis. In certain embodiments, the resulting acid / oil composition comprises lysophospholipids from partial hydrolysis of phospholipids in oil. Methods for obtaining extractants from biomass are described in 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 of which are incorporated herein by reference.

  In certain embodiments, the conversion (eg, hydrolysis, transesterification) of the oil in the feed or feed slurry can be performed in the fermentor by adding a catalyst to the fermentor. For example, a catalyst such as lipase may be added to the fermentor to convert the oil present in the feed or feed slurry to fatty acids and / or fatty esters. In certain embodiments, the conversion of the oil in the feed or feed slurry may be performed in a separate unit. For example, the feed or feed slurry may be sent to a unit, and a catalyst such as lipase may be added to the unit to convert the oil present in the feed or feed slurry to fatty acids. As another example, the feed or feed slurry may be sent to a unit, and a catalyst such as lipase and an alcohol (eg, ethanol, butanol, fusel alcohol) is added to the unit and the feed or feed slurry is added to the feed or feed slurry. Any oil present may be converted to a fatty ester. In certain embodiments, fatty acids and / or fatty esters may be added to the fermentor and used as an extractant for product alcohol recovery. In certain embodiments, the fatty acids and / or fatty esters may be added to an external extractor or extractant column and used as an extractant for product alcohol recovery.

  In certain embodiments, the oil may be separated from the raw slurry, the oil may be sent to a unit, and a catalyst such as a lipase may be added to the unit to produce a fatty acid stream. The fatty acid stream may be heated to inactivate the lipase, and the fatty acid stream may then be sent to an external extractor or storage tank. Fatty acids from the storage tank can be sent to an external extractor for extraction of product alcohol from the fermentation broth. In certain embodiments, oil separated from the raw slurry may be stored in a storage tank. A catalyst such as lipase can be added to the storage tank to produce a fatty acid stream. The fatty acid stream can be heated to inactivate the lipase, cooled, and then sent to an external extractor for extraction of the product alcohol from the fermentation broth. In certain embodiments, oil separated from the raw slurry may be sent to a unit, and a catalyst such as lipase may be added to the unit to produce a fatty acid stream. The fatty acid stream may be heated and cooled to inactivate the lipase, and then the fatty acid stream may be sent to a fermentor.

  In certain embodiments, one or more catalysts can be one or more enzymes, for example, a hydrolase enzyme. In certain embodiments, one or more catalysts can be one or more enzymes, for example, a lipase enzyme. Lipase enzymes are, for example, Absididia, Achromobacter, Aeromonas, Alcaligenes, Alternaria, Aspergillus, and Aspergillus. Achromobacter, Aureobasidium, Bacillus, Beauveria, Brochothrix, Candida, Chromobas, Chromobacter, Chromobacter Genus (Fusarium), genus Geotricum (Geotri) um), the genus Hansenula, the genus Humicola, the genus Hyphozyma, the genus Lactobacillus, the genus Metarhizium, the genus Mucor, and the genus Necapria (Nacria, Nacria) ), Genus Paecilomyces, genus Penicillium, genus Pseudomonas, genus Rhizoctonia, genus Rhizohorco, genus Rhizorhodiso, Rhizorhodzi Rhizorhodirhos, Rhizorhodos trophy ), Saccharomyces (S ccharomyces, Sus, Sporobolomies, Thermomyces, Thiarosporella, Trichoderma, Trichoderma or Verticillium, Verticillium or Verticillium ) Can be from any source. In certain embodiments, the source of the lipase is from Absidiah blakesleena, Absidiah corimbifera, Achromobacter iophagus, Alkalinea lipagens, Achromobacter alphaenas, and Achromobacter lipagna. Aspergillus flavus, Aspergillus flavus, Aspergillus niger, Aspergillus tubingensis, Aureobasidium ulphia ulbasipurans lans), Bacillus coagulans, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilus schocostrosco schocostrosco sacchos schocostrosco schocostrosco schocostrosco thasco schocostrosco serocho schocostrosco succinco sacro schocostrosco sasa baticos schocostrosco sasa baticos. (Burkholderia cepacia), Candida cylindracea (Candida rugosa), Candida paralipolytica (Candida paralipatica), Candida paralipoantica canta arctica) lipase A, Candida antarctica (Candida antarctica) lipase B, Candida Erunobi (Candida ernobii), Candida Deforumansu (Candida deformans), Candida rugosa (Candida rugosa), Candida parapsilosis (Candida parapsilosis), Chromobacter viscosum, Coprinus cinerius, Fusarium heterosporum, Fusarium oxysporum, Fusarium oxysporium, Fusarium oxysporum Potassium-Soranipishi (Fusarium solanipisi), Fusarium roseum, Kurumorumu (Fusarium roseum culmorum), Geotrichum-Kanjidumu (Geotrichum candidum), Geotrichum-Penishiratsumu (Geotricum penicillatum), Hansenula anomala (Hansenula anomala), Humicola Burebisupora (Humicola brevispora) And Humicola brevis var. Thermoidea (Humicola brevis var. thermoidea), Humicola insolens (Humicola insolens), Lactobacillus Kuruvatusu (Lactobacillus curvatus), Rhizopus oryzae (Rhizopus oryzae), Mucor Jabanikusu (Mucor javanicus), Neurospora crassa (Neurospora crassa), Nectria-Haematokoka (Nectria haematococca) , Penicillium cyclopium, Penicillium crustosum, Penicillium expansum, Penicillium rockforti roqueforti, Penicillium camemberti, Penicillium sp. I, Penicillium sp. II, Pseudomonas aerugonas monas genus moss, Pseudomonas aerugonas monas genus moss, Pseudomonas aerugonas shoganes Pseudomonas Pseudomonas s. Pseudomonas cepacia (also known as Burkholderia cepacia), fluorescent bacteria (Pseudomonas fluorescens), Pseudomonas fraggi (Pseudomonas fragia), and Pseudomonas pseudomona suffia maltophilia), Pseudomonas mendocina (Pseudomonas mendocina), Pseudomonas Mefitika lipolytica (Pseudomonas mephitica lipolytica), Pseudomonas Alcaligenes (Pseudomonas alcaligenes), Pseudomonas Purantari (Pseudomonas plantari), Pseudomonas shoe de alkali monocytogenes (Pseudomonas pseudoalcaligenes), Pseudomonas putida (Pseudomonas putida), Pseudomonas stutzeri, and Pseudomonas wisconsinensis (Pseudomona) wisconsinensis), rice sheath blight fungus (Rhizoctonia solani), Rhizomucor miehei (Rhizomucor miehei), Rhizopus Arizusu (Rhizopus arrhizus), Rhizopus Derema (Rhizopus delemar), Rhizopus japonicus (Rhizopus japonicus), Rhizopus Mikurosuporusu (Rhizopus microsporus, Rhizopus nodosus, Rhizopus oryzae, Rhodosporium torroides, Rhodostrologosa tortoloida, Rhodostrologosa tortoloida, Rhodotoroistrogul, Rhodotoroistrogul, Rhodotoroistrogul, Rhodotoroistrogula Cerevisiae (Saccharomyces cerevisiae), scan polo rags My Seth Shibatanusu (Sporobolomyces shibatanus), wild boar (Sus scrofa), Thermomyces lanuginosus (Thermomyces lanuginosus) (formerly Humicola lanuginosa (Humicola lanuginose)), Chiarosuporera-Faseorina (Thiarosporella phaseolina), It may be selected from the group consisting of Trichoderma harzianum, Trichoderma reesei, and Yarrowia lipolytica.

  In certain embodiments, the hydrolase and / or lipase can be expressed by a microorganism. In certain embodiments, the microorganism can be engineered to express a homologous or heterologous hydrolase and / or lipase. In certain embodiments, the hydrolase and / or lipase may be expressed by a microorganism that also produces the product alcohol. In certain embodiments, the hydrolase and / or lipase may be expressed by a microorganism that also expresses the butanol biosynthetic pathway. In certain embodiments, the butanol biosynthetic pathway can be a 1-butanol biosynthetic pathway, a 2-butanol biosynthetic pathway, an isobutanol biosynthetic pathway, or a 2-butanone biosynthetic pathway.

  Commercially available lipase preparations suitable as catalysts include, but are not limited to, Lipolase® 100 L, Lipex® 100 L, Lipoclean® available from Novozymes (Franklinton, NC). 2000T, Lipozyme (registered trademark) CALB L, Novozyme (registered trademark) CALA L, and Palatase 20000L, or fluorescent bacteria (Pseudomonas fluorescens pseudomonas s. Pseudomonas), available from Sigma Aldrich (St. Louis, MO). ), Mucor miehei, hog pancreas, Candida ki Candida cylindracea, Candida rugosa, Rhizopus niveus, Candida antarctica or Azriz izriz argis, Rhizopus niveus No. In certain embodiments, the lipase may be thermostable and / or thermostable, and / or solvent resistant.

  In certain embodiments, one or more catalysts can be a phospholipase. Phospholipases useful in the present invention can be from various biological sources, such as, but not limited to, Fusarium culmorum, Fusarium heterosporum, Fusarium solani, or Filamentous fungal species within the genus Fusarium, such as strains of Fusarium oxysporum; or Aspergillus awageri, Aspergillus spellus spelli, and Aspergillus sp. Aspergill s niger) or obtained from Aspergillus oryzae (Aspergillus oryzae) of the genus Aspergillus, such as strains (Aspergillus) in the filamentous fungi species. Thermomyces lanuginosus phospholipase mutants such as the product Lecitase® Ultra (Novozymes A'S, Denmark) are also useful in the present invention. One or more phospholipases can be applied as a lyophilized powder, immobilized, or dissolved in an aqueous solution.

  In certain embodiments, the phospholipase can be expressed by a microorganism. In certain embodiments, the microorganism can be engineered to express a homologous or heterologous phospholipase. In certain embodiments, the phospholipase may be expressed by a microorganism that also produces the product alcohol. In certain embodiments, the phospholipase can be expressed by a microorganism that also expresses the butanol biosynthetic pathway. In certain embodiments, the butanol biosynthetic pathway can be a 1-butanol biosynthetic pathway, a 2-butanol biosynthetic pathway, an isobutanol biosynthetic pathway, or a 2-butanone biosynthetic pathway.

  By-products of fermentation, such as isobutyric acid, phenylethanol, 3-methyl-1-butanol, 2-methyl-1-butanol, isobutyraldehyde, acetic acid, ketoisovaleric acid, pyruvic acid, and dihydroxyisovaleric acid are microbial May have an inhibitory effect on In certain embodiments, these by-products may be modified by esterification. For example, by-products can be esterified with carboxylic acids, alcohols, fatty acids, or other by-products. In certain embodiments, these esterification reactions can be catalyzed by lipases or phospholipases. As an example, lipase present in the fermentation broth can catalyze the esterification of by-products produced during the fermentation. Esterification of these by-products can minimize their inhibitory effect on microorganisms.

  Referring to FIG. 7A, raw material 12 is not described in detail as it may be processed as described in FIGS. The aqueous solution 22 may then be further processed to remove any residual oil. In certain embodiments, the aqueous solution 22 may be subjected to centrifugation, decantation, or any other method that may be used for oil removal. In certain embodiments, the aqueous solution 22 may be sent to a unit 25 (or bath) and a catalyst 23 (eg, a lipase) is added to the unit 25 to convert the oil present in the aqueous solution 22 to fatty acids. , Stream 27 may be generated. Stream 27 may then be sent to fermentation 30, and microorganisms 32 may also be added to fermentation 30 for production of product alcohol. After fermentation 30, stream 31 containing product alcohol and fatty acids may be sent to an external unit, for example, an external extractor or an external extraction loop, for product alcohol recovery.

  Referring to FIG. 7B, in certain embodiments, catalyst 23 may be deactivated, for example, by heating. In some embodiments, the stream 27 containing the catalyst 23 is heated (q) to deactivate the catalyst 23 prior to addition to the fermentation 30. Referring to FIG. 7C, in some embodiments, quenching can be performed in a separate unit, for example, the quenching unit. In certain embodiments, stream 27 may be sent to quench 28. After inactivation, stream 27 'may be sent to fermentation 30, and microorganisms 32 may also be added to fermentation 30 for production of product alcohol.

  By removing oil from the aqueous solution 22 by converting the oil to fatty acids, energy savings in the production plant due to more efficient fermentation, less fouling of the equipment due to removal of oil, energy requirements, e.g. A reduction in the energy required for drying and an improved operation of the evaporator or evaporation train are obtained. In addition, removal of the raw oil component is advantageous for product alcohol production because the oil present in the fermentor can be broken down into fatty acids and glycerin. Glycerin can accumulate in water and reduce the amount of water available for reuse throughout the system. Thus, the removal of the feed oil component increases the efficiency of product alcohol production by increasing the amount of water that can be recycled through the system. Also, stable emulsions are less likely to result from oil removal. In certain embodiments of the present invention, when an emulsion is formed, the emulsion can be readily broken down by mechanical treatment, addition of a protic solvent, or other conventional means.

  In another embodiment, referring to FIG. 7D, an aqueous solution 22 may be sent to the fermentation 30 and a catalyst 23 (eg, a lipase) is added to the fermentation 30 to remove the oil present in the aqueous solution 22 from fatty acids. And / or may be converted to a fatty ester. In certain embodiments, the fatty esters are obtained by a combination of fatty acids and alcohols. In certain embodiments, the alcohol can be any alcohol in fermentation 30, including product alcohol. In certain embodiments, the amount of oil in the aqueous solution 22 that is converted to fatty acids and / or fatty esters is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or It can be at least about 95%. In certain embodiments, the ratio of fatty esters and fatty acids produced by the conversion of the oil may be about 75:25. In certain embodiments, the ratio of fatty esters and fatty acids can be about 80:20. In certain embodiments, catalyst 23 may be added to fermentation 30 in an amount that maintains a particular oil conversion.

  After fermentation 30, stream 31 containing product alcohols, fatty acids, and fatty esters may be further processed for product alcohol recovery. For example, stream 31 can be sent to an external unit, for example, an external extractor or external extraction loop, for product alcohol recovery. In certain embodiments, the fatty acids and fatty esters in stream 31 can be used as extractants. In certain embodiments, the external unit may include an extractant. In certain embodiments, the extractant may include fatty acids and / or esters of fatty acids.

  The present invention also provides a method and system for recovering product alcohol produced by a fermentation process. One such process for product alcohol recovery is liquid-liquid extraction. The use of liquid-liquid extraction as an ISPR technique is best provided by a liquid-liquid extraction process that maximizes the net present value of the capital investment required to implement this technique. An aspect that maximizes the net present value of the liquid-liquid extraction process is to avoid the large capital and operating cost expenditures associated with separating the extractant from the fermentation broth.

  In one embodiment of the liquid-liquid extraction process, the extractant may be added directly to the fermentor, and the fermentation broth and the extractant perform mass transfer (eg, transfer of product alcohol from the fermentation broth to the extractant). Mixed together to provide a fermentation that may be able to promote high effective product alcohol titers. In such a process, if the mixing is too strong or too vigorous, the fermentation broth and extractant may need to be separated using a separation device such as a centrifuge. If the mixing is not too vigorous, phase separation can be performed by gravity settling caused by the density difference between the extractant and the fermentation broth. In either case, additional fermenters may be required to overcome the reduction in fermenter volume taken up by the extractant added to the fermenter. Direct addition of the extractant to the fermentor can be performed in a batch, semi-batch, or continuous manner, regardless of the phase separation in the fermenter. If a continuous mode is used and gravity separation of the fermentation broth and extractant is not possible, a separation device such as a centrifuge may be required for separation of the product alcohol from the extractant. If the separation process used to remove product alcohol from the extractant is such that the microorganisms present in the fermentation broth can survive the separation process, the fermentation broth from the product alcohol / extractant is Separation may not be necessary.

  Another embodiment of the liquid-liquid extraction process may include an external extractor or extraction column. For example, fermentation broth from a fermentor may be sent to an external extractor, where the fermentation broth is mixed with an extractant. The mixture of fermentation broth and extractant may then be separated to produce a product alcohol-lean fermentation broth stream and a product alcohol-rich extractant stream. The leaner fermentation broth stream can be returned to the fermentor. The richer extractant stream may be further processed to separate at least a portion of the product alcohol from the extractant for product alcohol recovery. In certain embodiments, the product alcohol recovery from the extractant stream can be set to a rate that maintains factory production. In certain embodiments, the liquid-liquid extraction process may include one or more external liquid-liquid extractors.

  In certain embodiments, the fermentation can be performed in a fermentor and an external extractor. The additional volume of fermentation broth present in the external extractor may serve to increase the overall fermenter volume and thus increase the overall production of product alcohol.

  The performance of the external extractor for removal of product alcohols depends on the surface area available for interfacial contact, the physical properties of the fermentation broth and extractant, the two phases present in the external extractor (eg, fermentation broth phase and (Extractant phase) and the concentration driving force difference between the fermentation broth phase and the extractant phase. Maximizing the efficiency of the external extractor at a given product alcohol concentration driving force can be achieved by reducing the droplet size of the phase dispersed in the external extractor, for example, by nozzle design, internal design, and / or stirring. Can be done by reducing. In certain embodiments, the design and operation of the external extractor is sufficient to provide sufficient product alcohol transfer between the fermentation broth phase and the extractant phase to maintain product alcohol productivity requirements. It can provide sufficient mixing.

In some cases, CO ₂ from the fermentation can be produced in an external extractor, leading to the formation of droplets that can interfere with phase separation. For example, a droplet of the fermentation broth may be attached to the CO ₂ rising through the extraction agent phase. In certain embodiments, the extractant phase may be maintained as a continuous phase to improve droplet aggregation. In certain embodiments, the external extractor may include an outlet port for the interior or CO _2. To improve the coagulation and recovery of the fermentation broth phase, for example, a coagulation pad may be added to an external extractor and / or an outlet port may be arranged.

  Conditions for separating the product alcohol from the fermentation broth can be detrimental to microorganisms present in the fermentation broth. In certain embodiments, the microorganisms can be separated from the fermentation broth before contacting the fermentation broth with the extractant. In certain embodiments, microorganisms can be separated from the mixture of fermentation broth and extractant prior to separation (or processing) of the mixture. Any separation method capable of separating microorganisms from fermentation broth or a mixture of fermentation broth and extractant, including, for example, centrifugation, may be used. Separating the microorganisms prior to contacting the fermentation broth with the extractant may allow for the use of higher temperatures and / or more stringent extraction conditions, such as non-biocompatible extractants. If a separation method that is not harmful to microorganisms is used, it may not be necessary to separate the fermentation broth and extractant prior to product alcohol removal.

  If the extractant and fermentation broth are not separated, the extractant may be included in the evaporator train feedstock and thus be formed during evaporation and possibly become a component of the syrup that is incorporated into animal feed. In certain embodiments, the extractant can be separated from the syrup using any separation means including, for example, centrifugation. Low boiling (eg, water equivalent) biocompatible extractants may not require such separation because the extractant and water can be reused for use in the production process.

  In a typical corn-based product alcohol production plant, by recycling the production plant water while distilling the recycled water in an evaporator train to remove salts and other dissolved solids of beer , The water balance of the whole production process can be maintained. The syrup obtained from the evaporator train may be mixed with the undissolved solid, and the mixture may be dried and sold as animal feed. Methods and systems for treating undissolved solids for animal feed are described, for example, in U.S. Patent Application Publication No. 2012/0164432; U.S. Patent Application Publication No. 2011/0315541; U.S. Patent Application Publication 2013. And PCT International Patent Application No. PCT / US2013 / 51571, the entire contents of each of which are incorporated herein by reference.

  As described herein, undissolved solids can be removed from the feed (or feed slurry) prior to addition of the feed to the fermentation. If undissolved solids are not removed upstream of the fermentation, centrifugation of the beer to remove undissolved solids may be required to avoid fouling of the evaporator. For example, in a commercial production plant for product alcohol from dry milled corn, the undissolved solids content in the evaporator train feed can operate at about 3% of the total suspended solids, It may be as high as 3.5-4% of the suspended solids. Upstream processes that remove enough solids to keep the percentage of total suspended solids below these percentage values may, for example, reduce the need for centrifugation before sending beer to an evaporator (or evaporative train). Can be gone. Eliminating this centrifugation would save the capital required to retrofit a plant for producing product alcohol from dry milled corn.

  By removing at least a portion of the undissolved solids present in the feed slurry prior to fermentation, the interfacial surface area between the fermentation broth phase and the extractant phase in the external extractor increases the undissolved solids at the interface. It can be increased by reducing the amount, promoting product alcohol transfer between the fermentation broth and the extractant, and providing a clear phase separation between the fermentation broth and the extractant. Also, clear phase separation can save capital expenditures, as no additional separation steps (eg, centrifugation) are required.

  Separation of the fermentation broth and the extractant leaving the external extractor is dependent on the particle size distribution of the solid content and the solid content in the fermentation broth, the gas content and cell size distribution in the fermentation broth, viscosity, density and surface tension. Can be affected by, but not limited to, the physical properties of the fermentation broth and extractant and the design and operation of the external extractor and the fermenter. These properties may determine the need for a separation device (eg, a centrifuge) to separate the fermentation broth and extractant leaving the external extractor or fermenter. Operation under conditions that eliminate the need for a separation device may minimize capital expenditures for performing liquid-liquid extraction ISPR. Furthermore, by minimizing the size of the extractor by maximizing the interfacial area between the fermentation broth phase and the extractant phase having the physical properties of a given set of fermentation broth and extractant, the fermentation The ability to phase separate broth and extractant at low cost can be maintained. By eliminating the capital and operating costs of separation devices such as centrifuges, the net present value of plants that produce product alcohol from dry milled corn using a liquid-liquid extraction ISPR process may be increased.

  In another embodiment of the methods and systems described herein, the extractor design, including the phase separation capability, can be adjusted to accommodate the physical properties of the fermentation broth and extractant. If undissolved solids are not removed from the raw slurry or if the concentration of product alcohol in the fermentation broth is too low, sufficient product alcohol to maintain the productivity of the factory using an extractor without a phase separator is provided. Sometimes it cannot be removed. Accordingly, the present invention provides methods and systems that include solids removal and product alcohol recovery using an external extractor, wherein the extractor enhances phase separation capacity for maximum product alcohol recovery. Designed to let.

  An exemplary process of the present invention is shown in FIG. Some processes and flows in FIG. 8 have been identified using the same names and numbers used in FIGS. 1-7, and are the same or similar processes described in FIGS. And flow.

  Feed 12 may be processed and the solids separated as described herein with reference to FIGS. 1-7 (100). Briefly, feedstock 12 may be liquefied to produce a feedstock slurry comprising undissolved solids, fermentable sugar (or fermentable carbon source), and, depending on the feedstock, oil. The raw slurry is then subjected to a separation method to remove suspended solids, producing a wet cake, an aqueous solution 22 (or centrifugate) containing dissolved fermentable sugars, and optionally an oil stream. obtain. For solid separation, decanter bowl centrifugation, three-phase centrifugation, disk stack centrifugation, filtration centrifugation, decanter centrifugation, filtration, vacuum filtration, belt filter, pressure filtration, membrane filtration, microfiltration, filtration using a sieve , Sieving, grids, porous grids, flotation, hydrocyclones, filter presses, screw presses, gravity sedimenters, vortex separators, or combinations thereof, but may be performed by any means.

  The aqueous solution 22 and the microorganisms 32 may be added to the fermentation 30, where the fermentable sugars are fermented by the microorganisms 32 to produce a stream 105 containing product alcohol. In certain embodiments, during fermentation, a portion of stream 105 may be transferred to extractor 120 (or extraction 120), where stream 105 is contacted with extractant 124. In certain embodiments, the extractant may be stored in an extractant storage tank or unit. In certain embodiments, stream 105 may be removed from fermentation 30 when the concentration of product alcohol and / or other metabolites reaches a predetermined concentration. In certain embodiments, the predetermined concentration may be a concentration of product alcohol and / or other metabolites that adversely affect microbial metabolism. In certain embodiments, stream 105 may be removed from fermentation 30 when the fermentation is started. In certain embodiments, stream 105 may be removed from fermentation 30 to minimize the effect of product alcohol on microorganisms 32. In certain embodiments, fermentation 30 may include one, two, three, four, five, six, seven, eight, or more fermenters.

  In certain embodiments, an extractant may be added to fermentation 30. In some embodiments, a portion of the fermentation broth containing the extractant may be transferred to the extractor 120, and in some embodiments, the extractant may be removed from the fermentation broth containing the extractant. By adding an extractant to fermentation 30, ISPR can be initiated at fermentation 30.

  Product alcohol, or a portion thereof, moves from stream 105 to extractant 124, and stream 122, which is richer in product alcohol, may be sent to separation 130. A stream 127 containing the fermentation broth, which is leaner in product alcohol, can be returned to fermentation 30. Separation 130 removes some of the product alcohol from stream 122, and stream 125 containing the leaner extractant can be returned to extractor 120. In some embodiments, extractor 120 can be external to fermentation 30. In certain embodiments, fermentation 30 may include an extractor. In certain embodiments, the extractant, fermentation broth, or both, may be at least partially immiscible. Stream 135 may be sent downstream for further processing including recovery of product alcohol (eg, distillation).

  During extraction, there may be some loss of the extractant or extractant. In certain embodiments, extractant 124 may be supplemented by the addition of extractant to extractor 120 or extractant storage unit. In certain embodiments, for example, where the extractant is obtained from a feed or feed slurry, the extractant 124 may be replenished by converting the oil in the feed or feed slurry to the extractant. For example, a catalyst may be added to fermentation 30 to convert the oil in aqueous solution 22 to fatty acids and / or fatty esters (see, eg, FIG. 7D), including product alcohols, fatty acids, and / or fatty esters. A portion of stream 105 may be transferred to extractor 120, where stream 105 may be contacted with extractant 124. Stream 122 containing the product alcohol-enriched extractant, fatty acid, and / or fatty ester may be sent to separation 130 to produce stream 125 containing the leaner extractant, fatty acid, and / or fatty ester. In some embodiments, stream 125 may be further processed before being returned to extractor 120. For example, the fatty esters present in stream 125 may be subjected to hydrolysis to produce a stream comprising product alcohols and fatty acids. This stream containing product alcohols and fatty acids may be sent to extractor 120, or the stream may be combined with stream 122, and the combined stream may be sent to separation 130. In another embodiment, this stream containing product alcohol and fatty acids may be sent to separation 130, or this stream may be sent to another separation unit to produce a product alcohol stream and a fatty acid stream. obtain. The fatty acid stream may be sent to extractor 120, and the product alcohol stream may be combined with stream 135 and may be further processed for product alcohol recovery.

  In certain embodiments, unacceptable levels of dispersed extractant remain in the fermentation broth returning to the fermentor, because phase separation of the fermentation broth and extractant after passing through the extractor may be insufficient. And / or unacceptable levels of fermentation broth droplets remain in the extractant going to distillation. In certain embodiments, phase separation of the fermentation broth and extractant may be facilitated by treating the heterogeneous mixture exiting the top or bottom of the extractor through one or more hydrocyclones or similar vortex devices. In certain embodiments, a static mixer may be used in place of the extractor to bring the fermentation broth and the extractant into contact with each other, and the heterogeneous mixture formed is composed of a water (eg, fermentation broth) phase and an organic phase. (Eg, extractant) may be pumped through one or more hydrocyclones or similar vortex devices to separate phases. In certain embodiments, one or more hydrocyclones or similar vortex devices can be used to remove liquids or droplets from a gas stream. In certain embodiments, the gas stream may be from a fermentor. In certain embodiments, the gas stream may be from a degassing device.

  In a batch or semi-batch fermentation process, when some of the fermentable sugars are metabolized by microorganisms 32, stream 103 containing beer may be sent downstream to separation 140 to separate product alcohol from beer. A stream 145 containing product alcohol may be sent downstream for further processing including recovery of product alcohol (eg, distillation). In a continuous fermentation process, stream 103 containing beer may be sent downstream to separation 140 to separate product alcohol from beer. A stream 142 containing the total distillation effluent may be sent downstream for further processing, including solids removal and the production of lean distillation effluent.

  In certain embodiments, fermentation 30 may include more than one fermentor, and stream 105 may include a combined plurality of streams from more than one fermentor. In some embodiments, the combined streams may be sent to extractor 120. In certain embodiments, stream 127 may be split and portions of stream 127 may be returned to multiple fermentors. In some embodiments, the extractor 120 can be a series of units connected together in parallel or in series.

  In certain embodiments, the extraction may be performed over a predetermined time period. Extraction may be performed, for example, until the concentration of product alcohol in fermentation 30 is low enough that separation 140 is not required. In certain embodiments, the extraction may be performed over an extended period.

  In certain embodiments of the methods and systems described herein, a decanter can be used for phase separation. In certain embodiments, a decanter may be used in combination with an extractor. In certain embodiments, the surface of the decanter can be modified to improve phase separation. For example, the surface of the decanter can be modified by the addition of hydrophilic and / or hydrophobic surfaces.

  In certain embodiments, oxygen, air, and / or nutrients may be added to stream 125 and / or stream 127. In certain embodiments, the nutrient may be soluble in the extractant. In certain embodiments, the concentration of oxygen may be measured in various flows and may be used as part of a control loop to change the flow of oxygen into the process. In certain embodiments, a mash may be added to the extractor 120 to allow for higher effective titers. In some embodiments, separations 130 and 140 can be extractors. In certain embodiments, these extractors can use water to extract the product alcohol from the extractant, after which the product alcohol can be separated from the aqueous phase. In certain embodiments, the extractant may be injected with a solute that promotes the ability to extract product alcohol from the fermentation broth. In certain embodiments, a surge tank may be placed between the extractor 120 and the separation 130 as a means to balance the concentration of the product alcohol in the extractant prior to the separation (eg, distillation).

In certain embodiments, extractor 120 can be designed to use a CO ₂ generated during the fermentation in order to mix the fermentation broth and extractant. In certain embodiments, extractor 120 can be designed to allow easy separation of CO ₂ in the fermentation broth. This design will facilitate control of the level of mixing by CO ₂ bubbles rising through the extractor 120. In some embodiments, the fermentation broth, in order to minimize the concentration of CO ₂ in stream 105 can be removed from the fermentation 30. In certain embodiments, the design of the extractor break-off area may include a surface to facilitate phase separation between the fermentation broth and the extractant. In certain embodiments, hydrophilic and / or hydrophobic surfaces can be attached to the detachment region to enhance phase separation. In certain embodiments, the external extractor may include an outlet port for the interior or CO _2. For example, a coagulation pad may be added to an external extractor.

In certain embodiments for minimizing CO ₂ mixing, the extractor may be designed to have a smaller diameter at the bottom of the extractor and gradually increase in diameter toward the top of the extractor (eg, cone form). In certain embodiments, the extractor may be designed such that the diameter increases in steps. For example, the extractor may include a first region of constant diameter, followed by a second region of constant diameter via a gradual increase in diameter. In certain embodiments, the extractor may further include a third region of constant diameter via a second gradual increase in diameter. In certain embodiments, the extractor may include one or more gradual increases in diameter. In certain embodiments, the extractor may include one or more regions of a fixed diameter.

During fermentation, the gas content of the fermentation broth (eg, CO ₂ ) changes, and these gases can be removed from the fermentation broth by using a gas stripper. The amount of gas removed from the fermentation broth can be adjusted by varying the flow through the gas stripper and / or the pressure of the gas stripper. In certain embodiments, the amount of CO ₂ in the fermentation broth may be reduced before transferring the fermentation broth to the extractor. For example, CO ₂ may be removed from the fermentation broth using a gas stripper or any means known to those skilled in the art. In certain embodiments, the removal of CO ₂ may be performed at or below ambient pressure. In some embodiments, the fermentation may be followed in the extractor, CO ₂ may be produced by microorganisms. In order to suppress CO ₂ mixture in the extractor to a minimum, in some embodiments, the residence time of the fermentation broth in the extractor may be reduced. In certain embodiments, the residence time can be reduced by adjusting the height of the extractor. In some embodiments, the height of the extractor can be reduced. By reducing the height of the extractor, the number of theoretical extraction stages can be reduced. In certain embodiments, to maintain the theoretical number of extraction stages, the extractor may be replaced with two or more extractors of reduced height. In certain embodiments, two or more extractors may be consecutive. In certain embodiments, two or more extractors may be linked. In certain embodiments, two or more extractors may be connected to maintain countercurrent. In certain embodiments, a degassing stage may be added to one or more extraction stages.

  Referring to FIG. 8, in one embodiment, the size of the dispersed phase droplets in the extractor 120 can be measured and adjusted by various means to increase the rate of mass transfer. For example, droplet size can be determined using a particle size analysis such as focused beam reflectometry (FBRM®) or particle microscopy (PVM®) technology (Mettler-Toledo, LLC, Columnus OH). Can be measured. In certain embodiments, the fermentation broth may be a dispersed phase and the extractant may be a continuous phase, and under these conditions, the solids present in the fermentation broth may not significantly interact with the extractant. Absent. In certain embodiments, the conditions of the separation 130 can be controlled to minimize the effects of oxidative and thermal instability on the extractant.

  In certain embodiments, the quality of the extractant can be monitored and the extractant can be replenished as often as necessary for good production of product alcohol. In certain embodiments, the extractant may be incorporated into the total distillation effluent solid. The total distillation effluent may be separated into a liquid (eg, dilute effluent) stream and a solids stream, and the solids may be washed to recover the extractant. In certain embodiments, the temperature of the extractor 120 can be adjusted to increase the efficiency of the overall process. In certain embodiments, the flow of fermentation broth and extractant to extractor 120 may be co-current or counter-current. In certain embodiments, a membrane can be used to minimize mixing of the fermentation broth and extractant. In certain embodiments, the extractant can be polymer beads or inorganic beads that absorb the product alcohol. In certain embodiments, the polymeric or inorganic beads can preferentially absorb the product alcohol.

  In certain embodiments, measurements such as in-line, on-line, at-line, or real-time measurements may be used to measure the concentration of product alcohol and / or metabolic by-products in various streams. These measurements can be used as part of a control loop to alter the flow between various units or vessels (eg, fermentation 30, extractor 120, separations 130 and 140, etc.) and improve the overall process.

  Another exemplary process of the present invention is shown in FIG. Some processes and flows in FIG. 9 are identified using the same names and numbers as used in FIGS. 1-8, and are the same or similar processes as described in FIGS. And flow.

  Feed 12 may be processed and the solids separated as described herein with reference to FIGS. 1-7 (100). In certain embodiments, feedstock 12 may be mixed with recycle water (eg, stream 162) generated by evaporation 160. As described herein, the raw slurry is subjected to a separation method to remove suspended solids, the wet cake 24, the aqueous solution 22 containing dissolved fermentable sugars (or centrifuge), Depending on the feedstock and the raw material, an oil may be produced. The wet cake 24 can be dried in a dryer 170 and used to produce DDGS. In certain embodiments, the wet cake 24 is reslurried with water (eg, recycled water / stream 162) and subjected to a separation to remove additional fermentable sugars and washed wet cake (eg, 74, 74 ′) described in FIGS. 4 and 5 can be generated. In certain embodiments, the wet cake streams 24, 74, and 74 'may be combined, and the combined wet cake streams may be dried in dryer 170 and used to produce DDGS.

  The aqueous solution 22 and the microorganism 32 may be added to the fermentation 30, where the fermentable sugars are metabolized by the microorganism 32 to produce a stream 105 containing product alcohol. In certain embodiments, enzymes may be added to fermentation 30. Stream 105 may be sent to extractor 120 and may be contacted with extractant 124. A stream 127 containing the fermentation broth, which is leaner in product alcohol, may be returned to fermentation 30, and a stream 122 containing the extractant, in which the product alcohol is richer, may be sent to separation 130. In certain embodiments, extractor 120 may be operated such that stream 122 contains the smallest cell mass and the smallest substrate. Separation 130 can damage microorganisms 32 or the substrate, thereby reducing the fermentation rate. By operating the extractor 120 with the smallest cell mass and substrate, the potential for damage from the separation 130 can be minimized. Stream 125 containing the leaner extractant may be returned to extractor 120. Stream 135 from separation 130 may be sent to purification 150 for further processing, including recovery of product alcohol. In certain embodiments, an extractant may be added to fermentation 30. In some embodiments, a portion of the fermentation broth containing the extractant may be transferred to the extractor 120, and in some embodiments, the extractant may be recovered from the fermentation broth containing the extractant. In certain embodiments, the flow rates of fermentation broth and extractant to the extractor can be adjusted to improve phase separation. For example, lowering the overall flow rate into the extractor at an early or late stage of the fermentation can improve the phase separation of the fermentation broth and extractant.

  As described herein, after a batch fermentation process or as a steady effluent stream in a continuous fermentation process, stream 103 containing beer is separated downstream to separate product alcohol from total distillation effluent 142. 140. Using an upstream solids removal process may reduce the amount of undissolved solids in the lean mash, so that it may not be necessary to centrifuge the total distillation effluent 142 to remove solids. Accordingly, the total distillation waste 142 can be sent directly to the evaporator 160. Syrup 165 produced by evaporation 160 can be mixed with wet cakes 24, 74, 74 'in dryer 170 to form DDGS.

  In certain embodiments, a backflow comprising total suspended solids (TSS) from the total distillation effluent can be used (or recycled) for the preparation of the raw slurry. In certain embodiments, the total distillation effluent or a portion of the total distillation effluent may be processed prior to evaporation by a solids separation system including, but not limited to, turbo filtration or ultracentrifugation, or Total distillation effluent or a portion of the total distillation effluent can be treated for self-cleaning water purification.

  In certain embodiments where coarse solids are removed from the liquefied mash, the total distillation effluent produced may contain fine solids and insoluble microbial fragments, and these dispersed solids may be turbocharged. It can be removed using a type filtration. Turbo filtration may involve subjecting the feed suspension to centrifugal movement through a filter that may retain fine solids. These fine solids when formed into a wet cake may contain some extractant that is absorbed both on the surface and inside the pores of the fine grain particles. In some cases, washing the wet cake with water is insufficient to recover the extractant from the wet cake. In certain embodiments, a concentrated product alcohol stream, such as an organic phase, may be used to recover the extractant from the total distillation effluent wet cake. In certain embodiments, the organic phase can be formed in a decanter. In certain embodiments, the wet cake washed with product alcohol can then be washed with water to recover product alcohol from the wet cake.

  In certain embodiments, the methods and systems described herein can include an extractant reservoir (or tank or tank). An extractant may be added to the extractant reservoir, which may be recycled to the extractor. In certain embodiments, the extractant may be sent to an extractor, and the flow from the extractor may be returned to the extractant reservoir. In certain embodiments, the extractant from the extractant reservoir can be recycled to the extractor and / or fermentor. In certain embodiments, the extractant stream may be circulated between the extractant reservoir, the extractor, and the fermentor. In certain embodiments, upon completion of the fermentation, the contents of the extractant reservoir, extractor, and / or fermenter may be further processed to recover product alcohol.

  Separation or extraction of the product alcohol from the extractant can be accomplished by methods known in the art, including, but not limited to, siphoning, decantation, centrifugation, gravity sedimentation, membrane phase separation, and the like. Can be performed using In certain embodiments, extraction can be performed, for example, using a mixer settler. A mixer settler is a stage-wise extractor, comprising a pump, stirrer, static mixer, mixing tee, impingement device, circulating screen, or It can be used with various elements for mixing, such as a raining bucket. Examples of mixer settlers are shown in FIGS. For example, FIG. 10A shows a mixer settler using a pump as a source of mixing. FIG. 10B shows a mixer settler using a mixer as the source of mixing. FIG. 10C shows a mixer settler using a static mixer as the source of mixing. FIG. 10D shows a mixer settler using a mixing tee as the source of mixing. FIG. 10E shows a mixer settler using an impingement mixer as the source of mixing. FIG. 10F shows a mixer settler using a laying bucket or mesh screen as the source of mixing. FIG. 10G shows a mixer settler using a centrifuge as the settler. FIG. 10H shows a mixer settler using a hydrocyclone or vortex separator as the settler. In certain embodiments, one or more mixing devices can be used in the methods and systems described herein.

  In certain embodiments, the mixer may include an agitator such as, for example, a flat blade, pitched blade turbine, or a curved propeller. The droplet size produced by the agitated mixer can be controlled by the agitator design, tank design, agitator speed, and operating mode. In the case of a static mixer, the droplet size can be controlled by the diameter and flow rate of the mixer. For example, the droplet size can be controlled by changing the flow rate through the mixer during fermentation. In certain embodiments, a gas and a mixer may be used in the mixing process.

  In certain embodiments, one or more mixer settlers may be used in the methods and systems described herein. In certain embodiments, one or more mixer settlers may be arranged in series or in countercurrent mode, as shown in FIGS. 10I and 10J. In certain embodiments, the mixer settlers can be stacked in a column arrangement to provide multiple mixing and sedimentation areas. In certain embodiments, the settler may include a hydrophilic or hydrophobic surface to facilitate phase separation.

  In another embodiment, a column or centrifugal extractor can be used in the methods and systems described herein. A column extractor is a differential extractor that provides conditions for mass transfer over the length of the extractor with a steadily changing concentration profile. Different types of differential extractors can be classified as non-mechanical, pulse-stirred, and rotary-stirred. Centrifugal extractors are another type of differential extractor with one such type, the Podbielniak® centrifugal contactor.

  In certain embodiments, non-mechanical spray towers can be used in the methods and systems described herein. An example of a non-mechanical spray tower is a non-mechanical spray tower that does not include the inside of the column. The number of nozzles and nozzle diameter can be used to determine droplet diameter. In some embodiments, the spray tower can have an interior. In certain embodiments, the spray tower may include spiral tubing. The helical tubing may allow for the rise of droplets and further mixing of fermentation broth and extractant. In certain embodiments, non-mechanical extractors such as packed towers, sieve trays, and baffle trays can be used in the methods and systems described herein. Examples of these extractors are shown in FIG. 10K. In certain embodiments, the filling of such extractors may be random or structured.

  In certain embodiments, a pulse agitated extractor may be used in the methods and systems described herein. Pulse agitated extractors also have a variety of designs, including reciprocating trays or diaphragms where the tray moves vertically. The overall packed tower and / or sieve tray column may also oscillate vertically to promote smaller dispersed phase droplets and more mass transfer. Examples of these extractors are shown in FIG. 10L. In certain embodiments, a rotating agitated or rotating disk contactor may be used in the methods and systems described herein. Examples of these extractors are shown in FIG. 10M.

  In certain embodiments, a stirred extractor can be used in the methods and systems described herein. For example, a stirred extractor with a centrifuge can provide high mass transfer rates and distinct phase separation. In certain embodiments, a stirred column may be used in the methods and systems described herein. For example, a stirred column with an interior can provide high mass transfer rates.

  One aspect of the liquid-liquid extraction process determines the good operating conditions of the extractor during a constantly changing fermentation. For example, a typical batch fermentation of corn to product alcohol is a first inoculation of a microorganism (or cell mass) that is added to a specific volume of fermentation broth in a fermenter, followed by up to a predetermined volume. An additional charge of the fermenter is used. Fermentation is continued until a predetermined amount of a fermentable carbon source (eg, sugar) has been consumed. During batch fermentation, the concentrations of cell clumps, reaction intermediates, by-products of the reaction, and substrate components change over time, as well as the physical properties of the fermentation broth, including viscosity, density, and surface tension. The extractor supplements the changing fermentation broth to improve the performance parameters of the fermentation, e.g., speed, titer, and yield parameters of production, and the economics of the plant, such as sales, return on investment, and profits. Can be driven in various ways. In addition, the characteristics of the kinetic fermentation can affect the size limit of the extractor. Proper integration of the operation of the extractor and fermenter can benefit from the use of a mechanical model of the process (see, for example, Daugulis and Kollerup, Biotechnology and Bioengineering 27: 1345-1356, 1985). It is also beneficial to reinforce the mechanical model, for example, to set key model parameters with experimental data. The design parameters of the differential extractor to take into account the improved speed, titer, and yield of the fermentation process include the maximum total flow to the extractor per cross-sectional area of the extractor column and the given fermentation broth The extractor height required to remove sufficient product alcohol at the extractant ratio is mentioned. It may be necessary to vary the maximum flow rate per unit area and the height of the extractor during batch fermentation. Another consideration of the differential extractor is the droplet size of the dispersed phase. A suitable droplet size can be a balance between small enough to provide sufficient mass transfer and large enough to allow a clear phase separation exiting the extractor. In a staged extractor, the mixing intensity required for efficient mass transfer, the corresponding time required for sedimentation, and / or the energy required to separate phases should be considered. It is a further factor. In either type of extractor, a staged extractor or a differential extractor, the ratio of fermentation broth to extractant supplied to the extractor plays a role in determining the size of the extractor.

  In certain embodiments, a fixed size extractor is used and the maximum allowable flow to avoid flooding the extractor is determined during the fermentation broth due to changes in the physical properties and concentration of the fermentation broth. If the fermentation is varied from a low value to a high value (eg, 1/3 to 2/3 of the maximum for a given extractor design), the fermentation is completed in a reasonable time while the flow to the extractor is Can be changed without exceeding. In certain embodiments, when the extractor is agitated, the speed of agitation may be varied during the fermentation to compensate for changes in the fermentation broth. Drop size may be measured in the extractor, and the speed to maintain a fixed drop size may be controlled throughout the fermentation to compensate for changes in the fermentation broth. The amount of mass transfer occurring at any given time measures the concentration of product alcohol in the inlet and outlet streams and adjusts conditions (eg, flow rates, flow ratios, agitation) to control mass transfer during fermentation Can be evaluated by:

  In certain embodiments, multiple extractors of various sizes may be used, such that the conditions (eg, flow rates, flow ratios, agitation) in each extractor provide improved control of the fermentation process. Can be adjusted. In certain embodiments, the ratio of fermentation broth to extractant increases the extraction efficiency, increases the concentration of product alcohol in the extractant (equivalent to increased efficiency), and reduces the required flow through the extractor. Can be adjusted to

  In further embodiments of the methods and systems described herein, there may be more than one fermentation broth or stream. An extractant phase having product alcohol absorbed from the first stream is contacted with a second stream containing less product alcohol or fermentation broth than the first stream to remove the second extract stream from the rich extractant phase. The transfer of the product alcohol to the aqueous phase may be possible. In certain embodiments, contacting the rich extractant with the dilute water stream may be performed with a multi-stage contact device or a static mixer, followed by a settler. In certain embodiments, contacting the rich extractant with the dilute water stream may be performed in the same device, wherein the dilute extractant is contacted with the fermentation broth. Extractors with perforated baffles may allow for downflow of both fermentation broth and dilute water streams in separate compartments, while extractants lean in product alcohol form a continuous phase across all compartments. obtain. The advantage of this configuration is the reduced amount of extractant that may be required in a production plant if the extractant remains limited to the closed volume of the extractor. Another advantage of this configuration is that the extractant can exhibit a longer service life because there is no possibility of decomposition during distillation. By transferring the product alcohol to a homogeneous water stream, the product alcohol can be sent to two or more strip columns by distribution of a dilute water stream, taking into account column capacity and thermal integration. If the product alcohol is extracted into the aqueous medium during or shortly after the product alcohol is extracted from the fermentation broth, the need to clean equipment exposed to the extractant may be reduced.

  In certain embodiments, product alcohol may be transferred from the fermentation broth to a second water stream or extractant across a selective barrier to the transfer of product alcohol. In certain embodiments, this barrier may be provided by a membrane material. The membrane material can be either organic or inorganic. Examples of membrane materials include polymers and ceramics. In certain embodiments, the product alcohol can be separated from the fermentation broth using a hydrogel. In certain embodiments, the hydrogel may include functional elements that facilitate interaction with the product alcohol, such as, but not limited to, hydroxyl functionality, hydrocarbon properties, and network size. In certain embodiments, the hydrogel may include a polymer network or polymer blend. Examples of polymer blends include, but are not limited to: acrylic acid, sodium acrylate, hydroxyethyl acrylate, methacrylate, hydroxybutyl acrylate, butyl acrylate, vinylated polyethylene oxide, vinylated propylene oxide, vinylated Examples include one or more of polytetramethylene oxide, acrylates and diacrylates of polyglycols, polyvinyl alcohol and hydrocarbon derivatized polyvinyl alcohol, and styrene and styrene derivatives. In certain embodiments, the hydrogel may include hydroxyethyl acrylate and methacrylate, hydroxybutyl acrylate and methacrylate, or butyl acrylate and methacrylate.

In another embodiment of the methods and systems described herein, the fermentation broth is removed from the bottom of the fermentor at a pressure above atmospheric pressure and passed through a first flash tank operating at atmospheric pressure, Dissolved gases such as CO ₂ can be released. The first flash tank may be a degassed cyclone, and steam from the first flash tank may be combined with steam from the fermenter and sent to a scrubber. In some embodiments, the fermentation broth from the first flash tank is passed through a second flash tank operating at a pressure less than atmospheric pressure, more soluble gases such as CO ₂ can be released. This second flash tank may be a degassed cyclone, and the steam from this second flash tank is recompressed to atmospheric pressure, cooled and partially condensed before the It can be combined with steam and sent to a scrubber. The fermentation broth leaving this second flash tank is injected into an extraction column operating at a pressure above atmospheric so that any residual or newly formed dissolved gas does not form a gas phase in the extraction column. obtain.

  In another embodiment of the methods and systems described herein, the fermentation broth is sent to an extractor and contacted with an extractant to produce a water stream and an organic stream comprising the extractant and product alcohol. Can be done. This organic stream can be sent to a flash tank (eg, a vacuum flash) for separation of the product alcohol from the extractant. In certain embodiments, the extractant stream from the flash tank can be recycled to the extractor and / or the fermentor. In some embodiments, the organic stream can be sent to a second extractor before the flash tank. This second extractor can be used, for example, to remove any residual water in the organic stream. The extractor can be a siphon, decanter, centrifuge, gravity settler, mixer settler, or a combination thereof. In certain embodiments, the extractant includes, but is not limited to, tallow, corn oil, canola oil, capric acid / caprylic triglyceride, castor oil, coconut oil, cottonseed oil, fish oil, jojoba oil, lard, linseed oil, beef oil, beef Oils such as foot oil, deer oil, coconut oil, peanut oil, rapeseed oil, rice oil, safflower oil, soybean oil, sunflower oil, drill oil, jatropha oil, and vegetable oil blends, or fatty acids derived therefrom.

  In certain embodiments of the methods and systems described herein, automatic self-cleaning filtration may be used for these methods and systems. The fermentation broth may be removed from the fermentor and may be cooled using a cooler (eg, an existing cooler in a fermentation production facility) before entering an automatic self-cleaning filter. Some particulate may be retained on the filter screen media as the purified mash passes through the filter. An additional filter may simultaneously backflush, where a portion of the refined mash may flow back through the screen carrying the particulates and a concentrated solids stream may be discharged. In certain embodiments, a portion of the purified mash may enter the top of the extractor, while the extractant is provided at the bottom of the extractor. The purified mash and extractant can be contacted either passively by density difference or using mechanical movement by means commonly used in the art (eg, a Karr® column). In certain embodiments, an organic liquid stream of extractant containing product alcohol exits the top of the extractor and is an aqueous liquid of a fermentation broth with at least partially reduced product alcohol compared to purified mash. A stream exits the bottom of the extractor. The aqueous liquid stream and the concentrated solid stream can be combined and returned to the fermentor. The product alcohol-rich extractant stream may be heated in a heat exchanger, which transfers heat from the product alcohol-lean extractant stream originating from the bottom of the extractor. After releasing some heat, the dilute extractant can be further cooled with water in a heat exchanger until a temperature suitable for fermentation is reached. The circulation of the fermentation broth may include pathways through heat transfer and mass transfer devices, allowing removal of heat and product alcohol each time it passes through an external cooling loop. Further, in some embodiments, the rate of heat and product alcohol removal regulates the circulation flow through the external cooling loop, regulates the flow of cooling fluid in the heat exchanger, and / or regulates the flow of extractant. By adjusting, the rate of heat and product alcohol production during fermentation can be balanced.

  In certain embodiments of the methods and systems described herein, phase separation of the extractant from the fermentation broth may be facilitated by adjusting the temperature and / or pH of the process. For example, the process may be operated at a different temperature and / or pH than the temperature and / or pH of the fermentor. In certain embodiments, the process may be operated at a reduced pH as compared to the fermentor. In certain embodiments, the process may be operated at a higher temperature compared to the fermentor. In certain embodiments, the process may be operated at a reduced pH and higher temperature compared to the fermentor. Higher temperatures can improve the kinetics of product alcohol mass transfer between the aqueous and organic phases, with extractant droplets dispersed in the aqueous phase and water droplets dispersed in the organic phase. Can improve the dynamic characteristics of aggregation. In certain embodiments, the temperature inside the extractor containing the fermentation broth and extractant can be increased by heating the fermentation broth and / or extractant entering the extractor. The fermentation broth can be heated directly by injection of steam or steam or indirectly via a heat exchanger. In certain embodiments, the extractant supplied to the extractor may result from a distillation whose temperature may already be raised. In certain embodiments, the extractant may be cooled to a temperature above the fermentation temperature.

In certain embodiments, the reduced pH can minimize the solubility and dispersibility of the extractant in the aqueous broth phase. In certain embodiments, the extractant can be a fatty acid with a known and relevant pKa value. In certain embodiments, the pH of the fermentation broth can be reduced below the pKa of the extractant such that the carboxylic acid groups of the fatty acid are substantially protonated. In certain embodiments, the pH can be lowered by introducing CO ₂ gas into the fermentation broth or by injecting a small amount of liquid acid into the fermentation broth, such as sulfuric acid or any other organic or inorganic acid. In certain embodiments, the pH of the fermentation broth after separation from the extractant can be adjusted to the pH of the fermentation.

  In certain embodiments, where the extractant phase is a continuous phase, the aqueous phase may be partitioned or dispersed in the extractant phase. For example, a fermentation broth containing product alcohol can be sent to an extractor (eg, an external extractor) by a distributor or dispersion device. In certain embodiments, the distributor or dispersion device can be a nozzle, such as a spray nozzle. In certain embodiments, the distributor or dispersion device can be a spray tower. As an example, the droplets of the fermentation broth may be passed through an extractant, and the product alcohol is transferred to the extractant. Drops of fermentation broth can aggregate at the bottom of the extractor and be returned to the fermentor. The extractant comprising product alcohol may be further processed for recovery of the product alcohol described herein. In addition, at the completion of the fermentation, the product alcohol remaining in the fermentor can be further processed for product alcohol recovery. In certain embodiments, the extractant phase may be countercurrent.

  In certain embodiments, where the extractant phase is a continuous phase and the aqueous phase is a dispersed phase, the mass transfer rate can be enhanced using electrostatic spraying to disperse the aqueous phase in the extractant phase. In some embodiments, one or more spray nozzles can be used for electrostatic spraying. In certain embodiments, one or more spray nozzles can be an anode. In some embodiments, one or more spray nozzles can be a cathode.

  In certain embodiments, extractor effluent can be used to promote phase separation. For example, a portion of the rich extractant from the top of the extractor (ie, the product alcohol-rich extractant) may be returned to the top of the extractor as reflux, while the remaining rich extractant is It can be further processed for recovery of the product alcohol. Also, a portion of the lean fermentation broth from the bottom of the extractor may be returned to the bottom of the extractor as reflux, and the remaining lean fermentation broth may be returned to the fermentor. In another embodiment, the rich extractant exits the top of the extractor and enters the decanter, where it can be separated into a heavy phase and a light phase. The heavy phase from the decanter can be sent to the top of the extractor to facilitate phase separation. The light phase from the decanter can be further processed for product alcohol recovery.

  In certain embodiments of the methods and systems described herein, multiple extractant flows may be used for product alcohol recovery. For example, multiple fermenters and extractors at different points in the fermentation cycle of each fermentor may be used. Referring to FIG. 11A as an example, fermentation apparatus 300 is at an earlier time as compared to fermentation apparatus 400, and fermentation apparatus 400 is at an earlier time as compared to fermentation apparatus 500. A fermentation broth containing the product alcohol 302 from the fermentor 300 may be contacted with the extractant 307 in the extractor 305, and the product alcohol is transferred to the extractant and the product alcohol-enriched extractant 309 Can be generated. Product alcohol-enriched extractant 309 from extractor 305 may be sent to extractor 405. Fermentation broth containing product alcohol 402 from fermentation apparatus 400 may be sent to extractor 405 to produce product alcohol-enriched extractant 409. Product alcohol-enriched extractant 409 may be sent to extractor 505. The fermentation broth containing the product alcohol 502 from the fermenter 500 can be sent to the extractor 505. Product alcohol enriched extractant 509 from extractor 505 can be processed for product alcohol recovery. Product alcohol-lean fermentation broth (304, 404, 504) may be returned to fermentors 300, 400, and 500, respectively. The number of fermenters and extractors can vary depending on the operating equipment. The advantages of this process are, for example, total extractant treatment and a reduction in the size of the extractor.

  In another embodiment of this example, there may be a further fermenter F 'and a further extractor E' (FIG. 11B). In this embodiment, when the fermentor 500 (at a later point in time compared to the fermenters 300 and 400) completes fermentation, the fermenter 500 may be taken off-line, In certain embodiments, the fermentation apparatus 500 may perform sanitary and / or sterilization procedures, such as clean-in-place (CIP) and sterilization-in-place (SIP) procedures. When the fermentation apparatus 500 is deactivated, the fermentation apparatus F 'can be run on-line. In this embodiment, the fermentation device F 'is at an earlier time than the fermentation device 300, and the fermentation device 300 is at an earlier time as compared to the fermentation device 400. Similar to the description for FIG. 11A, a fermentation broth containing product alcohol F′-02 from fermentation apparatus F ′ may be contacted with an extractant in extractor E ′, wherein the product alcohol is added to the extractant. To produce a product alcohol-enriched extractant E'-09. Product alcohol-enriched extractant E'-09 from extractor E 'may be sent to extractor 305. A fermentation broth containing product alcohol 302 from fermentation apparatus 300 may be sent to extractor 305 to produce product alcohol-enriched extractant 309. Product alcohol-enriched extractant 309 may be sent to extractor 405. Fermentation broth containing product alcohol 402 from fermentation apparatus 400 may be sent to extractor 405. Product alcohol-enriched extractant 409 from extractor 405 can be processed for product alcohol recovery. The product alcohol-lean fermentation broth (F'-04, 304, 404) may be returned to fermenters F ', 300, and 400, respectively. In certain embodiments, the process comprises multiple cycles, for example, at least one, at least two, at least three, at least four, at least five, at least ten, at least fifteen, at least twenty, or more. It can be repeated over a cycle. In certain embodiments, the process of deactivating the fermentor and operating additional fermenters may be manual or automated. The advantage of this process is the reduced extractor flow to product recovery (eg, distillation).

  In certain embodiments, the extractant can reduce the flash point (ie, flammability) of the product alcohol. Flash point refers to the lowest temperature at which flame propagation occurs over the surface of a liquid. The flash point can be measured, for example, using the ASTM D93-02 method ("Standard Test Methods for Flash Point by Pensky-Martens Closed Tester"). The lower flash point of the product alcohol can improve the safety conditions of the alcohol production plant, for example, by minimizing the risk of fire of potentially flammable product alcohol. By improving safety conditions, the risk of injury as well as the risk of property damage and loss of revenue is minimized. In certain embodiments where microbial inactivation is required, the extractant may enhance microbial inactivation.

  In certain embodiments, the processes described herein are integrated, for example, using online, in-line, at-line, and / or real-time measurements of fermentation broth and extractant concentrations and other physical properties. It can be an extractive fermentation process. These measurements can be used, for example, in a feedback loop to adjust and control fermentation conditions and / or extractor conditions. In certain embodiments, the concentration of product alcohol and / or other metabolites and substrates in the fermentation broth is measured using any suitable measurement device for online, in-line, at-line, and / or real-time measurements. obtain. In certain embodiments, the measuring device is: a Fourier transform infrared spectrometer (FTIR), a near infrared spectrometer (NIR), a Raman spectrometer, a high pressure liquid chromatography (HPLC), a viscometer, a density meter, a tensiometer. , A droplet size analyzer, a pH meter, a dissolved oxygen (DO) probe, and the like. In certain embodiments, off-gas released from the fermentor can be analyzed, for example, by an in-line mass spectrometer. Measurement of the off-gas released from the fermentor can be used as a means to identify chemical species present during the fermentation reaction. The concentration of product alcohol and other metabolites and substrates dissolved in the extractant 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 a control system, wherein the conditions within the fermentor (temperature, pH, nutrients, enzyme and / or substrate concentrations) may include And / or conditions in the extractor (fermentation broth flow, fermentation broth to extractant flow, agitation speed, droplet size, temperature, pH, DO content) can be modified. Similarly, the conditions in the extractor can be modified to maintain the concentration and / or concentration profile in the fermentor. 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 the fermentation can be provided by varying concentrations of components (eg, sugars, enzymes, nutrients, etc.) in the fermenter, varying conditions in the extractor, or both.

  As an example of an isobutanol fermentation process, the efficiency of isobutanol extraction in a Karr® column changes continuously as the concentrations of starch, sugars and isobutanol in the fermentation broth change. To maximize the efficiency of the extractor, it may be advantageous to change the rate at which isobutanol is removed from the fermentation broth to match the production profile of the isobutanol fermentation. The isobutanol concentration in the extractant is maximized, resulting in a more energy efficient distillation operation.

  As part of a process control method, real-time measurement of isobutanol in fermentation broth (eg, column feed) can be combined with real-time measurement of isobutanol in extractants and dilute fermentation broth. These measurements can be used to adjust the ratio (flow rate) of fermentation broth to extractant to the extractor. The flexibility to match the rate of isobutanol extraction with the rate of isobutanol production allows the extractor to operate efficiently throughout the extraction. Furthermore, by maintaining a high concentration of isobutanol in the extractant, the volume flow to the distillation column can be minimized, saving energy for the distillation operation. Phase separation can also be monitored using real-time measurements, for example, by monitoring the rate of phase separation, extractant droplet size, and / or fermentation broth composition. In certain embodiments, phase separation may be monitored by conductivity measurements, dielectric constant measurements, viscoelastic measurements, or ultrasonic measurements. In certain embodiments, an automated phase separation detection system can be used to monitor phase separation. This automated system, for example, adjusts the flow of fermentation broth and extractant to and from the extractor after mixing the fermentation broth and extractant, and / or adjusts the droplet size of the extractant. Can be used for With these real-time monitoring systems, a clear phase separation of the aqueous and organic phases is obtained.

  As another example of a process control method, a droplet diameter is measured using a process particle analyzer (JM Canty, Inc., Buffalo, NY), a focused beam reflection measurement method (FBRM (registered trademark)), or a particle microscopic image measurement method. (PVM®) technology (Mettler-Toledo, LLC, Columnus OH). In certain embodiments, these measurements can be real-time in situ particle system characterization. By monitoring the droplet size in real time, changes in droplet shape and size can be detected, and process steps can be adjusted to adjust the droplet size and promote the rate of mass transfer. For example, droplet size can be used to monitor the amount of extractant in a fermentation broth. After phase separation, some extractant may be present in the fermentation broth, and in certain embodiments where the fermentation broth is recycled to the fermenter, monitoring of the droplet size may be performed during the fermentation broth returning to the fermenter. Will provide a means of minimizing the amount of extractant of the liposome. If the amount of extractant in the fermentation broth is too high, phase separation can be achieved, for example, by adjusting the droplet size of the extractant in the extractor and / or by adjusting the flow rate of fermentation broth and extractant to the extractor. Can be improved. These adjustments during the process steps can minimize the amount of extractant in the fermentation broth, as well as minimize the amount of extractant in the distillate waste liquor and DDGS.

  In one embodiment of this control method, the isobutanol in the fermentation broth will not exceed a concentration or set point at which the concentration of isobutanol is detrimental to microorganisms. The isobutanol fermentation broth set point may be adjusted higher or lower as the fermentation proceeds based on the course of the fermentation. For example, a continuous comparison of the concentration of isobutanol in the fermentation broth and the set point concentration of isobutanol is used to adjust the ratio of fermentation broth to extractant or the flow of fermentation broth and extractant to the extractor. obtain. To control the concentration of isobutanol in the fermentation broth, in situ measurements of the fermentation broth were performed using Fourier Transform Infrared Spectroscopy (FTIR), Near Infrared Spectroscopy (NIR), and / or Raman Spectroscopy. Can be. Further, the measurement of the fermenter headspace can be performed using FTIR, Raman spectroscopy, and / or mass spectrometry.

  In certain embodiments, efficient extractor operation can be performed to just before flooding of the extractor. The use of real-time process control using concentration data from inlet and outlet streams allows the extractor to be easily operated to just before flooding. In certain embodiments, real-time extractant monitoring can be used to detect the distribution of by-products or contaminants from the fermentation broth into the extractant. By-products such as alcohols, lipids, oils, and other fermentation components can reduce the extraction efficiency of the extractant. Many process monitoring techniques include, but are not limited to, Fourier Transform Infrared Spectroscopy (FTIR), Near Infrared Spectroscopy (NIR), High Performance Liquid Chromatography (HPLC), and Nuclear Magnetic Resonance (NMR). Applicable to measurement. The analytical technique chosen to monitor the extractant in the presence of by-products or contamination can be a different technique than that used for real-time alcohol measurements. The real-time data can be used to initiate remediation of the contaminated extractant or removal of the contaminated extractant from the process. These techniques and gas chromatography (GC) and supercritical fluid chromatography (SFC) can also be used to monitor the thermal decomposition of the extractant.

  Referring to FIG. 12, the systems and methods of the present invention may include means for online, in-line, at-line, and / or real-time measurements (circles represent measurement devices, dotted lines represent feedback loops). FIG. 12 is similar to FIG. 9 except for the addition of measurement devices for on-line, in-line, at-line, and / or real-time measurements, and will not be described again in detail.

  As an example, on-line measurements of water stream 22 can be used to monitor the concentration of fermentable carbon sources (eg, polysaccharides), oil, and / or dissolved oxygen. For example, FTIR may be used to monitor the dispersion of oil in stream 22 and process imaging may be used to monitor the concentration in stream 22 and the size of oil droplets. In certain embodiments, on-line measurements of fermentation 30 may be used to monitor product alcohol removal rates. Measurement of the fermentable carbon source, dissolved oxygen, product alcohol, and by-products can be used to adjust the rate of product alcohol removal to maintain the concentration of product alcohol in the fermentation 30 that can withstand microorganisms. Can be used. By maintaining the set point product alcohol concentration, product inhibition and toxicity can be minimized.

  The on-line measurements of stream 105 and stream 122 can be used to operate a process control feedback loop. For example, the concentration of product alcohol in stream 105 may be used to control the flow rate of this stream to extractor 120; And can be used to set the ratio of fermentation broth to extractant. Further, online measurements of stream 105 and stream 122 can also be used to establish a real-time product alcohol mass balance. A process control feedback loop for the extractor 120 and the separation 130 can be used to monitor the quality of the extractant and the phase separation of the fermentation broth. For example, an on-line measurement device may be used to detect the balance of the separation of the extractant and the fermentation broth, and thus the feed rate of the extractant and the fermentation broth may be adjusted to improve the phase separation. An on-line device, such as an optical device, may be used to detect in the presence of a lag layer (eg, a mixture of oil, aqueous solution, and solid), for example, in the extractor 120, the fermentation broth versus extraction The ratio of the agents can be adjusted to minimize the formation of a lag layer. An on-line measurement of stream 135 from separation 130 can be used to monitor the presence of fermentation broth in this stream, and the presence of fermentation broth in stream 135 can indicate poor phase separation. If the concentration of fermentation broth in stream 135 exceeds a certain set point, process changes such as adjusting the flow rate or adjusting the ratio of fermentation broth to extractant may be implemented to improve phase separation. Further, the concentration of product alcohol in stream 135 can be used as a process control feedback loop to ensure efficient operation of separation 130.

  As another example, online measurement of the concentration of product alcohol in stream 127 can be used to monitor extraction efficiency and maintain the concentration of product alcohol in fermentation 30 that can withstand microorganisms. In addition, stream 127 may be monitored for the presence of extractant as a means of minimizing the amount of extractant returning to fermentation 30. For example, spectroscopic imaging and process imaging techniques can be used to monitor the presence of extractant in stream 127. Further, a particular concentration of extractant in stream 127 may be maintained to improve extraction efficiency and phase separation.

  In another embodiment, stream 135 from separation 130 may be sent to purification 150 for further processing, including recovery of product alcohol and extractant 152. Extractant 152 may be sent to extractor 120. Online measurement can be used to monitor the contaminant and decomposition product stream 152. By monitoring stream 152, the potential for contamination of extractor 120 and fermentation 30 is minimized. If there is an increase in contaminants in stream 152, this stream may be further processed to remove these contaminants, for example, by absorption or chemical reaction.

  During the extraction process, a lag layer may form at the interface between the aqueous and organic phases, and the lag layer composed of solids and the extractant (eg, droplets of the extractant) may accumulate, possibly forming a phase. May hinder separation. Stirring of the aqueous and organic phases can be used to reduce the formation of a lag layer. For example, vanes can be used to disperse the lag layer at the aqueous-organic phase interface. Also, fluid flow or vibration / oscillation, such as a recirculation loop, can be used to disrupt lug formation. 13A and 13B illustrate an exemplary process for mitigating the formation of a lag layer. FIG. 13A illustrates the use of a static mixer combined with a stirring unit downstream of the settler or decanter for treatment of the lag layer, and FIG. 13B illustrates the use of a static mixer upstream of the settler or decanter for treatment of the lag layer. 2 illustrates the use of a static mixer combined with a stirring unit. In certain embodiments, other devices such as a coalescer or ultrasonic agitation may be used to disperse the lag layer. In certain embodiments, these devices may be integrated into a settler or decanter.

  The methods and systems described herein can be performed using batch, fed-batch, or continuous fermentation. Batch fermentation is a closed system in which the composition of the fermentation broth is established at the start of the fermentation and is not artificially modified in the fermentation process. In certain embodiments of batch fermentation, an extractant may be added to the fermentor. In certain embodiments, the volume of the extractant can be from about 20% to about 60% of the fermentor working volume.

Fed-batch fermentation is a form of batch fermentation in which a substrate (eg, fermentable sugar) is gradually added during the fermentation process. If catabolic repression can inhibit microbial metabolism, fed-batch systems are useful, in which case it is desirable to have a limited amount of substrate in the medium. In certain embodiments, substrate and / or nutrient concentrations can be monitored during fermentation. In certain embodiments, parameters such as pH, dissolved oxygen, and gas (eg, CO ₂ ) can be monitored during fermentation. From these measurements, the rate or amount of addition of substrate and / or nutrient can be determined. In certain embodiments, as the level or amount of fermentation broth decreases during fermentation, additional mash maintains the level or amount of fermentation broth, e.g., maintains the level or amount of fermentation broth at the start of the fermentation process. To be added to the fermentor. In certain embodiments of fed-batch fermentation, an extractant may be added to the fermentor.

  Continuous fermentation is an open system in which fermentation broth is continuously added to a fermentor and a quantity of fermentation broth is removed for further processing (eg, recovery of product alcohol). In certain embodiments, the addition and removal of the fermentation broth may be simultaneous. In certain embodiments, an equal amount of fermentation broth may be added and removed from the fermentor. In certain embodiments of continuous fermentation, an extractant may be added to the fermentor. In certain embodiments, the volume of the extractant can be from about 3% to about 50% of the effective volume of the fermentor. In certain embodiments, the volume of the extractant can be from about 3% to about 20% of the fermentor effective volume. In certain embodiments, the volume of the extractant can be from about 3% to about 10% of the effective volume of the fermentor.

  In certain embodiments of the methods and systems described herein, gas stripping may be used to remove product alcohol from the fermentation broth. Gas stripping may be performed by providing one or more gases, such as air, nitrogen, or carbon dioxide, to the fermentation broth, thereby forming a product alcohol-containing gas phase. For example, gas stripping can be performed by sparging one or more gases through a fermentation broth. In certain embodiments, the gas may be provided by a fermentation reaction. As an example, carbon dioxide may be provided as a by-product of the metabolism of a fermentable carbon source by a microorganism. In certain embodiments, gas stripping may be used simultaneously with the extractant to remove product alcohol from the fermentation broth. The product alcohol is prepared using methods known in the art, such as using a chilled water trap to condense the product alcohol, or washing the gas phase with a solvent. Can be recovered from the product alcohol-containing gas phase.

Recombinant microorganisms and biosynthetic pathways While not wishing to be bound by theory, the methods described herein are directed to alcohol-producing microorganisms, in particular, recombinant microorganisms that produce alcohol at a titer that exceeds their level of resistance. It is considered useful in connection with any microorganism capable of producing a fermentation product comprising.

  Alcohol producing microorganisms are known in the art. For example, the fermentative oxidation of methane by methane assimilating bacteria (eg, Methylosinus trichosporium) produces methanol and produces the yeast strain CEN. Ethanol is produced by PK113-7D (CBS 8340, the Central Buro voor Shimmelculeure; van Dijken, et al., Enzyme Microb. Techno. 26: 706-714, 2000). Recombinant microorganisms that produce alcohol are also known in the art (see, for example, Ohta, et al., Appl. Environ. Microbiol. 57: 893-900, 1991; Underwood, et al., Appl. Environ. Microbiol. Shen and Liao, Metab. Eng. 10: 312-320, 2008; Hahnai, et al., Appl. Environ. Microbiol. 73: 7814-7818, 2007; U.S. Patent No. 5,514. U.S. Patent No. 5,712,133; PCT Application Publication No. WO 1995/028476; Feldmann, et al., Appl. 38, 354-361, 1992; Zhang, et al., Science 267: 240-243, 1995; U.S. Patent Application Publication No. 2007/0031918 A1; 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; PCT Application Publication No. WO 2010/075241, all of which are incorporated herein by reference).

  In addition, the microorganism can be modified using recombinant techniques to produce a recombinant microorganism capable of producing product alcohols such as ethanol and butanol. Microorganisms that are modified by recombinant techniques to produce product alcohols through the biosynthetic pathway include Clostridium, Zymomonas, Escherichia, Salmonella, and Serratia. (Serratia), Erwinia, Klebsiella, Shigella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Lactobacillus and Lactobacillus. (Enterococcus), Alcaligenes, Klebsiella (K ebsiella), the genus Paenibacillus, the genus Arthrobacter, the genus Corynebacterium, the genus Brevibacterium, the genus Schizosaccharomyces, and the genus Schizosaccharomyces Mycobacterium. Examples include members of the genus Yarrowia, the genus Pichia, the genus Candida, the genus Hansenula, the genus Issatchenkia, or the genus Saccharomyces. In certain embodiments, the recombinant microorganism is Escherichia coli, Lactobacillus plantarum, Kluyveromyces lactis, Kluyveromyces marcianus cervaceae and Kluyveromyces cerevisiae cerevisiae. (Saccharomyces cerevisiae). In certain embodiments, the recombinant microorganism is a yeast. In certain embodiments, the recombinant microorganism is a genus Saccharomyces, Zygosaccharomyces, Schizosaccharomyces, Dekkera, Toruloptis, Toruloptis, Toruloptis and Toruloptis. Crabtree positive yeast selected from several species of the genus Candida. Crabtree positive yeast species include, but are not limited to, Saccharomyces cerevisiae, Saccharomyces kluyveri, Schizosaccharomyces saccharomyces pombe, Mickey (Saccharomyces mikitae), Saccharomyces paradoxus (Saccharomyces paradoxus), Zygosaccharomyces rouxii (Zygosaccharomyces rouxii), and Candida glabrad (Candida glabrida) ata).

  Saccharomyces cerevisiae is known in the art and is available from the American Type Culture Collection (Rockville, Texas, G.C., France). , Ferm Solutions, North American Bioproducts, Martrex, and Lalemand, and are available from a variety of sources. Examples of Saccharomyces cerevisiae include, but are not limited to, BY4741 and CEN. PK 113-7D, Ethanol Red® yeast, Ferm Pro ™ yeast, Bio-Ferm® XR yeast, Gert Strand Prestige Batch Turbo alcohol yeast, Gert Strand Pot distillers yeast, Gerd Striller stirrser Gerdgersertgersertgersertgersger 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 can be immobilized using alginate, calcium alginate, or polyacrylamide gels, or by inducing 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 may be used in combination with immobilized or encapsulated microorganisms. This combination may improve productivity such as specific volume productivity, metabolic rate, product alcohol yield, and resistance to product alcohol. In addition, immobilization and encapsulation can minimize the effects of process conditions such as shear on microorganisms.

Production of butanol using fermentation and microorganisms producing butanol are described, for example, in US Patent No. 7,851,188 and US Patent Application Publication Nos. 2007/0092957; 2007/0259410. No. 2007/0292927; No. 2008/0182308; No. 2008/0274525; No. 2009/0155870; No. 2009/0305363; and No. No. 2009/0305370, the entire contents of each of which are incorporated herein 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 to a fermentation product. In certain embodiments, the biosynthetic pathway converts pyruvate as well as amino acids into fermentation products. 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 substrate-product conversion of the pathway are encoded by heterologous polynucleotides in the microorganism. In certain embodiments, the polypeptide catalyzing the substrate-product conversion of acetolactate to 2,3-dihydroxyisovaleric acid and / or the polypeptide catalyzing the substrate-product conversion of isobutyraldehyde to isobutanol is: It is possible to use reduced nicotinamide adenine dinucleotide (NADH) as a cofactor.

Biosynthetic pathways Biosynthetic pathways for the production of isobutanol that may be used include those described in US Pat. No. 7,851,188, which is incorporated herein by reference. In one embodiment, the isobutanol biosynthesis pathway involves 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 acetohydroxyacid reductoisomerase;
c) 2,3-dihydroxyisovaleric acid to α-ketoisovaleric acid, which can be catalyzed, for example, by acetohydroxyacid dehydratase;
d) isobutylaldehyde from α-ketoisovaleric acid, which can be catalyzed by, for example, branched α-keto acid decarboxylase; and e) isobutanol from isobutyraldehyde, which can be catalyzed by, for example, branched alcohol dehydrogenase.

In another embodiment, the isobutanol biosynthesis pathway involves 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 dihydroxyacid dehydratase;
d) valine from α-ketoisovaleric acid, which can be catalyzed, for example, by transaminases or valine dehydrogenases;
e) valine to isobutylamine, which can be catalyzed, for example, by valine decarboxylase;
f) isobutylamine to isobutyraldehyde, which can be catalyzed, for example, by omega transaminase; and g) isobutyraldehyde to isobutanol, which can be catalyzed, for example, by branched alcohol dehydrogenase.

In another embodiment, the isobutanol biosynthesis pathway involves 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 acetohydroxyacid reductoisomerase;
c) 2,3-dihydroxyisovaleric acid to α-ketoisovaleric acid, which can be catalyzed, for example, by acetohydroxyacid dehydratase;
d) isobutyryl-CoA from α-ketoisovaleric acid, which can be catalyzed, for example, by branched-chain ketoacid dehydrogenase;
e) isobutyryl-CoA to isobutyraldehyde, which can be catalyzed, for example, by acylated aldehyde dehydrogenase; and f) isobutyraldehyde to isobutanol, which can be catalyzed, for example, by branched alcohol dehydrogenase.

Biosynthetic pathways for the production of 1-butanol that may be used include those described in U.S. Patent Application Publication No. 2008/0183088, which is incorporated herein by reference. In one embodiment, the 1-butanol biosynthetic pathway involves the following substrate-product conversion:
a) acetyl-CoA to acetoacetyl-CoA, which can be catalyzed, for example, by acetyl-CoA acetyltransferase;
b) acetoacetyl-CoA to 3-hydroxybutyryl-CoA, which can be catalyzed, for example, by 3-hydroxybutyryl-CoA dehydrogenase;
c) For example, 3-hydroxybutyryl-CoA to crotonyl-CoA, which can be catalyzed by crotonase;
d) Crotonyl-CoA to butyryl-CoA, which can be catalyzed by, for example, butyryl-CoA dehydrogenase;
e) butyryl-CoA to butyraldehyde, which can be catalyzed, for example, by butyraldehyde dehydrogenase; and f) butyraldehyde to 1-butanol, which can be catalyzed, for example, by butanol dehydrogenase.

Biosynthetic pathways for the production of 2-butanol that can be used include those described in U.S. Patent Application Publication No. 2007/0259410 and U.S. Patent Application Publication No. 2009/0155870, which are incorporated herein by reference. What is described is mentioned. In one embodiment, the 2-butanol biosynthesis pathway involves the following substrate-product conversion:
a) pyruvate to α-acetolactate, which can be catalyzed, for example, by acetolactate synthase;
b) acetoin from α-acetolactate, which can be catalyzed, for example, by acetolactate decarboxylase;
c) 3-amino-2-butanol from acetoin, 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-butanol from 2-butanone, which can be catalyzed by, for example, butanol dehydrogenase.

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

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

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

The terms “acetohydroxyacid synthase”, “acetolactate synthase”, and “acetolactate synthetase” (abbreviated as “ALS”) have enzymatic activity that catalyzes the conversion of pyruvate to acetolactate and CO ₂ It may be used interchangeably herein to refer to a polypeptide. Examples of acetolactate synthase are known by EC number 2.2.1.6 (Enzyme Nomenclature 1992, Academic Press, San Diego). These unmodified enzymes are Bacillus subtilis (GenBank (GenBank) numbers: 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) ) Numbers: AAA25161 (SEQ ID NO: 5), L16975 (SEQ ID NO: 6)), but are not limited thereto. Available from several sources.

  The terms "keto acid reductoisomerase" ("KARI"), "acetohydroxy acid isomero reductase", and "acetohydroxy acid reductoisomerase" are used interchangeably and refer to (S) -acetolactate from 2,3 Refers to a polypeptide having enzymatic activity which catalyzes a reaction to dihydroxyisovalerate. An example of a KARI enzyme may be categorized as EC number EC 1.1.1.16 (Enzyme Nomenclature 1992, Academic Press, San Diego), and Escherichia coli (GenBank number: NP_418222 (sequence). No. 7), NC_000913 (SEQ ID NO: 8)), Saccharomyces cerevisiae (GenBank (GenBank) No .: NP_013459 (SEQ ID NO: 9), NC_001144 (SEQ ID NO: 10)), Methanococcus maripardis (GenBank (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)), but are not limited thereto. It is. KARIs include the Anaerotipes caccae KARI variants "K9G9" and "K9D3" (SEQ ID NOS: 15 and 16, respectively). The keto acid reductoisomerase (KARI) enzyme is disclosed in U.S. Patent Application Publication Nos. 2008/0261230, 2009/0163376, and 2010/0197519, which are incorporated herein by reference. And PCT Application Publication No. WO 2011/041415. Examples of KARIs disclosed in these documents include Lactococcus lactis, Vibrio cholera, Pseudomonas aeruginosa PAO1, and PF5 mutants derived from Pseudomonas fluorescens derived from Pseudomonas fluorescens. Is what you do. In some embodiments, the KARI uses NADH. In certain embodiments, the KARI uses reduced form of nicotinamide adenine dinucleotide phosphate (NADPH).

  The terms "acetohydroxyacid dehydratase" and "dihydroxyacid dehydratase" ("DHAD") refer to a polypeptide having enzymatic activity that catalyzes the conversion of 2,3-dihydroxyisovalerate to [alpha] -ketoisovalerate. An example of acetohydroxyacid dehydratase is known by EC number 4.2.1.9. Such enzymes include Escherichia coli (GenBank No .: YP_026248 (SEQ ID NO: 17), NC000913 (SEQ ID NO: 18)), Saccharomyces cerevisiae (GenBank No. 25_P_N_01). (SEQ ID NO: 19), NC 001142 (SEQ ID NO: 20), M. maripaludis (GenBank (GenBank) number: CAF29874 (SEQ ID NO: 21), BX957219 (SEQ ID NO: 22)), Bacillus subtilis (B. subtilis) (GenBank (GenBank) numbers: CAB14105 (SEQ ID NO: 23), Z99115 (SEQ ID NO: 24)), Lactococcus lactis (L. actis, and N. crassa, and are available from a wide variety of microorganisms, including, but not limited to, U.S. Patent Application Publication No. 2010/0081154, and the United States, incorporated herein by reference. Patent No. 7,851,188 describes a 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”) 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 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 (GenBank) numbers: 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. caseolyticus (SEQ ID NO: 33), and L. grayi (SEQ ID NO: 34). It is possible.

The term "branched alcohol dehydrogenase"("ADH") refers to a polypeptide having enzymatic activity that catalyzes the conversion of isobutyraldehyde to isobutanol. An example of a branched alcohol dehydrogenase is known by EC number 1.1.1.265, but is not compatible with 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 include Saccharomyces cerevisiae (GenBank number: NP_010656 (SEQ ID NO: 35), NC_001136 (SEQ ID NO: 36), NP_014051 (SEQ ID NO: 37), NC_001145 (SEQ ID NO: 38)), Escherichia coli (GenBank (GenBank) numbers: NP_417484 (SEQ ID NO: 39), NC_000913 (SEQ ID NO: 40)), C.I. Acetobutylicum (GenBank numbers: NP_349892 (SEQ ID NO: 41), NC_003030 (SEQ ID NO: 42); NP_3498991 (SEQ ID NO: 43), NC_003030 (SEQ ID NO: 44)) Available from such sources. U.S. Patent Application Publication No. 2009/0269823 describes SadB, an alcohol dehydrogenase (ADH) derived from Achromobacter xylosoxidans. Alcohol dehydrogenase can be obtained from horse liver-derived ADH and Beijerinckia (as described in US Patent Application Publication No. 2011/0269199, which is incorporated herein by reference).
indica) ADH.

  The term "butanol dehydrogenase" refers to a polypeptide (or polypeptide) having enzymatic activity that catalyzes the conversion of isobutyraldehyde to isobutanol or 2-butanone and 2-butanol. Butanol dehydrogenase is a subset of the broad family of alcohol dehydrogenases. Butanol dehydrogenase may be NAD- or NADP-dependent. The NAD-dependent enzyme is known as EC 1.1.1.1 and is available, for example, from Rhodococcus ruber (GenBank number: CAD36475, AJ491307). The NADP-dependent enzyme is known as EC 1.1.1.2 and is available, for example, from Pyrococcus furiosus (GenBank number: AAC25556, AF013169). In addition, butanol dehydrogenase is available from Escherichia coli (GenBank number: NP 417484, NC_000913), and cyclohexanol dehydrogenase is available from 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 number: NP_149325, NC_001988; this enzyme has both aldehyde and alcohol dehydrogenase activity); NP_349891, NC_003030; and NP_349892, NC_003030) and E. coli (Escher) GenBank number: NP_417-484, NC_000913).

The term “branched ketoacid dehydrogenase” refers to the conversion of α-ketoisovalerate to isobutyryl-CoA (isobutyryl-coenzyme A), usually using NAD ⁺ (nicotinamide adenine dinucleotide) as 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 ketoacid dehydrogenase is composed of four subunits, and the sequences from all the subunits are Bacillus subtilis (GenBank No .: 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 (GenBank) numbers: 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 the enzymatic activity of catalyzing the conversion of isobutyryl-CoA to isobutyraldehyde, usually using either NADH or NADPH as the 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 number: AAD31841 (SEQ ID NO: 61), AF157306 (SEQ ID NO: 62)), Clostridium acetobutyricum (Clonidium acetobutylicum Genbank (G)). : 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 sa 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 enzymatic activity that catalyzes the conversion of α-ketoisovalerate 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 Nos .: YP-0226231 (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 number: YP_026247 (SEQ ID NO: 75), NC_000913 (SEQ ID NO: 76)), Saccharomyces. Saccharomyces cerevisiae (GenBank (GenBank) number: NP — 012682 (SEQ ID NO: 77), NC — 00142 (SEQ ID NO: 78)) and Methanobacterium thermoautotrophim (Metanobacterium themoautogenbank (Ganbank. SEQ ID NO: 79) and NC_000916 (SEQ ID NO: 80).

  The term "valine dehydrogenase" refers to a polypeptide having an enzymatic activity that catalyzes the conversion of α-ketoisovalerate to L-valine, usually using NAD (P) H as the electron donor and ammonia as the amine donor. Point to. Examples of valine dehydrogenases are known by EC numbers 1.4.1.8 and 1.4.1.9, and such enzymes are known as Streptomyces coelicolor (GenBank). No .: NP — 628270 (SEQ ID NO: 81), NC — 003888 (SEQ ID NO: 82)) and Bacillus subtilis (GenBank No .: CAB14339 (SEQ ID NO: 83), Z99116 (SEQ ID NO: 84)). It is available from a number of non-limiting sources.

The term "valine decarboxylase" refers to a polypeptide having an enzyme activity that catalyzes the conversion of isobutylamine and CO ₂ from L- valine. An example of a valine decarboxylase is known by the EC number 4.1.1.14. Such enzymes include, for example, Streptomyces genus (Streptomyces) such as Streptomyces viridifaciens (GenBank (GenBank) No .: AAN10242 (SEQ ID NO: 85), AY116644 (SEQ ID NO: 86)). ).

  The term "omega transaminase" refers to a polypeptide having enzymatic activity that catalyzes the conversion of isobutylamine to isobutyraldehyde using a suitable amino acid as an amine donor. Examples of omega transaminases are 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 oneidenisis (GenBank number: NP — 91546N) (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 enzymatic activity that catalyzes the conversion of two molecules from acetyl-CoA to acetoacetyl-CoA and coenzyme A (CoA). Examples of acetyl-CoA acetyltransferases are acetyl-CoA acetyltransferases having short-chain acyl-CoA and substrate selectivity (forward reaction) for acetyl-CoA. C. 2.3.1.9 [Enzyme Nomenclature 1992, Academic Press, San Diego], but enzymes with a wider substrate range (EC 2.3.3.16) function similarly. Acetyl-CoA acetyltransferase is available from several sources, for example, Escherichia coli (GenBank number: NP_416728, NC_000913; NCBI) amino acid sequence, NCBI nucleotide sequence, Clostridium acetobutylum (Clostridium acetobutylum) GenBank (GenBank) numbers: NP_349476.1, NC_003030; NP_149242, NC_001988, Bacillus subtilis (GenBank numbers: NP_390297, NC_000964), and Saccharomyces cerevisiae siec (Saccharomyces cerevisiae siec) Enbanku (GenBank) number: NP_015297, it is available from NC_001148).

  The term "3-hydroxybutyryl-CoA dehydrogenase" refers to a polypeptide having 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 in each case E.I. C. 1.1.1.35 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. Respectively. C. 1.1.1.157 and E.I. C. Classified as 1.1.1.36. 3-Hydroxybutyryl-CoA dehydrogenase is available from a number of sources, such as Clostridium acetobutylicum (GenBank numbers: NP_349314, NC_003030), Bacillus subtilisenBan (Gen) Nos .: AAB09614, U29084), Ralstonia eutropha (GenBank numbers: YP_294481, NC_007347), and Alcaligenes eutrophus (Alcaligenes eutrophus) (GenBaA 7A, available from GenBank 7A, available from GenBank 7A, 19A, from 73A, GenA (7A) to GenA (73A), available from GenA, 7A, GenA (98A, GenA), and AA from GenA (7A), available from GenA, 7A, JAA, 7A, 8A, and 8A), available from GenBank (GenA). It is.

The term "crotonase" refers to a polypeptide having an enzyme 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.P. C. 4.2.1.55. Crotonase can be obtained from several sources, for example, Escherichia coli (GenBank number: NP_415911, NC_000913), Clostridium acetobutylicum (GenBank number 93, N_C0030; It is available from Bacillus subtilis (GenBank number: CAB13705, Z99113), and Aeromonas cavier (GenBank number: BAA21816, D88825).

  The term "butyryl-CoA dehydrogenase" refers to a polypeptide having enzymatic activity that catalyzes the conversion of crotonyl-CoA to butyryl-CoA. Examples of butyryl-CoA dehydrogenases may be NADH-dependent, NADPH-dependent, or flavin-dependent, each being E. coli. C. 1.3.1.44, E.C. C. 1.3.1.38, and E.I. C. It may be classified as 1.3.99.2. Butyryl-CoA dehydrogenase is available from a number of sources, for example, Clostridium acetobutylicum (GenBank numbers: NP_347102, NC_003030), Euglena gracilis (EuGnaB8A, UKA5B) and EuganaB8A (E90). ), Streptomyces collinus (GenBank number: AAA92890, U37135), and Streptomyces coelicolor (GenBank number available from GenBank, CA9327). 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 described in C. Known as 1.2.1.57, for example, Clostridium beijerinkii (GenBank numbers: AAD31841, AF157306) and Clostridium acetobutylicum (Genbank numbers). NP.sub .-- 149325, NC.sub .-- 001988).

The term "isobutyryl-CoA mutase" refers to a polypeptide having 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 5.4.9.13. These enzymes are Streptomyces cinnamonensis (GenBank number: AAC08713 (SEQ ID NO: 95), U67612 (SEQ ID NO: 96); CAB59633 (SEQ ID NO: 97), AJ246605 (SEQ ID NO: 98)). Streptomyces coelicolor (GenBank (GenBank) number: CAB70645 (SEQ ID NO: 99), AL939123 (SEQ ID NO: 100); CAB92663 (SEQ ID NO: 101), AL939121 (SEQ ID NO: 102)), and Streptomyces Seth 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 “acetolactate decarboxylase” refers to a polypeptide having enzymatic activity that catalyzes the conversion of α-acetolactate to acetoin. Examples of acetolactate decarboxylase are known as EC 4.1.1.5 and include, for example, Bacillus subtilis (GenBank numbers: AAA22223, L04470), Klebsiella terrigena (GenBank number: AAA25054, L04507) and Klebsiella pneumoniae (GenBank number: AAU43774, AY722205).

  The terms "acetoin aminase" or "acetoin transaminase" refer to a polypeptide having enzymatic activity that catalyzes the conversion of acetoin to 3-amino-2-butanol. The 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 amino donors. NADH- and NADPH-dependent enzymes may use ammonia as a second substrate. A suitable example of a NADH-dependent acetoin aminase, also known as an amino alcohol dehydrogenase, is 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 referred to as amine: pyruvate transaminase) described by Shin and Kim (J. Org. Chem. 67: 2848-2853, 2002). .

  The term "acetoin kinase" refers to a polypeptide having enzymatic activity that catalyzes the conversion of acetoin to phosphoacetoin. Acetoin kinase may use ATP (adenosine triphosphate) or phosphoenolpyruvate as the 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 enzymatic activity that catalyzes the conversion of phosphoacetoin to 3-amino-2-butanol O-phosphate. The 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. Pyridoxal phosphate-dependent enzymes may use amino acids such as alanine or glutamate. NADH- and NADPH-dependent enzymes may use ammonia as a second substrate. There is no report of an enzyme that catalyzes this reaction in phosphoacetoin, but there is a pyridoxal phosphate-dependent enzyme that has been proposed to perform a similar reaction on a similar substrate, serinol phosphate (Yasuta, et al.). al., Appl.Environ.Microbial.67: 4999-5009, 2001).

  The term "aminobutanol phosphate phospholyase", also called "amino alcohol O-phosphate lyase", refers to a polypeptide having enzymatic activity that catalyzes the conversion of 3-amino-2-butanol O-phosphate to 2-butanone. Point. Aminobutanol phosphate phospho-lyase may use the cofactor pyridoxal 5'-phosphate. There is a report of an enzyme that catalyzes a similar reaction on a similar substrate, 1-amino-2-propanol phosphate (Jones, et al., Biochem J. 134: 167-182,1973). U.S. Patent Application Publication No. 2007/0259410 describes an aminobutanol phosphate phospho-lyase from the organism Erwinia carotovora.

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

The term "butanediol dehydrogenase", also known as "acetoin reductase", refers to a polypeptide having enzymatic activity that catalyzes the conversion of acetoin to 2,3-butanediol. Butanediol dehydrogenase is a subset of the broad family of alcohol dehydrogenases. The butanediol dehydrogenase enzyme may have specificity for producing (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. The butanediol dehydrogenase specific for (R) is known as EC 1.1.1.4, for example, Bacillus cereus (GenBank number NP 830481, NC — 004722; AAP07682, AE017000). And Lactococcus lactis (GenBank number AAK04995, AE006323).

  The term "butanediol dehydratase", also known as "diol dehydratase" or "propanediol dehydratase", refers to a polypeptide having 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 and includes, for example, Klebsiella oxytoca (GenBank number: AA08099 (α subunit), D45071; BAA08100 ( and BBA08101 (γ subunit), D45071 (note that all three subunits are required for activity), and Klebsiella pneumonia (GenBank) No .: AAC98384 (α subunit), AF102064; GenBank No .: AAC98385 (β subunit), AF102064, GenBank (GenBank) k) Number: AAC98386 (γ subunit), available from AF102064) Other suitable diol dehydratases include, but are not limited to, Salmonella typhimurium (GenBank number: AAB84102). (Large subunit), AF026270; GenBank (GenBank) number: AAB84103 (medium-sized subunit), AF026270; GenBank (GenBank) number: AAB84104 (small subunit), AF026270); and Lactobacillus collinoides (GenBank (GenBank) numbers: CAC82541 (large subunit), AJ2977 3; GenBank number: CAC82542 (medium subunit); AJ297723; B12-dependent diol dehydratase available from GenBank number: CAD01091 (small subunit), AJ297723); and Lactobacillus brevis ( Lactobacillus brevis) (especially 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 diol dehydratase gene isolation are well known in the art (eg, US Pat. No. 5,686,276).

  The term "pyruvate decarboxylase" refers to a polypeptide having enzymatic activity that catalyzes the decarboxylation of pyruvate to acetaldehyde and carbon dioxide. Pyruvate dehydrogenase is known by the 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 microorganisms comprising the isobutanol biosynthetic pathway provided herein may further comprise one or more additional modifications. U.S. Patent Application Publication No. 2009/0305363, which is incorporated by reference, modifies yeast for expression of acetolactate synthase restricted to cytosol and substantial loss of pyruvate decarboxylase activity. Thus, an increased conversion of pyruvate to acetolactate is 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 United States 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. 5,030,363, incorporated herein by reference, and / or U.S. Patent Application Publication. Modifications that provide an equilibrium of increased carbon flux or reduced equivalents via the Entner-Doudoroff pathway as described in US Patent Application No. 2010/0120105, which is incorporated herein by reference. Other modifications include incorporation of at least one polynucleotide that encodes a polypeptide that catalyzes a step in a pyruvate-based biosynthetic pathway. Other modifications include at least one deletion, mutation, and / or substitution in an endogenous polynucleotide encoding a polypeptide having acetolactate reductase activity. In certain embodiments, the polypeptide having acetolactate reductase activity is YMR226C of Saccharomyces cerevisiae (SEQ ID NOs: 127, 128) or a homolog 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 homolog thereof. A genetic modification having the effect of reducing glucose repression, wherein the yeast producing host cell is pdc-, is described in U.S. Patent Application Publication No. 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 can include deletion or down regulation of a polynucleotide encoding a polypeptide that catalyzes the conversion of glyceraldehyde-3-phosphate to glyceric acid 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 has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% of the nucleotide or polypeptide sequences described herein. %, Or 99%. The term "percent identity" as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. The identities and similarities are calculated by using the Molecular Molecular Biology (Lesk, AM, Ed.) Oxford University: NY (1988); Biocomputing: Informatics and Genome Prod. 1993); Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, Eds.) Humania: NJ (1994); Sequence Analysis in Medicine, Inc. .) Academic (1987); and Sequence Analysis Primer (Gribskov, M. and Deverex, J., Eds.) Stockton: NY (1991).

  Methods for determining identity can be designed to give the best match between the sequences tested. Methods for determining identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations can be performed using the MegAlign ™ program of the LASERGENE Bioinformatics Computing Suite (DNASTAR Inc., Madison, WI). Multiple alignments of sequences are 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). And 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 and calculation of percent identity using the Clustal method are KTUPLE = 1, GAP PENALTY = 3, WINDOW = 5 and diagonal saved ( DIAGONALS SAVED) = 5. These parameters for nucleic acids are KTUPLE = 2, GAP PENALTY = 5, WINDOW = 4, and DIAGONALS SAVED = 4. After alignment of the sequences 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, the Clustal W alignment method is available, and an alignment method named Clustal W (Higgins and Sharp, CABIOS. 5: 151-153, 1989; Higgins, et al., Comput. Appl. Biosci. 8: 189-189). 191, 1992) and are found in the MegAlign ™ v6.1 program of the LASERGENE Bioinformatics Computing Suite (DNASTAR Inc.). Default parameters for multiple alignments (gap penalty (GAP PENALTY) = 10, gap length penalty (GAP LENGTH PENALTY) = 0.2, delay divergent sequences (Delay Divergen Seqs) (%) = 30, DNA transition weights (DNA) (Transition Weight) = 0.5, Protein Weight Matrix = Gonnet series, DNA Weight Matrix = IUB). After alignment of the sequences 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. Press, Cold Spring Harbor, 1989, referred to herein as Maniatis) and Ausubel et al. (Ausubel, et al., Current Protocols in Molecular Biology, pub.by Greene Publishing. Examples of methods for constructing microorganisms that include the butanol biosynthetic pathway are described, for example, in U.S. Patent No. 7,851,188 and U.S. Patent Application Publication No. 2007/0092957. No. 2007/0259410; No. 2007/0292927; No. 2008/0182308; No. 2008/0274525; No. 2009/0155870; No. 2009 / No. 0305363; and US 2009/0305370, each of which is incorporated herein by reference in its entirety.

  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 changes in form and detail of the invention can be made without departing from the spirit and scope of the invention. Therefore, the breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

  All publications, patents, and patent applications mentioned herein 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 hereby incorporated by reference. To the extent that it is specifically and individually indicated herein by reference.

  The following non-limiting examples further illustrate the invention. While the following examples include corn as a feedstock, it should be understood that other biomass sources, such as sugarcane, can be used in the feedstock without departing from the invention. Further, while the examples below include ethanol and butanol, other alcohols or fermentation products may be produced without departing from the invention.

  Abbreviations have the following meanings: "atm" means atmospheric pressure, "ccm" means cubic centimeters / minute, "g / L" means grams / liter, and "g" means grams. Where "gpl" means grams / liter, "gpm" means gallons / minute, "h" or "hr" means hours, "HPLC" means high performance liquid chromatography, "Kg" means kilogram, "L" means liter, "min" means minute, "mL" means milliliter, "ppm" means parts per million, "psig" "Means pounds per square inch (gauge pressure) and"% by weight "means percent by weight.

Example 1
Methods for the Production and Recovery of Butanol Produced by Fermentation The methods described herein can be demonstrated using computational modeling, such as Aspen modeling (eg, US Pat. No. 7,666,282). See specification). For example, the commercially available modeling software Aspen Plus® (Aspen Technology, Inc., Burlington, Mass.) Uses the American Institute to create Aspen models for integrated butanol fermentation, purification, and water management processes. of Chemical Engineers, Inc. It can be used with a physical property database such as DIPPR available from (New York, NY). This process modeling can perform many basic engineering calculations, such as mass and energy balances, gas / liquid equilibrium, and kinetic calculations. To generate the Aspen model, information inputs include, for example, experimental data, water content and ingredient composition, temperatures for mash cooking and flashing, saccharification conditions (eg, enzyme ingredients, starch conversion, temperature, pressure). , Fermentation conditions (eg, microbial feed, glucose conversion, temperature, pressure), degassing conditions, solvent towers, preflash towers, condensers, evaporators, centrifuges, and the like.

  The Aspen model shows that 53400 kg / h of corn is mashed and fermented to produce isobutanol, with most of the isobutanol being produced in a tight mass and energy balance that is extracted and distilled during fermentation. Was. This model included an approximation of a series of batch fermentations as a continuous process. An example of this fermentation, extraction, and distillation process is shown in FIG.

  Liquefied corn mash 601 purified to contain 1.5% by weight suspended solids was injected at 170.7 ton / hr and 85 ° C. via a heat exchanger and water cooler, Feed to fermentation 600 at ° C. A steam stream 602 having an average continuous molar composition of 95.8% carbon dioxide, 3.4% water, and 0.8% isobutanol is fed from fermentation 600 at atmospheric pressure at 17.2 tonnes / hour. Released to scrubber. An average beer stream 603 containing 12.6 gpl isobutanol was continuously discharged from fermentation 600 and preheated by a mash 601 via a heat exchanger before distilling for isobutanol recovery.

  Stream 604 having a combined average flow rate of 3875 tons / hour is removed from fermentation 600 at an average isobutanol concentration of 11.1 gpl and an average temperature of 32 ° C., and extractor 610 is used for partial removal of isobutanol. Circulate through. The emerging aqueous broth 605 containing 7.9 gpl of isobutanol is cooled to 30 ° C. by heat exchange with cooling tower water (CTW) before re-entering the fermentation 600. Solvent containing diisopropylbenzene enters extractor 610 and exits as stream 606 containing 30.1 gpl of isobutanol. Extractor 610 effectively provides five theoretical liquid-liquid equilibrium stages for contacting the fermentation broth with the solvent. Stream 606 passes through a heat exchanger at 340 tons / hour and enters the center of 12 theoretical stages of distillation column 620. The reboiler operates at 0.6 atmospheres (atm) and 183 ° C. using 150 psig steam to produce a solvent stream 607 containing diisopropylbenzene and substantially free of isobutanol. 607 exchanges heat with solvent stream 606 via a heat exchanger and is further cooled by cooling water CTW before re-entering extractor 610. The overhead vapor of distillation column 620 is cooled (CTW), condensed (630) and refluxed at 23.1 ton / hr, residual vapor off-gas at 608 ton / hr 608, and 99.2% iso-gas. A 13.2 ton / h distillation product 609 comprising butanol, 0.6% water, and 0.2% diisopropylbenzene is formed.

Example 2
Method for the Recovery of Ethanol Using an Extractant Column A 1-inch diameter Karr® extraction tower (Koch Modular Process Systems, Paramus, NJ) is used to treat the fermentation broth produced during ethanol fermentation. did. The column includes a series of plates that extend down the length of the column and are attached to a central axis. The shaft is mounted on a drive capable of moving the perforated plate (1/4 inch diameter perforations) up and down in a reciprocating motion. The frequency of movement was variable during the test, but both the stroke length of the vibration (0.75 inches) and the tray spacing (2 inches) were fixed. The column used had a plate stack height of 3000 mm.

  At the top of the column, an aqueous raw material consisting of a fermentation broth was provided, while at the bottom of the column, a raw material of corn oil fatty acid (COFA) as an extractant was provided. The two feeds flow countercurrent to each other through the column, and they were collected as products on opposite sides of the column.

The fermentation broth was obtained using a fermentation protocol for the production of ethanol from liquefied and saccharified corn mash, where some of the solids had been removed by centrifugation. Sometimes, while performing part of testing while maintaining the CO ₂ gas emission maximum or nearly maximum, to perform another portion when the gas discharge is stopped effectively and extracted test over several days . The COFA used for this operation was a distillation grade from Emery Oleochemicals (Cincinnati, OH).

  Some experiments were performed using COFA as the continuous phase in the column, while others were performed using the continuous aqueous phase. Experiments were also performed with or without the interior of a given location. Two types of interior were tested: stainless steel and polytetrafluoroethylene (PTFE). The flow range was examined to determine the flow regime in which the column could be operated without flooding.

Effect of Dynamic Feed from Fermentation During the tests, it was found that in some cases the performance of the column changed as the fermentation progressed. At the beginning of the fermentation, the fermentation broth containing the raw material is high in sugar content, and at an intermediate time a significant amount of CO ₂ (which can affect the fluid stream) is generated from the fermentation broth, while at a later time the fermentation broth The concentration of ethanol in the broth is high. This change in feed over time was reflected in changes in the capacity of the extraction column.

Under conditions using a PTFE plate and a continuous COFA phase (without agitation), the fermentation is at near peak gas evolution ("intermediate broth") and at the end of fermentation ("final broth (" A difference in performance was shown when using fermentation broth collected in "end broth"). With the final broth, a liquid throughput rate of 14 gpm / ft ² (Sample 3E) was achieved without flooding the column. The maximum throughput of the intermediate broth obtained before flooding was less than 9 gpm / ft ² (sample 4D), with significant differences in water droplet size and appearance. The droplet size of the aqueous phase was larger (with droplet formation) in the final broth compared to the intermediate broth.

Continuous phase Maximum column throughput was also affected by the nature of the continuous phase. By the end of the fermentation conditions, operating in a continuous aqueous phase and inside stainless steel (S. Steel), a total liquid volume of about 14 gpm / ft ² was obtained (sample 2B). Within the continuous organic phase and PTFE, the rate was less than 9 gpm / ft ² (Sample 4D). The results are shown in Table 2. The abbreviation AQ refers to the aqueous phase and the abbreviation ORG refers to the organic phase. Referring to Table 2, the phases are continuous, the sample refers to the operating conditions, the interior refers to the material inside, and Nom. AQ refers to the nominal aqueous flow rate, Nom. ORG refers to the nominal organic flow, total flow (ccm) refers to the total flow of aqueous and organic raw materials, and total flow (gpm / ft ² ) refers to the total flow per unit cross-sectional area.

When the column was operated without internals at the end of the fermentation using a feed containing fermentation broth, the choice of the continuous phase affected the capacity of the column. With a continuous aqueous phase, it was possible to operate at about 25 gpm / ft ² (samples 2G and 2H). However, the continuous COFA phase caused flooding problems at 18 gpm / ft ² (Sample 2I). The results are shown in Table 3.

Example 3
Effect of Fermentation Conditions on Extraction Column Performance The nature of the fermentation broth is not unchanged and changes as the fermentation process proceeds. During fermentation, the carbohydrate concentration decreases as the carbohydrate is metabolized by microorganisms. This change in composition of the fermentation broth will change physical parameters such as viscosity and surface tension of the fermentation broth that affect the extraction process. At intermediate times, in addition to the change in concentration, significant amounts of CO ₂ are generated; this CO ₂ will affect the flow of aqueous and organic liquids through the column.

The fermentation broth from the ethanol fermentation was processed using a 1-inch diameter glass Karr® extraction tower (Koch Modular Process Systems, Paramus, NJ) equipped with a PTFE interior. At some point during the fermentation the treatment was performed. Organic extractant (COFA) is the continuous phase in the column and the fermentation broth passes through the column as droplets. Before the introduction of the fermentation broth to the column, the fermentation broth was passed through a T-tube in the conduit (line) (tee), wherein the CO ₂ bubbles present in the raw material, was removed through the vent hole.

In the stationary interior (without agitation), when using fermentation broth taken during peak gassing ("intermediate broth"), compared to broth taken near the end of fermentation ("final broth"), Performance differences were indicated. With an intermediate broth, a liquid throughput rate of 14 gpm / ft ² was obtained. (Before flooding of the column) maximum throughput of the final broth was less than 9gpm / ft ^2. There were significant differences in the size and appearance of the drops. The droplet size of the aqueous phase was clearly larger in the final broth compared to the intermediate broth.

Example 4
Effect of Isobutanol Concentration on Extraction Column Efficiency During a typical fermentation process, product levels change over time. This dynamic change in concentration can affect mass transfer during the extraction process.

To demonstrate the effect of isobutanol concentration, about 3 g / g using a 1 inch diameter glass Karr® extraction tower (Koch Modular Process Systems, Paramus, NJ) equipped with a stainless steel interior. The fermentation broth from the fermentation containing L of isobutanol was processed. While the fermentation broth was formed in the continuous phase in the extractor, the organic extractant (COFA) passed through the column as droplets. Although the CO ₂ production was substantially stopped, the fermentation broth was passed through a T-tube in the line where any CO ₂ bubbles present in the feed were removed before the feed entered the extraction column. .

  Samples of feed and outgoing streams were analyzed for isobutanol by liquid chromatography (LC) or gas chromatography (GC). The results are shown in Table 4. Mass balance was performed and the height of the equilibrium transfer stage (HETS) was calculated using the Kremser equation. HETS values were 10 and 13 feet for two data points of the raw fermentation broth.

  Next, isobutanol was added to the fermentation broth to a concentration of 20 g / L. An extraction test was performed and the data indicated that the HETS was 18 feet. This value is about 50% higher than that obtained with the neat broth and is consistent with the data obtained with dilute effluent containing about 20 g / L isobutanol (see FIG. 15).

Example 5
ISPR using external extraction tower
The fermentation broth from the isobutanol fermentation (10 liter scale) was circulated to a 5/8 inch diameter benchtop Karr® column. The extraction solvent (COFA) was recycled from the extractant reservoir to the Karr® column. A control fermentation was performed and a volume of COFA was added to the fermentor to continuously extract isobutanol from the fermentation broth.

During the fermentation, the Karr® column was run twice. The first run was at 4-7 hours of fermentation and the second run was at 22-33 hours of fermentation. For both fermentation was monitored parameters such as pO ₂ and pH. The measured pO ₂ was lower in the run where the Karr® column was used, as compared to the control run without the Karr® column. Absolute pH values were similar for the Karr® column and control, but the pH profile was different for the two runs. For one mild peak of the control, the pH in the Karr® column peaked early, flattened out, and then peaked again.

  Two aliquots of the extraction solvent (1.8 liters each) were analyzed from a Karr® column. Samples were taken from each aliquot and analyzed for isobutanol content. The amount of isobutanol produced during the fermentation using the Karr® column was comparable to the amount of isobutanol produced during the control fermentation. Fermentation using a Karr® column yielded a total of 82.4 grams of isobutanol: about 34 grams were in the 3.6 liter organic phase and 48 grams were in the aqueous phase. . The control (30% by volume of organic phase added to the fermentor) produced 90 g / L, 60 grams were in the 3 liter organic phase and 30 grams were in the aqueous phase. The isobutanol concentration in the aqueous phase was lower in the control due to the presence of COFA in the control fermenter from time zero, relative to the non-zero onset of extraction during Karr® column operation. At Karr® at 22 hours, isobutanol was extracted from the fermentor faster than it was produced. Glucose profiles were generated for control and Karr® columns. The profiles were similar, indicating that cell growth and metabolism were comparable. The results are shown in FIGS. 16A and 16B. In parentheses indicate when the Karr® column was operating (4-7 hours and 22-33 hours).

Example 6
ISPR using a mixer settler
Using an external mixer-settler system, isobutanol was continuously removed from the active fermentation broth containing the isobutanol-producing microorganisms (ie, isobutanologen). The test used about 100 liters of fermentation broth inoculated with an isobutanol producing microorganism (ie, isobutanologen). The contents of the fermenter were recycled from the fermentor through a mixer-settler extraction system. An extractant containing distilled COFA without isobutanol was used in a once-through basis.

  Two static mixers were tested. Most of the tests used a Kenics® stainless steel static mixer (１ ／ inch diameter with 36 mixing elements). During the 12-24 hour run, a plastic mixer was used (StaMixCo HT-11-12.6-24, StaMixCo LLC, Brooklyn, NY). The fermentation broth and COFA were fed to the other side of the tee, from which the mixture flowed through a static mixer. The material exiting the static mixer was fed to a settler. The settler was made from a 5 liter glass tank. The dip tube passed over the top of the settler, near the periphery, and extended down almost half the settler. The organic phase was withdrawn through the port at the top of the settler, while the fermentation broth was removed from the bottom of the settler. The settler was equipped with a stirrer that provided gentle mixing to the aqueous-organic phase interface to assist in the separation of the two liquid phases, thereby minimizing the accumulation of solids at the interface. . The data collected during the run is presented in Table 5, and FIG. 17 shows the isobutanol removal achieved during the fermentation. As can be seen from the data, the isobutanol levels in the aqueous broth remained relatively constant, indicating that isobutanol was removed from the fermentation broth at about the same rate as it was produced. . Referring to Table 5, the elapsed time is the time from the start of fermentation, the AQ flow rate is the aqueous raw material flow, the ORG flow is the organic raw material flow, and iB in the AQ raw material is Isobutanol, iB in the ORG product is isobutanol in the rich organic product.

Example 7
Online, at-line, and real-time measurements A mash stream prepared from corn feed was sent to a three-phase centrifuge to produce three streams: mash, corn oil, and wet cake. Online or at-line process measurements are used, for example, to improve starch / sugar recovery and corn oil quality, and to maximize the amount of starch / sugar extracted from wet cakes. Real-time measurements are used to control backflow, cooking water, or water addition to the slurry tank, for example, to maintain a starch / sugar concentration set point. The minimum amount of added water is used to maximize the amount of starch / sugar extracted from the wet cake while reducing the hydraulic load on the three-phase centrifuge.

  The corn mash samples were analyzed using Fourier Transform Infrared Spectroscopy (FTIR) with a diamond attenuated total reflection (ATR) probe allowing the measurement of the presence of solids. The FTIR was calibrated by collecting the spectra of the standard samples and the total starch / sugar measurement using HPLC was completed. A multivariate partial least squares (PLS) model for FTIR was created using the HPLC data. FTIR spectra were collected to generate total starch concentration. FIG. 18 shows the range of starch concentrations used to calibrate FTIR.

  Corn mash having an average starting concentration of 250 g / L was fed to a three-phase centrifuge. The subsequent wet cake was reslurried and the starch concentration was measured on two samples: 80 g / L and 70 g / L. Next, this slurry was separated using a three-phase centrifuge, and the wet cake was reslurried. When the starch concentration of this slurry was measured, it was 28.9 g / L. The results are shown in FIG. Using these measurements, the exact amount of water was measured and the wet cake was reslurried at each stage. Optimization of the water addition maximized the starch concentration and minimized the hydraulic load on the separation process. The moisture content of the wet cake was measured using near infrared spectroscopy (NIR).

  The quality of the corn oil is monitored in real time and the data is used to control three-phase centrifuge variables (eg, feed rate, gravity, inlet flow, scroll speed). The quality of the corn oil produced by the three-phase centrifuge was measured by monitoring the concentration of water carried into the corn oil during the separation. Corn oil spectra were collected as the corn oil exited the three-phase centrifuge using FTIR with a diamond ATR probe. The detection limit for water using the diamond ATR probe technique was about 500 ppm. Lower detection limits were achieved by using flow cells with longer effective path lengths.

  FIG. 20 includes a series of infrared spectra of corn oil containing water concentrations in the range of more than percent level concentrations up to the order of 100 ppm (100's of ppm). Water concentration was measured using the -OH extension region from 3700 cm-1 to 3050 cm-1. The data indicated that the process FTIR was used to generate real-time water concentrations in the oil data. The real-time water concentration data was used to control process variables (eg, feed rate, gravity, inlet flow rate, scroll speed) of the three-phase centrifuge. Operation of the three-phase centrifuge can be controlled to obtain the highest quality corn oil or to maximize throughput without exceeding the water set point.

  Real-time extractant monitoring was used to detect and monitor the thermal decomposition of the extractant. The detection of these pyrolysis products in real time is used to begin improving the extractant or removing contaminated extractant from the process.

  FIG. 21 is an example of real-time measurement of isobutanol-enriched COFA. Data was collected using a Meter-Toledo ReactIR ™ 247 using a diamond ATR sampling probe in the flow cell. The COFA stream was collected from the outlet of a 1 inch diameter Karr® column and injected into the FTIR using a peristaltic pump. FTIR was calibrated by generating a COFA standard with isobutanol and generating a multivariate PLS model.

Example 8
Analysis of Droplet Size This example describes the analysis of liquid extractant droplets after a process stream containing fermentation broth and extractant (COFA) has been sent to a static mixer. About 24 hours after the process stream exited the static mixer, a PVM® probe (Mettler-Toledo, LLC, Columbus OH) was inserted into the process stream. Images were collected every 2 minutes during the fermentation run using a PVM® probe. The images showed the presence of both COFA droplets ranging in size from 50 to 80 μm in diameter and CO ₂ bubbles ranging in size from 200 to 400 μm in diameter. With the monitoring of the droplet size in the process stream containing the fermentation broth and COFA after the static mixer, to ensure good mass transfer of isobutanol into the COFA droplets, the droplets must be of a specific size. Ensure that it is kept below the average diameter.

  Also, using a PVM® probe, COFA droplets in a dilute broth stream were imaged before the flow returned to the fermentor. Detection of COFA droplets in this stream indicates the amount of COFA returning to the fermentor. Flow images were collected every two minutes during the fermentation using a PVM® probe. Unlike the stream leaving the static mixer, the lean broth stream had fewer and smaller droplets (10-40 μm). These measurements demonstrate the feasibility of using process imaging to monitor the amount of COFA returning to the fermentor.

  Real-time average droplet size data from both sample points were used to monitor the phase separation of fermentation broth and COFA. An increase in the concentration or number of small COFA droplets detected in the recycle stream of dilute fermentation broth (after isobutanol extraction) can be an indicator of the phase separation of the fermentation broth, where the COFA is degraded, Too much COFA is leaving the extractor. The average COFA droplet size is increased after the static mixer to improve the quality of the phase separation and reduce the number or concentration of COFA droplets returning to the fermentor in the lean broth stream.

  Additional process variables that can affect the average COFA droplet size include the concentration of polysaccharide in the fermentation broth, the ratio of fermentation broth to COFA, and the total flow through the static mixer. As the fermentation progresses, the flow rate and / or the ratio of fermentation broth to COFA can be varied to maintain a constant average COFA droplet size.

Ｕ＝単位面積当たりの流量（ガロン／分／平方フット）
Ｆ＝抽出器ユニットへの発酵ブロスおよび抽出剤の総流量（ガロン／分）
Ａ＝流れの方向における断面積（平方フィート）
抽出塔の場合、これは、下式によって示される。

Ｄ＝カラム直径（フィート）。 Example 9
Extractor Design This example describes a method for designing a large extractor unit. The data from the pilot size extraction is used to estimate the size of the large extractor unit. The effect of flow rate, agitation speed, and the presence or absence of the interior on the phase separation of the extractor unit stream from the pilot scale extraction is determined. The total flow rate and the ratio of fermentation broth flow rate to extractant flow rate are varied at a fixed temperature during the fermentation and conditions are observed where the phase separation is interrupted. Record the maximum achievable flow rate to the extractor unit per square foot of extractor unit flow surface area. Determine the flow per unit area using the following equation:

U = flow rate per unit area (gallon / minute / square foot)
F = total flow of fermentation broth and extractant to the extractor unit (gal / min)
A = cross section in the direction of flow (square feet)
In the case of an extraction column, this is represented by the following equation:

D = column diameter (feet).

Ｆ_大規模＝大規模な抽出器への発酵ブロスおよび抽出剤の総流量（ガロン／分）。 The diameter of the large extractor unit is estimated by the expected flow rates of fermentation broth and extractant to the extractor unit using the following equation:

F _Large = Total flow of fermentation broth and extractant to the large extractor (gal / min).

Ｆ_ブロス＝抽出器ユニットへのブロスの流量（ガロン／分）
Ｆ_抽出剤＝抽出器ユニットへの抽出剤の流量（ガロン／分）
ｍ＝発酵ブロスおよび抽出剤相中の生成物アルコールの分配係数（ｇ／Ｌ当たりのｇ／Ｌ）
Ｘｆ＝発酵ブロス原料中の生成物アルコールの濃度（ｇ／Ｌ）
Ｘｎ＝抽出器ユニットを出る発酵ブロス中の生成物アルコールの濃度（ｇ／Ｌ）
Ｙｓ＝抽出器ユニットに入る抽出剤中の生成物アルコールの濃度（ｇ／Ｌ）
ｎ＝抽出器ユニットの高さによって得られる理論段の数
式３は、Ｅ≠１である場合のみ有効である。 The height of the pilot scale extractor unit is measured at different flow ranges, including different flow rates with and without internals, different stirring speeds, and different concentrations of product alcohol. Using this data, the number of theoretical stages obtained by the height of the extractor unit, Kuremuza of formula (Kremser Equation) estimated (Seader and Henley ^{with, Separation Process Principles, 2 nd edition} , John Wiley & Sons, 2006, pp. 358-359):

F _broth = flow rate of broth to the extractor unit (gal / min)
F _extractant = flow rate of extractant to extractor unit (gal / min)
m = partition coefficient of product alcohol in fermentation broth and extractant phase (g / L per g / L)
Xf = concentration of product alcohol in fermentation broth raw material (g / L)
Xn = concentration of product alcohol in the fermentation broth leaving the extractor unit (g / L)
Ys = concentration of product alcohol in extractant entering extractor unit (g / L)
n = number of theoretical plates obtained by the height of the extractor unit Equation 3 is valid only when E ≠ 1.

（式中、’は、大規模な抽出器ユニットの条件を示す）で大規模に予測される運転条件を用いて推定される。 The theoretical plate height of the extractor unit is indicated by dividing the height of the extraction column used for pilot scale extraction by the number of theoretical plates realized in a given experiment. The number of theoretical plates required to achieve large-scale separation is given by Equation 4:

(Where 'denotes the condition of the large-scale extractor unit) and is estimated using the large-scale predicted operating conditions.

  The product of the number of theoretical plates and the height of the theoretical plate measured for similar flow conditions estimates the overall height of the large extractor unit. The flow rates and concentrations expected in large-scale extractor units are estimated using dynamic fermentation models (eg, Daugulis, et al., Biotech. Bioeng. 27: 1345-1356, 1985).

  While various embodiments of the present 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 changes in form and detail of the invention can be made without departing from the spirit and scope of the invention. Therefore, the breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

  All publications, patents, and patent applications mentioned herein 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 hereby incorporated by reference. To the extent that it is specifically and individually indicated herein by reference.

Claims (30)

Providing a fermentation broth comprising a microorganism that produces a product alcohol;
Contacting said fermentation broth with at least one extractant forming an extractant phase, wherein said extractant phase comprises product alcohol ;
Contacting said extractant phase with a water stream forming an aqueous phase, wherein said product alcohol is transferred from said extractant phase to said aqueous phase;
Recovering the product alcohol.
The extraction of the product alcohol is monitored by real-time measurement of phase separation by measuring the rate of phase separation and / or droplet size of the extractant, wherein the real-time measurement comprises conductivity measurement, dielectric measurement, A method for recovering product alcohol from a fermentation broth, which is a viscoelasticity measurement or an ultrasonic measurement.   2. The method of claim 1, wherein the step of contacting the fermentation broth with at least one extractant is performed in the fermentation device, an external unit, or both.   3. The method according to claim 2, wherein the external unit is an extractor.   The extractor is selected from a siphon, a decanter, a centrifuge, a gravity settler, a phase separator, a mixer settler, a column extractor, a centrifugal extractor, a stirred extractor, a hydrocyclone, a spray tower, or a combination thereof. 4. The method of claim 3, wherein: Wherein the at least one extraction agent, fatty alcohols C ₇ -C _22, fatty acids C ₇ -C _22, esters of fatty acids of C ₇ -C _22, fatty aldehydes of C ₇ -C _22, of C ₇ -C ₂₂ The method of claim 1, wherein the method is selected from fatty amides, and mixtures thereof. The at least one extractant is oleyl alcohol, behenyl alcohol, cetyl alcohol, lauryl alcohol, myristyl alcohol, stearyl alcohol, oleic acid, lauric acid, linoleic acid, linolenic acid, myristic acid, stearic acid, octanoic acid, decanoic acid, undecane. Acid, methyl myristate, methyl oleate, 1-nonanol, 1-decanol, 2-undecanol, 1-nonanal, 1-undecanol, undecanal, laurinaldehyde, 2-methylundecanal, oleamide, linoleamide, palmitoamide, stearylamide 2. The method of claim 1, wherein the method is selected from: 2-ethyl-1-hexanol, 2-hexyl-1-decanol, 2-octyl-1-dodecanol, and mixtures thereof.   7. The method of claim 6, wherein a hydrophilic solute is added to the fermentation broth.   The method according to claim 7, wherein the hydrophilic solute is selected from the group consisting of a polyhydroxylated compound, a polycarboxylic acid, a polyol compound, an ionic salt, or a mixture thereof.   The method according to claim 1, wherein the step of contacting the fermentation broth with at least one extractant is performed in two or more external units.   The method according to claim 1, wherein the step of contacting the fermentation broth with at least one extractant is performed in two or more fermenters.   The method of claim 10, wherein the fermentor includes an interior or device for improving phase separation.   12. The method of claim 11, wherein the interior or device is selected from the group consisting of a coalescer, baffle, perforated plate, well, lamella separator, cone, or a combination thereof.   2. The method according to claim 1, wherein the step of providing a fermentation broth comprising microorganisms is performed in two or more fermenters.   The method of claim 1, wherein the product alcohol is selected from ethanol, propanol, butanol, pentanol, hexanol, and fusel alcohol.   2. The method of claim 1, wherein said microorganism comprises a butanol biosynthetic pathway. 16. The method of claim 15 , wherein the butanol biosynthesis pathway is a 1-butanol biosynthesis pathway, a 2-butanol biosynthesis pathway, or an isobutanol biosynthesis pathway. Wherein the microorganism is a recombinant microorganism, The method of claim 1 5. Providing a raw slurry comprising a fermentable carbon source, an undissolved solid, an oil, and water;
Separating the raw slurry, thereby forming (i) an aqueous solution containing a fermentable carbon source, (ii) a wet cake containing solids, and (iii) an oil;
Adding the aqueous solution to the fermentation broth.   19. The method of claim 18, wherein the oil is hydrolyzed to form a fatty acid.   20. The method of claim 19, wherein the fermentation broth is contacted with the fatty acid.   20. The method of claim 19, wherein the oil is hydrolyzed by an enzyme.   22. The method of claim 21, wherein said enzyme is one or more lipases or phospholipases.   19. The method of claim 18, wherein the raw slurry is produced by hydrolysis of a raw material.   24. The method of claim 23, wherein the feedstock is selected from rye, wheat, corn, sugarcane, barley, cellulosic or lignocellulosic materials, or a combination thereof.   The raw material slurry is 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, grid 20. The method of claim 18, wherein the separation is by a porous grid, a flotation method, a hydrocyclone, a filter press, a screw press, a gravity settler, a vortex separator, or a combination thereof.   19. The method of claim 18, wherein said separating the feedstock is a single step process.   19. The method of claim 18, wherein said wet cake is combined with said aqueous solution.   19. The method of claim 18, further comprising contacting the aqueous solution with a catalyst to convert oils in the aqueous solution to fatty acids.   29. The method of claim 28, wherein said aqueous solution and fatty acids are added to said fermentation broth.   29. The method of claim 28, wherein said catalyst is deactivated.

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