JP2015528309A - Method and system for production of fermentation products - Google Patents

Abstract

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

Description

  This application is based on US Provisional Patent Application No. 61 / 699,976, filed September 12, 2012; US Provisional Patent Application No. 61 / 712,385, filed October 11, 2012. Benefits of 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 are hereby incorporated by reference.

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

  The present invention relates to the production of fermentation products such as alcohols including ethanol and butanol, and to 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 a variety of 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 fragrance industry. The production of butanol or butanol isomers from materials such as plant-derived materials can minimize the use of petrochemicals and will lead to progress in the art. Furthermore, the generation 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, for example, in the production of butanol, some microorganisms that produce butanol in high yield also have a low butanol toxicity threshold. Removing butanol from the fermentation as it is produced is a means to address these low butanol toxicity thresholds. Accordingly, there is a continuing need to develop efficient methods and systems for producing butanol in high yields despite the low butanol toxicity threshold of butanol producing microorganisms.

  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, thereby allowing the microorganism to It becomes possible to produce butanol in a high yield. One ISPR method for removing fermented alcohol described in the art is liquid-liquid extraction (see, for example, Patent Document 1). In general, for butanol fermentation, the fermentation broth containing the microorganism is contacted with the extractant at a time before the butanol concentration reaches, for example, a toxic level. Butanol is distributed in the extractant to reduce the concentration of butanol in the fermentation broth containing the microorganism, thereby reducing microbial exposure to inhibitory butanol.

  In order to be technically and economically feasible, liquid-liquid extraction is the contact of the extractant with the fermentation broth for efficient mass transfer of 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 a reduction in the extractant's partition coefficient over a long period of operation is minimal. The extractant can become contaminated over time each time it is reused, for example by the accumulation of lipids present in the biomass used as fermentation feedstock, which can be accompanied by a decrease in the partition coefficient of the extractant. .

  Furthermore, the presence of undissolved solids during extractive fermentation can adversely affect the efficiency of alcohol production. For example, the presence of undissolved solids can reduce the mass transfer coefficient, impede phase separation, cause oil accumulation from the undissolved solids in the extractant, and lead to a decrease in extraction efficiency over time, May slow the detachment of extractant droplets from the fermentation broth, may reduce fermenter volumetric efficiency, and the extractant will be trapped in a solid and finally finished as a soluble cereal-containing dry cereal distiller (DDGS) May increase the loss of extractant.

US Patent Application Publication No. 2009/030305370

  Accordingly, 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 reduction in extractant partition coefficient. The present invention meets 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 recovering the fermentation product. The present invention relates to a method for recovering fermentation products from In certain embodiments, the step of contacting the fermentation broth with at least one extractant is performed in a fermentation apparatus, an external unit, or both. In certain embodiments, the external unit is an extractor. In certain embodiments, the extractor comprises a siphon, a decanter, a centrifuge, a gravity settler, a phase splitter, a mixer settler, a column extractor, a centrifugal extractor, a stirred extractor, a hydrocyclone, a 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 Of 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 Selected from amide, 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 is performed in two or more external units. In certain embodiments, the step of contacting the fermentation broth with at least one extractant is performed in two or more fermentation apparatuses. In certain embodiments, the fermenter includes an interior or device for improving phase separation. In certain embodiments, the interior or device is selected from coalescers, baffles, perforated plates, wells, lamellar separators, cones, and combinations thereof. In certain embodiments, real-time measurements are used to monitor the fermentation product extraction. In certain embodiments, the fermentation product extraction is monitored by real-time measurement of phase separation. In certain embodiments, phase separation is monitored by measuring the rate of phase separation, extractant droplet size, and / or the composition of the fermentation broth. In certain embodiments, phase separation is monitored by conductivity measurements, dielectric constant measurements, viscoelastic measurements, and / or ultrasonic measurements. In certain embodiments, the step of providing a fermentation broth comprising microorganisms is performed in two or more fermentation apparatuses. In certain embodiments, the fermentation product can 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 includes providing a raw slurry comprising a fermentable carbon source, undissolved solids, oil, and water; and separating the raw slurry into the following three streams: (i) fermentable An aqueous solution comprising a carbon source, (ii) a wet cake comprising a solid, and (iii) forming 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 enzymatically. In certain embodiments, the enzyme is one or more lipases or phospholipases. In certain embodiments, the raw slurry is produced by hydrolysis of the raw material. In certain embodiments, the raw material is selected from rye, wheat, corn, sugar cane, barley, cellulosic or lignocellulosic materials, and combinations thereof. In some embodiments, the raw slurry is decanter bowl centrifuge, three-phase centrifuge, disc stack centrifuge, filtration centrifuge, decanter centrifuge, filtration, membrane filtration, microfiltration, vacuum filtration, belt filter, pressure filtration, By filtration through a sieve, sieving separation, grating, porous grid, floatation, hydrocyclone, filter press, screw press, gravity settling, vortex separator, or combinations thereof To be separated. In certain embodiments, the step of separating the feedstock is a single step process. In certain 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 the oil in the aqueous solution to fatty acids. In certain embodiments, the aqueous solution and fatty acid are added to the fermentation broth. In certain embodiments, the catalyst is deactivated.

  The present invention includes one or more fermenters: one or more fermenters including an inlet for receiving a raw slurry; and an outlet for discharging a fermentation broth containing fermentation products; and one or more extractions A first inlet for receiving the fermentation broth; a second inlet for receiving the extractant; a first outlet for discharging the dilute fermentation broth; and for discharging the rich extractant And a system including one or more extractors including a second outlet. 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 certain embodiments, the separation means comprises decanter bowl centrifugation, three-phase centrifugation, disc 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, grid, porous grid, flotation method, hydrocyclone, filter press, screw press, gravity settler, vortex separator, and combinations thereof. In certain embodiments, the system also includes an online measurement device. In certain embodiments, the online measurement device is a particle size analyzer, Fourier transform infrared spectrometer, near infrared spectrometer, Raman spectrometer, high pressure liquid chromatography, viscometer, density meter, tension meter, droplet size analyzer, pH Selected from a total, a dissolved oxygen probe, and combinations thereof.

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

Figure 2 schematically illustrates an exemplary method and system of the present invention where undissolved solids are removed by separation after liquefaction and prior to fermentation. 1 schematically illustrates an exemplary method and system of the present invention in which ISPR is performed downstream of the fermentation. Fig. 4 schematically illustrates another exemplary alternative method and system of the present invention in which an oil stream is discharged. Fig. 4 schematically illustrates another exemplary alternative method and system of the present invention in which a wet cake is subjected to a wash cycle. Fig. 4 schematically illustrates another exemplary alternative method and system of the present invention in which the oil stream is drained and the wet cake is subjected to a wash cycle. Another exemplary alternative method of the present invention, wherein the aqueous solution and wet cake are combined and sent to fermentation (FIG. 6A), and the aqueous solution, oil, and wet cake are combined and sent to fermentation (FIG. 6B) The system is shown schematically. Another exemplary alternative method of the present invention, wherein the aqueous solution and wet cake are combined and sent to fermentation (FIG. 6A), and the aqueous solution, oil, and wet cake are combined and sent to fermentation (FIG. 6B) The system is shown schematically. FIG. 2 schematically illustrates an exemplary alternative method and system of the present invention in which an aqueous solution is subjected to conversion (eg, hydrolysis, transesterification) and / or deactivation. FIG. 2 schematically illustrates an exemplary alternative method and system of the present invention in which an aqueous solution is subjected to conversion (eg, hydrolysis, transesterification) and / or deactivation. FIG. 2 schematically illustrates an exemplary alternative method and system of the present invention in which an aqueous solution is subjected to conversion (eg, hydrolysis, transesterification) and / or deactivation. FIG. 2 schematically illustrates an exemplary alternative method and system of the present 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. Figure 2 schematically shows a multiple extractant flow system. Figure 2 schematically shows 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 lag layer. 2 schematically illustrates an exemplary method of the present invention including a fermentation, extraction, and distillation process. The influence of the ratio of fermentation broth to extractant (aqueous phase / organic phase (aq / org)) on the extraction tower efficiency is shown. A and B show the effect of ISPR using an external extraction tower on isobutanol concentration and glucose profile. The influence of ISPR using a mixer settler on the isobutanol removal rate is shown. Figure 5 shows an FTIR spectrum in the range of starch concentrations using in-line measurement. Figure 5 shows the FTIR spectrum of starch concentration of wet cake during corn mash processing. Figure 2 shows FTIR spectrum of corn oil during corn mash processing. 2 shows 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. Also, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. All publications, patents, and other references mentioned herein are hereby incorporated by reference in their entirety for all purposes.

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

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

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

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

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

  “Biomass” as used herein refers to corn, sugarcane, wheat, cellulosic or lignocellulosic materials, and cellulose, hemicellulose, lignin, starch, oligosaccharides, disaccharides, and / or monosaccharides, and It refers to a natural product containing fermentable sugars and / or hydrolyzable polysaccharides that provide starch, including any sugars and starches derived from natural resources such as materials including mixtures thereof. Biomass can also include additional components such as proteins and / or lipids. The biomass may be derived from one source, or the biomass may include a mixture derived from two or more sources. For example, the biomass may comprise a mixture of corn cobs and corn stover, or a mixture of grass and leaves. Biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, paper sludge, garden waste, wood waste and forestry waste (eg, forest thinning) It is done. Examples of biomass include, but are not limited to, crop residues such as corn, corn cobs, corn hulls, corn stover, grass, wheat, rye, wheat straw, spelled wheat, rye wheat, barley, Barley straw, oats, pasture, rice, rice straw, switchgrass, potato, sweet potato, cassava, chrysanthemum, sugar cane pomace, sorghum, sugar cane, sugar beet, feed beet, soy, palm, coconut, rapeseed, safflower, Sunflower, millet, eucalyptus, cocoon, ingredients obtained from grinding grains, trees (eg branches, roots, leaves), wood chips, sawdust, shrubs and shrubs, vegetables, fruits, flowers, animal manure, and mixtures thereof Is mentioned. For example, mash, juice, molasses, or hydrolyzate can be formed from the biomass by any process known in the art for processing 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 2007/0031918, which is hereby incorporated by reference. It can be processed to obtain a hydrolyzate containing fermentable sugars by any method. Enzymatic saccharification of cellulosic and / or lignocellulosic biomass typically involves degrading cellulose and hemicellulose to produce a hydrolyzate containing sugars including glucose, xylose, and arabinose (eg, an enzyme mixture (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).

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

  As used herein, “fermentable sugar” refers to one or more sugars that can be metabolized by the microorganisms disclosed herein for the production of fermentation products.

  “Raw material” as used herein refers to a raw material in a fermentation process, the raw material contains a fermentable carbon source with or without undissolved solids and oil, and where applicable, the raw material is: The fermentable carbon source is removed from the starch or contains the fermentable carbon source before or after being obtained from the degradation of the complex saccharide by further processing, such as by liquefaction, saccharification, or other processes. The feedstock can include or be derived from biomass. Suitable raw materials include, but are not limited to, rye, wheat, corn, corn mash, sugar cane, sugar cane mash, barley, cellulosic material, lignocellulosic material, or mixtures thereof. When referring to “feed oil”, it will be understood that this term encompasses oil produced from a given feed.

  “Fermentation broth” as used herein refers to water, a fermentable carbon source (eg, sugars), dissolved solids, and optionally a microorganism that produces a fermentation product (eg, product alcohol); Optionally refers to a mixture of the 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 fermenter made by metabolism of a fermentable carbon source by a microorganism. In some cases, the term “fermentation broth” as used herein may be used interchangeably with “fermentation medium” or “fermented mixture”. In certain embodiments, the fermentation broth containing the product alcohol may be referred to as a fermented beer or beer.

  A “fermentor” or “fermentor” as used herein undergoes a fermentation reaction whereby fermented products (eg, product alcohols such as ethanol or butanol) are produced from a fermentable carbon source. Refers to the unit to be played. The term “fermenter” may be used interchangeably with “fermentor” herein.

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

  A “glycation unit” as used herein refers to a unit that undergoes saccharification (ie, degradation of oligosaccharides into 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 starch, dextran, glycogen, cellulose, pentosan, and carbohydrates including sugars.

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

  As used herein, “non-dissolved solid” refers to a non-fermentable portion of a raw material that does not dissolve in an aqueous medium, such as germ, fiber, gluten, and any additional ingredients. For example, the non-fermentable portion of the feed includes the portion that remains as a solid of the feed and can absorb liquid from the fermentation broth.

  “Oil” as used herein refers to lipids obtained from plants (eg, biomass) or animals. Examples of oils include but are not limited to beef tallow, corn oil, canola oil, capric / caprylic triglycerides, castor oil, coconut oil, cottonseed oil, fish oil, jojoba oil, lard, flaxseed oil, cow leg oil, Examples include oil of deer, coconut oil, peanut oil, rapeseed oil, rice oil, safflower oil, soybean oil, sunflower oil, kiri oil, jatropha oil, and vegetable oil blends.

A “product alcohol” as used herein refers to any alcohol that can be produced by a microorganism in a fermentation process that uses biomass as the fermentable carbon substrate source. Product alcohols include, but are not limited to, C ₁ -C ₈ alkyl alcohols. In certain embodiments, the product alcohol is an alkyl alcohol of _C 2 -C _8. In other embodiments, the product alcohol is an alkyl alcohol of C ₂ -C _5. It will be appreciated that C ₁ -C ₈ alkyl alcohols include, but are not limited to, methanol, ethanol, propanol, butanol, pentanol, and hexanol. Similarly, the alkyl alcohol C ₂ -C _8, but are not limited to, ethanol, propanol, butanol, pentanol, and hexanol and the like. In certain embodiments, the product alcohol may also include fusel alcohol (or fusel oil) and glycerol. “Alcohol” may also be used herein in connection with the product alcohol.

  “Butanol” as used herein refers to butanol isomers 1-butanol (1-BuOH), 2-butanol (2-BuOH), tert-butanol (t-BuOH), and / or isobutanol ( iB), 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 isomers 1-pentanol, 3-methyl-1-butanol, 2-methyl-1-butanol, 2,2-dimethyl-1-propanol, It 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 biological, such as fermentation, to control product concentration in a biological process as the product is produced. Refers to the selective removal of a specific product from the process.

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

  As used herein, “carboxylic acid” refers to a carbon atom bonded to an oxygen atom by a double bond to create a carbonyl group (—C═O), and a hydroxyl group (—OH by a single bond. ) Is an organic compound represented by the general chemical formula —COOH. The carboxylic acid may exist in the form of a protonated carboxylic acid, a salt of a carboxylic acid (eg, an ammonium salt, a sodium salt, or a potassium salt), or 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 derived from biomass. obtain.

As used herein, a “fatty acid” is a carbon having from C _{4 to} C ₂₈ carbon atoms (most commonly C _{12 to} C ₂₄ carbon atoms), which is either saturated or unsaturated. Refers to an acid (eg, an aliphatic monocarboxylic acid). The fatty acids can also be branched or unbranched. Fatty acids can 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 are obtained by hydrolysis of fats or synthetically. The term fatty acid can refer to a single chemical species or a mixture of fatty acids. Furthermore, the term fatty acid also encompasses free fatty acids.

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

  “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. A three-phase mixture aqueous phase, a two-phase or three-phase mixture aqueous phase in which the aqueous phase contains a certain 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 that includes a fermentation extraction, the term “fermentation broth” may in this case refer to the aqueous phase in a two-phase fermentation extraction.

  As used herein, “organic phase” refers to the non-existence 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 water phase. In some cases, the term “organic phase” as used herein may be used interchangeably with “extractant phase”.

  “Effective titer” 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 connection with a process stream refers to a composition of a stream that includes the entire stream as well as any one or more components of the stream, including all components of the stream. Refers to any fractional part of the stream to be retained.

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

  The present invention provides a method and system 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 can be initiated by introducing the raw material 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 material, lignocellulosic material, or mixtures thereof. These raw materials can be processed using methods such as dry grinding or wet grinding. In certain embodiments, prior to introduction into the fermenter, the feed may be liquefied to produce a feed slurry that may include undissolved solids, fermentable carbon sources (eg, sugars), and oil. The liquefaction of the raw material can be performed by any known liquefaction process including, but not limited to, an acid process, an enzymatic process (eg, α-amylase), an acid-enzymatic process, or a combination thereof. In certain embodiments, liquefaction can be performed in one or more liquefaction units.

  When the raw slurry is fed directly to the fermenter, undissolved solids and / or oils can 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, microparticles) leads to the extractant by preventing contact between the extractant and the fermentation broth. Reducing the mass transfer rate of the product alcohol; generating or promoting an emulsion in the fermentor, thereby preventing phase separation of the extractant and fermentation broth; at least a portion of the extractant and product alcohol being Reducing the efficiency of recovering and reusing extractants because they are “trapped” by solids that can be removed as soluble cereal-containing dry cereal distillers (DDGS); there is a solid that occupies volume in the fermenter Reducing the fermenter volumetric efficiency because of and slower release of the extractant from the fermentation broth; and the lifetime 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 “trapped” by DDGS can deviate from DDGS values and requirements for selling as animal feed. Thus, to avoid and / or minimize these problems, at least a portion of the undissolved solids can be removed from the raw slurry prior to adding the raw slurry to the fermentor. The extraction activity and efficiency of product alcohol production can be enhanced when extraction is performed in a fermentation broth from which undissolved solids have been removed.

  A method and system for processing a raw material to produce a raw slurry, separating the raw slurry, and producing a water phase containing a fermentable carbon source and a solid phase (eg, wet cake) Reference is made herein. As shown in FIG. 1, in one embodiment, the system includes a liquefaction 10 configured to liquefy the feedstock to produce a feed slurry. For example, the feedstock 12 can be introduced into the liquefaction 10 (eg, via the inlet of the liquefaction unit). Raw material 12 includes fermentable carbon sources such as sugar and / or starch, including but not limited to barley, oats, rye, sorghum, wheat, rye wheat, spelled wheat, millet, sugar cane, corn, or combinations thereof. Can be any suitable biomass material known in the art. Water can also be introduced into the liquefaction 10.

  The process of liquefying the raw material 12 includes hydrolyzing the starch in the raw material 12 into a water-soluble sugar. Any known liquefaction process used in the art, including but not limited to acid processes, enzyme 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 and a suitable enzyme 14, such as α-amylase, is introduced into the liquefaction 10. Examples of α-amylases that can be used in the systems and methods of the present invention are US Pat. No. 7,541,026; US Patent Application Publication No. 2009/0209026; US Patent Application Publication No. 2009/0238923. US Patent Application Publication No. 2009/0252828; US Patent Application Publication No. 2009/0314286; US Patent Application Publication No. 2010/02278970; US Patent Application Publication No. 2010/0048446 U.S. Patent Application Publication No. 2010/0021587, the entire contents of each of which are 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 US Pat. No. 7,498,159; US Patent Application Publication No. 2012/0003701; US Patent Application Publication No. 2012/0129229; PCT International International Publication No. 2010/096562 pamphlet; and PCT International Publication No. 2011/153516 pamphlet, the entire contents of each of which are incorporated herein by reference. In certain embodiments, enzymes for liquefaction and / or saccharification may be expressed by microorganisms that also produce product alcohol. In certain embodiments, an enzyme for liquefaction and / or saccharification may be expressed by a microorganism that also expresses a butanol biosynthetic pathway. In certain embodiments, the butanol biosynthesis pathway can be the 1-butanol biosynthesis pathway, the 2-butanol biosynthesis pathway, the isobutanol biosynthesis pathway, or the 2-butanone biosynthesis pathway.

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

  Separation 20 has an inlet for receiving raw slurry 16 and may be configured to remove undissolved solids from raw slurry 16. Separation 20 may also be configured to remove oil and / or oil and undissolved solids. Separation 20 may stir or rotate 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, sugars in the form of oligosaccharides, and water. The aqueous solution 22 may include at least about 10% by weight oligosaccharide, at least about 20% by weight oligosaccharide, or at least about 30% by weight oligosaccharide. The aqueous solution 22 can be discharged from the separation 20 via the outlet. In certain embodiments, the outlet may be located near the top of the separation 20.

  The wet cake 24 may include undissolved solids. The wet cake 24 can be discharged from the separation 20 via the outlet. In certain embodiments, the outlet may be located near the bottom of the separation 20. The wet cake 24 may also contain some sugar and water. The wet cake 24 may be washed with additional water in the separation 20 after the aqueous solution 22 has been drained from the separation 20. Alternatively, the wet cake 24 can be washed with additional water by an additional separation device. By washing the wet cake 24, sugars (for example, oligosaccharides) present in the wet cake are recovered, and the recovered sugar and water can be reused in the liquefaction 10. After washing, the wet cake 24 can be further processed to form a solubilized dry cereal distiller (DDGS) by any suitable known method. There are several advantages to forming DDGS from the wet cake 24 formed in the separation 20. Because undissolved solids are not sent to the fermentor, DDGS is not subjected to fermentor conditions. For example, since 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), the microorganisms and / or other materials are , Not captured by DDGS. These effects provide an advantage for subsequent processing and marketing of DDGS as animal feed, for example.

  Separation 20 can be any conventional used in the art including, for example, a centrifuge such as a decanter bowl centrifuge, a three-phase centrifuge, a disk 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 performed by filtration, vacuum filtration, belt filter, pressure filtration, membrane filtration, microfiltration, sieving filtration, sieving separation, grate or It can be performed by a grid, porous grid, flotation method, hydrocyclone, filter press, screw press, gravity settler, vortex separator, or any method or device that can be used to separate solids from liquids. In certain 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 oligosaccharide aqueous solution 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, for example, if a three-phase centrifuge is used to remove solids from the raw slurry 16, a third stream containing oil can be generated. Thus, several product streams can be produced using different separation techniques or combinations thereof.

  Three-phase centrifuges are used for three-phase separation of raw slurry, such as raw slurry separation, to produce two liquid phases (eg, water and oil streams) and solid phase (eg, solid or wet cake) (See, eg, Flottweg Tricanter®, Flottweg 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 is removed via a separate discharge system. obtain.

  In certain embodiments using corn as a feedstock, a three-phase centrifuge can be used to simultaneously remove solids and corn oil from the liquefied corn mash. The solid can be an undissolved solid that remains after the starch has been hydrolyzed to soluble oligosaccharides during liquefaction. Corn oil can be removed from corn kernel germs during grinding and / or liquefaction. In certain embodiments, a three-phase centrifuge may have one feed stream and three outlet streams. The feed stream can consist of liquefied corn mash produced during liquefaction. The mash can consist of an aqueous solution of oligosaccharides (eg, liquefied starch); a non-dissolved solid composed of insoluble non-starch components derived from corn; and corn oil composed of glycerides and free fatty acids. The three outlet streams from the three-phase centrifuge are a wet cake containing the majority of undissolved solids from the mash; a heavy centrifuge stream containing the majority of the liquefied starch from the mash; And may be a light centrate stream containing the majority of corn oil from the mash. The heavy centrifuge stream can be fed to the fermentation. The wet cake can be washed with process reclaimed water such as the evaporator condensate and / or backset described herein to recover soluble starch from the wet cake. The light centrifuge stream can 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 can be used as an extractant.

  With reference to FIG. 1, a fermentation 30 (or fermenter 30) configured to ferment an aqueous solution 22 to produce product alcohol has an inlet for receiving the aqueous solution 22. Fermentation 30 may be any suitable fermenter known in the art. Fermentation 30 may include a fermentation broth. In certain embodiments, simultaneous saccharification and fermentation (SSF) can occur within fermentation 30. Any known saccharification process used in the art may be used, including but not limited to acid processes, enzyme processes, or acid-enzyme processes. In certain embodiments, enzyme 38 (eg, glucoamylase, etc.) can be introduced at the entrance of fermentation 30 to hydrolyze the oligosaccharides in aqueous solution 22 to form monosaccharides. Examples of glucoamylases that can be used in the systems and methods of the present invention are US Pat. No. 7,413,887; US Pat. No. 7,723,079; US Patent Application Publication No. 2009/0275080. U.S. Patent Application Publication No. 2010/0267114; U.S. Patent Application Publication No. 2011/0014681; and U.S. Patent Application Publication No. 2011/0020899. Is incorporated herein by reference. In certain embodiments, the glucoamylase can be expressed by a microorganism. In certain embodiments, the glucoamylase can be expressed by a microorganism that also produces product alcohol. In certain embodiments, the glucoamylase can be expressed by a microorganism that also expresses the butanol biosynthetic pathway. In certain embodiments, the butanol biosynthesis pathway can be the 1-butanol biosynthesis pathway, the 2-butanol biosynthesis pathway, the isobutanol biosynthesis pathway, or the 2-butanone biosynthesis pathway.

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

  Microorganisms 32 can be introduced into the fermentation 30. In certain embodiments, microorganism 32 may be included in the fermentation broth. In certain embodiments, the microorganism 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 can be used in the methods and systems described herein. In certain embodiments, the breeding tank can be about 2% to about 5% of the size of the fermenter. In certain embodiments, the breeding tank may contain one or more of the following mashes, water, enzymes, nutrients, extractants, and microorganisms. In certain embodiments, product alcohol may be produced in the breeding tank.

  In certain embodiments, the microorganism 32 may be a bacterium, cyanobacteria, filamentous fungus, or yeast. In certain embodiments, the microorganism 32 metabolizes the sugar in the aqueous solution 22 to produce a product alcohol. In certain embodiments, the microorganism 32 may be a recombinant microorganism. In certain embodiments, the microorganism 32 can be immobilized by adsorption, covalent bonding, crosslinking, confinement, encapsulation, and the like. Methods for confining cells are known in the art, for example, as described in US Patent Application Publication No. 2011/0306116, which is hereby incorporated by reference.

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

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

In certain embodiments, the interior or device of the fermentation apparatus can be used to improve the phase separation between the fermentation broth and the extractant. For example, the interior or device can serve as a coalescer to facilitate phase separation between the fermentation broth and the extractant and / or can serve as a physical barrier to improve phase separation. The interior or device of these fermenters also prevents solids from settling in the extractant phase (or layer), promotes the aggregation of water droplets that can be incorporated into the extractant layer, and off-gas (eg, CO ₂ , (Air) removal, thereby minimizing the extractant phase and / or liquid-liquid interface disturbances. 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, lamellar separators, cones, and the like. In certain 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 certain embodiments, the interior may rotate. In certain embodiments, the interior or device may be located at or near the liquid-liquid interface level 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 agglomeration and recovery of the aqueous phase.

In certain embodiments, stream 35 may be discharged from the outlet of fermentation 30 prior to completion of ISPR and / or fermentation. The discharged stream 35 may contain microorganisms 32. The microorganism 32 can be separated from the stream 35 by, for example, centrifugation or membrane filtration. In certain embodiments, by removing the microorganisms prior to the addition of the extractant to the fermentation broth, the microorganisms are not exposed to the extractant and thus are not subject to any adverse effects that the extractant can 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 the use of extractants with improved properties but lower biocompatibility) can be used to recover the product alcohol. In certain embodiments, the microorganisms 32 may be recycled to the fermentation 30 that may increase the production rate of product alcohol, thereby improving the efficiency of product alcohol production.

  Referring to FIG. 2, in some embodiments, ISPR may be performed downstream of fermentation 30. In certain embodiments, stream 33 comprising product alcohol and microorganisms 32 may be discharged from the outlet of fermentation 30 and sent downstream, for example, to an extraction tower for product alcohol recovery. In certain embodiments, stream 33 can be processed by separating microorganism 32 prior to ISPR. For example, removal of microorganisms 32 from stream 33 can be performed by centrifugation, filtration, vacuum filtration, belt filter, pressure filtration, membrane filtration, microfiltration, sieving filtration, sieving separation, grate or lattice, porous lattice Can be performed by 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 microorganism 32, stream 33 may be sent to an extraction tower for product alcohol recovery.

  Further embodiments of the methods and systems described herein are shown in FIGS. 3-6, including the option of adding an extractant to the fermentor (eg, producing stream 36) or extraction performed downstream of the fermentor (eg, producing stream 33) are shown in FIGS. 1 and 2, respectively. Therefore, it will not be described again in detail.

  Referring to FIG. 3, the system and method of the present invention may include the discharge of oil 26 from the outlet of the separation 20. Raw slurry 16 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 separation 20. Can be separated. In certain embodiments, the separation of the raw slurry 16 into the first liquid phase, the second liquid phase, and the solid phase can be performed in a single step. In certain embodiments, feedstock 12 is corn and oil 26 is corn oil. In certain embodiments, oil 26 may be sent to a storage tank or any unit suitable for storing oil. Any suitable separation device, such as a three-phase centrifuge, can be used to drain the aqueous solution 22, wet cake 24, and oil 26. In certain embodiments, when the raw material is corn, some of the oil from raw material 12, such as corn oil, remains in wet cake 24. In certain embodiments, when the oil 26 is removed from the 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 raw material or raw 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. Oil extraction aids such as surfactants, demulsifiers, or flocculants as well as enzymes can be used to improve the removal of oil from the raw material or raw material slurry. Examples of oil extraction aids include, but are not limited to, non-polymeric liquid surfactants; talcum powder; fine talcum powder; salt (NaOH); calcium carbonate; and Pectinex® Ultra SP-L, Enzymes such as Celluclast® and Viscozyme® L (Sigma-Aldrich, St. Louis, MO), and NZ 33095 (Novozymes, Franklinton, NC).

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

  In certain embodiments, separation 70 may be, for example, decanter bowl centrifugation, three-phase centrifugation, disk stack centrifugation, filtration centrifugation, decanter centrifugation, filtration, vacuum filtration, belt filter, pressure filtration, membrane filtration, sieving. Capable of separating solids and liquids, including filtration, sieving separation, grids, porous grids, flotation methods, hydrocyclones, filter presses, screw presses, gravity settlers, vortex separators, or combinations thereof Any separation device can be used.

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

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

  In certain embodiments, a portion of the undissolved solid can be sent to fermentation 30. In certain embodiments, this portion of the undissolved solid can have a smaller particle size (eg, a fine powder). In certain embodiments, this portion of the undissolved solid may form a total distillation waste. In certain embodiments, the total distillation waste can be treated to form a thin distillation waste and a wet cake. In certain embodiments, the wet cakes and wet cakes 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 drained separately from the wet cake, and the oil is included as part of the wet cake and is ultimately present in the DDGS. When corn is used as a raw material, corn oil contains triglycerides, diglycerides, monoglycerides, fatty acids, and phospholipids, which provide a source of metabolic energy for animals. The presence of oil (eg, corn oil) in the wet cake and ultimately DDGS provides the desired animal feed, eg, high fat content animal feed.

  In certain embodiments, the oil can be separated from the wet cake and DDGS and converted to an ISPR extractant for subsequent use in the same or different alcohol fermentation processes. Methods for obtaining extractants from biomass are described in US Patent Application Publication No. 2011/0312044; US Patent Application Publication No. 2011/0312043; and US Patent Application Publication No. 2012/0156738. The entire contents of each are incorporated herein by reference. The oil can 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 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 can be subjected to a drying process to remove any residual water. The treated wet cake can be used to produce DDGS. Treated DDGS can be used as supplementary feed for animals such as dairy and beef cattle, poultry, pigs, livestock, horses, aquaculture, and pets.

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

  In certain embodiments, when corn is used as a raw material, xanthophyll is isolated from corn and / or undissolved solids, as a pigment component in DDGS or animal feed, or as a supplement for pharmaceutical and dietary supplement applications Can be used as Methods for isolating xanthophyll 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, the entire contents of each of which are incorporated herein by reference. 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, xanthophyll can be isolated from corn and / or undissolved solids and added to COFA. In certain embodiments, COFA and / or xanthophyll can be used in food, pharmaceutical, and dietary supplement applications.

  After extraction from 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 so that the solvent can be evaporated, collected and reused. The recovered oil can be converted to ISPR extractant for subsequent use in the same or different alcohol fermentation processes.

  Since the oil present in the fermentor can be broken down into fatty acids and glycerin, removal of the oil component of the feed is advantageous for product alcohol production. Glycerin can accumulate in water and reduce the amount of water available for reuse throughout the system. Thus, removal of the feed oil component 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 can 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 containing oil 26, and a solid phase or wet cake 24 using, for example, a three-phase centrifuge. The wet cake 24 can be further processed to recover fermentable sugar and oil. Wet cake 24 may be sent to mixing 60 and combined with water or other solvent to form wet cake mixture 65. In certain embodiments, the water can be backflow, cooking water, process water, distilled residual water, water collected from evaporation, or any water source available at a fermentation treatment facility, or any combination thereof. The wet cake mixture 65 may be sent to a separation 70 (eg, a three-phase centrifuge) to produce a washed centrifuge 75 that includes fermentable sugar, oil stream 76, and wet cake 74. Washed centrifuge liquid 75 can be reused for liquefaction 10.

  As described herein, the wet cake may be subjected to one or more cleaning cycles or cleaning systems. In certain embodiments, the wet cake 74 may be sent to the second mixing 60 'to form a wet cake mixture 65'. The wet cake mixture 65 'can be sent to the second separation 70' to produce a wash centrifuge 75 ', an oil stream 76' and a wet cake 74 '. The washed centrifuge liquid 75 ′ can be reused for liquefaction 10. In certain embodiments, the washed centrifuge 75 'may be combined with the washed centrifuge 75 and reused for liquefaction 10. In certain embodiments, the wet cake 74 'can be combined with the wet cake 74 for further processing as described below. In certain embodiments, oil stream 76 'and oil 26 can be combined and further processed to produce an extractant that can be used in a fermentation process, or oil stream 76' and 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 syrup and then dried to form DDGS using any suitable process. There are several advantages to forming DDGS from wet cake 74. Since 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 fermentor. These advantages make it easier to process DDGS as animal feed, for example. As described herein, in certain embodiments, the wet cakes 74, 74 ', and the wet cake formed from the total distillation waste liquor can be combined and further processed to produce DDGS.

  As shown in FIG. 6A, the aqueous solution 22 and wet cake 24 can be combined, cooled, and sent to the fermentation 30. The raw slurry 16 can be separated into a first liquid phase or aqueous solution 22, a second liquid phase containing oil 26, and a solid phase or wet cake 24 using, for example, a three-phase centrifuge. In certain embodiments, oil 26 may be sent to a storage tank or any unit suitable for storing oil. The aqueous solution 22 and wet cake 24 can be sent to the mix 80 and reslurried to form an aqueous solution / wet cake mixture 82. Mixture 82 may be sent to cooler 90 to produce cooled mixture 92, which may be sent to fermentation 30. In certain embodiments, when oil 26 is removed from feed 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) to include 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 portions thereof, can be sent to fermentation 30. In certain embodiments, the aqueous solution 22, wet cake 24, and oil 26, or portions thereof, may be combined, for example, by mixing to form an aqueous solution, wet cake, and oil mixture, the mixture being fermented 30 can be sent. In certain embodiments, aqueous solution 22 and wet cake 24 may be combined to form an aqueous solution and wet cake mixture, and then oil 26 is added to the mixture to provide the aqueous solution, wet cake, and oil mixture. May be formed and this mixture may be sent to fermentation 30. In certain embodiments, the aqueous solution 22 and wet cake 24 may be combined to form an aqueous solution and wet cake mixture, and the mixture and oil 26, or portions thereof, are sent to the fermentation 30 as a separate stream. 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 liquefaction 10 and separation 20 or between separation 20 and fermentation 30. In certain embodiments, liquefaction and / or saccharification is performed using raw starch enzymes or cold hydrolyzing enzymes such as Stargen ™ (Genencor International, Palo Alto, Calif.) And BPX ™ (Novozymes, Franklinton, NC). Can be done. In certain embodiments, 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 may 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 separation unit and the second separation unit may 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, disc stack centrifugation, filtration centrifugation, decanter centrifugation, filtration, vacuum filtration, belt filter, pressure filtration, membrane filtration, filtration using sieve, sieving separation, This can be done by several means including but not limited to grids, porous grids, flotation methods, hydrocyclones, filter presses, screw presses, gravity settlers, vortex separators, or combinations thereof.

  The absence or minimal of undissolved solids in the fermentation broth has several advantages. For example, the need for operating units in downstream processing is eliminated, thereby increasing the efficiency of product alcohol production. Also, some or all of the centrifuge used to treat the total distillation waste can be omitted as a result of less undissolved solids in the fermentation broth exiting the fermenter. 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 further description of methods and systems for separating undissolved solids from feed slurries, see, for example, US 2012/0164302 and PCT International, each of which is incorporated herein by reference in its entirety. See patent application PCT / US2013 / 51571.

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

  In certain embodiments, the extractant can be selected based on the particular properties and / or characteristics described herein. For example, the viscosity of the extractant can affect the mass transfer characteristics of the system, that is, the efficiency with which product alcohol can be extracted from the aqueous phase to the extractant phase (ie, the organic phase). The density of the extractant can affect the phase separation. In certain embodiments, selectivity refers to the relative amount of product alcohol relative to water taken up by the extractant. 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 will separate the butanol while minimizing the need for a pyrolysis or side reaction of the extractant or a deep vacuum in the distillation process. Should be low enough to allow

  The extractant can be biocompatible with the microorganism, i.e., it is 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 rate 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 the ability of a microorganism to utilize a fermentable carbon source. For example, with a non-biocompatible extractant, the microbe is more than about 25%, greater than about 30%, greater than about 35%, greater than about 40%, greater than about 45%, or about 50% of the rate in the absence of the extractant. Glucose cannot be used at a rate exceeding%.

  One skilled in the art can select the extractant to maximize the desired properties and / or characteristics described herein and to optimize the recovery of the product alcohol. One skilled in the art can also appreciate 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. In addition, the extractant mixture can be used to adjust and optimize the physical properties of the extractant, such as density, boiling point, and viscosity. For example, an extractant with a sufficient partition coefficient of product alcohol and sufficient biocompatibility to allow an appropriate combination to allow economical use of the extractant to remove product alcohol from the fermentation broth. Can be provided.

In certain embodiments, the extractant useful in the methods and systems described herein can be an organic solvent. In certain embodiments, the 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 It can be an organic extractant selected from the group consisting of ₂₂ fatty aldehydes, C ₁₂ -C ₂₂ fatty amides, and 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 ~ It can be an organic extractant selected from the group consisting of C ₂₂ fatty aldehydes, C ₄ -C ₂₂ fatty amides, and mixtures thereof. In certain embodiments, the fatty acid may be a C _{4 to} C ₂₄ fatty acid and / or the ester may be an ester of a C _{4 to} C ₂₄ fatty acid. 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 It can be an organic extractant selected from the group consisting of ₁₈ fatty aldehydes, C ₁₂ -C ₁₈ fatty amides, and 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 It can be an organic extractant selected from the group consisting of ₁₈ fatty aldehydes, C ₁₄ -C ₁₈ fatty amides, and 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 can include a carboxylic acid. In certain embodiments, the ester of a fatty acid can be a combination of a fatty acid and an alcohol (eg, a fatty ester). In certain embodiments, the alcohol can be a product alcohol. In certain embodiments, the ester can be a methyl ester, ethyl ester, propyl ester, butyl ester, pentyl ester, hexyl ester, 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 _A second extractant selected from ₂₂ fatty aldehydes, C ₁₂ -C ₂₂ fatty amides, and mixtures thereof may be included. 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 a second extractant selected from C ₁₂ -C ₂₂ fatty alcohols, C ₁₂ -C ₂₂ fatty acids, esters of C ₁₂ -C ₂₂ fatty acids, and 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 a second extractant selected from C ₁₂ -C ₁₈ fatty alcohols, C ₁₂ -C ₁₈ fatty acids, esters of C ₁₂ -C ₁₈ fatty acids, and 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 a second extractant selected from C ₁₄ -C ₁₈ fatty alcohols, C ₁₄ -C ₁₈ fatty acids, esters of C ₁₄ -C ₁₈ fatty acids, and 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 fatty amides, and first extraction agent selected from mixtures thereof; fatty _alcohols, fatty acids C 7 _{-C 11} and of _C 7 _{-C 11,} esters of fatty acids of _C 7 ~C _11, C 7 ~C _A second extractant selected from ₁₁ fatty aldehydes, and mixtures thereof.

  In certain embodiments, the extractant is oleyl alcohol, behenyl alcohol, cetyl alcohol, lauryl alcohol (also called 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, undecanal, laurinaldehyde, 2-methyl Undecanal, oleamide, linoleamide, palmitoamide, stearylamide, 2-ethyl-1-hexanol, 2-hexyl-1-decanol, 2-octyl-1-dodecanol, and their It may be an organic extractant such compounds. In certain embodiments, the extractant may include one or more of the following oleic acid, lauric acid, linoleic acid, linolenic acid, myristic acid, palmitic acid, stearic acid, octanoic acid, decanoic acid, and undecanoic acid. . In certain embodiments, the extractant may include one or more of the following oleic acid, linoleic acid, linolenic acid, myristic acid, palmitic acid, and stearic acid. In certain embodiments, the extractant can include one or more of the following oleic acid, linoleic acid, palmitic acid, and stearic acid. In certain embodiments, the extractant comprises one or more of the following oleic acid, lauric acid, linoleic acid, linolenic acid, myristic acid, palmitic acid, stearic acid, octanoic acid, decanoic acid, and undecanoic acid, and olein One or more esters of acid, lauric acid, linoleic acid, linolenic acid, myristic acid, palmitic acid, stearic acid, octanoic acid, decanoic acid, and undecanoic acid may be included. In certain embodiments, the extractant is one or more of the following oleic acid, linoleic acid, linolenic acid, myristic acid, palmitic acid, and stearic acid, and oleic acid, linoleic acid, linolenic acid, myristic acid, palmitic acid. 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 acid, linoleic acid, palmitic acid, and stearic acid, and one or more esters of oleic acid, linoleic acid, palmitic acid, and stearic acid. 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, undecanal, 2-ethyl-1-hexanol, 2-hexyl-1-decanol, One or more of 2-octyl-1-dodecanol may be included.

  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 US 2009/0305370 and US 2011/0097773; see the entire contents of each. Is incorporated herein by reference. In certain embodiments, the biocompatible extractant can have a high atmospheric pressure boiling point. For example, the biocompatible extractant can have an atmospheric boiling point that is higher than the atmospheric boiling point of water.

  In certain embodiments, a hydrophilic solute can be added to the fermentation broth that is contacted with the extractant. Due to the presence of hydrophilic solutes in the aqueous phase, phase separation can be improved and the proportion of product alcohol that is separated into the organic phase can be increased. Examples of hydrophilic solutes include, but are not limited to, polyhydroxylated compounds, polycarboxylic acid compounds, polyol compounds, and dissociated ionic salts. Sugars 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. Examples of the polycarboxylic acid compound include citric acid, tartaric acid, maleic acid, succinic acid, polyacrylic acid, and sodium, potassium, or ammonium salts thereof. Ionic salts that can be used as hydrophilic solutes in the fermentation broth 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 is from the aqueous phase (eg, fermentation broth) to the organic phase (eg, extractant) without adversely affecting the growth and / or productivity of the microorganism that produces the product alcohol. Can be selected by those skilled in the art to maximize the migration of the product alcohol. High levels of hydrophilic solutes can impart osmotic stress and / or toxicity to microorganisms. One skilled in the art can use a number of known methods for determining the optimal amount of hydrophilic solute to minimize the effects of osmotic stress and / or toxicity on microorganisms.

  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, an extractant that does not provide hydrogen bonds to water selectively absorbs alcohol. In certain embodiments, the extractant can 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). It may also include alkyl-substituted benzenes, including but not limited to, meta-diisopropylbenzene, para-diisopropylbenzene, triethylbenzene, ethylbutylbenzene, and tert-butylstyrene. The advantage of using an alkyl-substituted benzene is a relatively high butanol affinity compared to other hydrocarbons. Furthermore, isopropyl substituted or isobutyl substituted benzene may provide certain advantages for butanol affinity over other substituted benzenes. Another advantage is lower viscosity, lower surface tension, lower density, higher thermal stability, and higher chemical stability, which aids phase separability and long term reuse. 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 hydrogen bonds to the hydroxyl portion of butanol, whereby the mixture is 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 operating under vacuum. This distillation can operate at reflux to maintain a high purity butanol distillate that contains little extractant. The bottoms can include a portion of butanol contained in the distillation feed so that the reboiling temperature under vacuum is suitable for supplying heat indirectly from the available steam. The distillation may be carried out using a condensing device, where only the reflux liquid is condensed and the substantially butanol composition vapor distillate is decanted from the condensed beer tower overhead vapor. The butanol stream can be sent directly to the bottom of a rectification column fed simultaneously. The advantage of this type of distillation is that the heat that entrains 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 can be produced from a raw material. For example, oil such as corn oil present in the feedstock can be used to produce an extractant for extractive fermentation. The glycerides in the oil chemically or into reaction products such as fatty acids and / or fatty esters (eg, ethyl esters, butyl esters, fusel esters) that can be used as extractants for the recovery of product alcohol It can be converted enzymatically. Using corn oil as an example, corn oil triglycerides are reacted with a base such as ammonia hydroxide to give fatty amides and glycerol. In certain embodiments, the oil in the feed can 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 acylglycerides in the oil. In certain embodiments, the resulting acid / oil composition comprises glycerol, an acylglyceride hydrolysis byproduct. In certain embodiments, the resulting acid / oil composition comprises lysophospholipids from partial hydrolysis of phospholipids in the oil. Methods for obtaining extractants from biomass are described in US Patent Application Publication No. 2011/0312044; US Patent Application Publication No. 2011/0312043; and US Patent Application Publication No. 2012/0156738. The entire contents of each of which are hereby incorporated by reference.

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

  In certain embodiments, the oil may be separated from the feed slurry, the oil 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 to deactivate the lipase and then the fatty acid stream may 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, the oil separated from the raw slurry can 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 quench the lipase, cooled, and then 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 sent to the 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 the fermenter.

  In certain embodiments, the one or more catalysts can be one or more enzymes, eg, hydrolase enzymes. In certain embodiments, the one or more catalysts can be one or more enzymes, such as a lipase enzyme. The lipase enzymes are, for example, Absidia, Achromobacter, Aeromonas, Alcaligenes, Alternaria, Aspergillus, Aspergillus, Achromobacter, Aureobasidium, Bacillus, Beauveria, Brochothrix, Candida, Cromobacter, Chromobacter, Chromobacter Genus (Fusarium), Geotricum (Geotri) um), Hansenula, Humicola, Hyphozyma, Lactobacillus, Metarhidium, Mucor, Nectria, Nectria, and Nectria ), Pecilomyces, Penicillium, Pseudomonas, Rhizoctonia, Rhizomucor, Rhizodrum ), Saccharomyces (S ccharomyces), wild boar (Sus), sporoboromyces, Thermomyces, Thiarosporelella, Trichoderma il / Virticia wi ) From any source. In certain embodiments, the source of lipase is Absidia blackesleena, Absidia corimbifera, Achromobacter iophagas, Alkalinea sp. brassicola, Aspergillus flavus, Aspergillus niger, Aspergillus tubingensis, Aureobasiul pulid (Aureobasiul pulidus) lans), Bacillus coagulans, Bacillus pumilus, Bacillus stearothermophilus, rothia thorax (Bacillis subtilis) (Burkholderia cepacia), Candida cylindracea (Candida rugosa), Candida paralipolytica (Candida paralipolitica), Candida antida arctica lipase A, Candida antarctica lipase B, Candida ernobii, Candida deformos, Candida rosida Chromobacter viscosum, Corinus cinerius, Fusarium heterosporum, Fusarium oxysporum, 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) , Humicola brevis var. Thermoidea (Humicola brevis var. thermoidea, Humicola insolens, Lactobacillus curtotus, Rhizopus auricae, Mucor javnicus (Mucor javanics) Penicillium cyclopium, Penicillium crustosum, Penicillium expansum, Penicillium rockforti roqueforti, Penicillium camembertii, Penicillium sp. II, Penicillium sp. al, Pseudomonas aeruginosa Pseudomonas cepacia (also known as Burkholderia cepacia), Pseudomonas fluorescens, Pseudomonas fragii, Pseudomonas on multoria 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, Rhodosporidium toruloides, Rhodotorus glutinosto 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 can 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 homologous or heterologous hydrolases and / or lipases. In certain embodiments, the hydrolase and / or lipase can be expressed by a microorganism that also produces product alcohol. In certain embodiments, the hydrolase and / or lipase can be expressed by a microorganism that also expresses the butanol biosynthetic pathway. In certain embodiments, the butanol biosynthesis pathway can be the 1-butanol biosynthesis pathway, the 2-butanol biosynthesis pathway, the isobutanol biosynthesis pathway, or the 2-butanone biosynthesis 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 (R) CALB L, Novozyme (R) CALA L, and Palatase 20000L, or available from Sigma Aldrich (St. Louis, MO), Pseudomonas fluorescens, ), Mucor miehei, hog pancreas, Candida ki Candida cylindracea, Candida rugosa, Rhizopus niveus, Candida antarctica, genus sp. Can be mentioned. In certain embodiments, the lipase may be heat stable and / or heat resistant and / or solvent resistant.

  In certain embodiments, the one or more catalysts can be a phospholipase. Phospholipases useful in the present invention can be of various biological sources, such as, but not limited to, Fusarium culmorum, Fusarium heterosporum, Fusarium solani, or Filamentous fungi within the genus Fusarium, such as the strain of Fusarium oxysporum; 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 variants such as 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, phospholipases can be expressed by microorganisms that also produce 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 biosynthesis pathway can be the 1-butanol biosynthesis pathway, the 2-butanol biosynthesis pathway, the isobutanol biosynthesis pathway, or the 2-butanone biosynthesis pathway.

  Byproducts 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 microorganisms May have an inhibitory effect on In certain embodiments, these by-products can 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, the lipase present in the fermentation broth can catalyze the esterification of by-products produced during fermentation. Esterification of these by-products can minimize their inhibitory effects on microorganisms.

  Referring to FIG. 7A, raw material 12 is not described in detail because it can be processed as described in FIGS. The aqueous solution 22 can then be further processed to remove any residual oil. In certain embodiments, the aqueous solution 22 can be subjected to centrifugation, decantation, or any other method that can be used for oil removal. In certain embodiments, the aqueous solution 22 may be sent to a unit 25 (or tank) and a catalyst 23 (eg, 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 microorganism 32 may also be added to fermentation 30 for production of product alcohol. After fermentation 30, stream 31 containing product alcohol and fatty acids can be sent to an external unit, such as 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 certain embodiments, stream 27 containing catalyst 23 is heated (q) to deactivate catalyst 23 prior to addition to fermentation 30. Referring to FIG. 7C, in certain embodiments, deactivation may be performed in a separate unit, eg, a deactivation unit. In certain embodiments, stream 27 may be sent to deactivation 28. After deactivation, stream 27 'may be sent to fermentation 30 and microorganism 32 may also be added to fermentation 30 for production of product alcohol.

  By removing the oil from the aqueous solution 22 by converting the oil into a fatty acid, energy savings of the production plant by more efficient fermentation, less fouling of the equipment by removing the oil, energy requirements such as cereal distillation lees A reduction in the energy required to dry and an improved operation of the evaporator or evaporation train is obtained. Furthermore, since the oil present in the fermenter can be broken down into fatty acids and glycerin, removal of the raw oil component is advantageous for product alcohol production. Glycerin can accumulate in water and reduce the amount of water available for reuse throughout the system. Thus, 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, a stable emulsion is less likely to result from oil removal. In certain embodiments of the invention, when an emulsion is formed, the emulsion can be easily broken down by mechanical processing, addition of protic solvents, or other conventional means.

  In another embodiment, referring to FIG. 7D, the aqueous solution 22 may be sent to the fermentation 30 and a catalyst 23 (eg, lipase) is added to the fermentation 30 so that the oil present in the aqueous solution 22 is a fatty acid. And / or can be converted to fatty esters. In certain embodiments, the fatty ester is obtained by a combination of a fatty acid and an alcohol. In certain embodiments, the alcohol can be any alcohol in fermentation 30 including the product alcohol. In certain embodiments, the amount of oil in 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 ester and fatty acid produced by oil conversion can be about 75:25. In certain embodiments, the ratio of fatty ester and fatty acid 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 comprising product alcohol, fatty acid, and fatty ester can be further processed for product alcohol recovery. For example, stream 31 may be sent to an external unit, such as an external extractor or an 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 can include fatty acids and / or esters of fatty acids.

  The present invention also provides a method and system for recovering product alcohol produced by the 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. The 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 fermenter and the fermentation broth and extractant perform mass transfer (eg, transfer of product alcohol from the fermentation broth to the extractant). Mixed together to result, fermentation may be able to promote a high effective product alcohol titer. In such a process, if the mixing is too strong or intense, the fermentation broth and extractant may need to be separated using a separation device such as a centrifuge. If the mixing is not very intense, phase separation can be done by gravity settling caused by the density difference between the extractant and the fermentation broth. In either case, additional fermenter may be required to overcome the reduction in fermenter volume taken up by the extractant added to the fermenter. The direct addition of the extractant to the fermenter can be done in batch, semi-batch, or continuous mode regardless of phase separation in the fermenter. If a continuous system 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 the separation of the product alcohol from the extractant. If the separation process used to remove the product alcohol from the extractant is such that the microorganisms present in the fermentation broth can survive during the separation process, the fermentation broth from the product alcohol / extractant Separation may not be necessary.

  Another embodiment of the liquid-liquid extraction process may include an external extractor or extraction tower. For example, fermentation broth from the fermenter may be sent to an external extractor, where the fermentation broth is mixed with an extractant. The mixture of fermentation broth and extractant can then be separated to produce a fermentation broth stream that is lean in product alcohol and an extractant stream that is rich in product alcohol. A more dilute fermentation broth stream can be returned to the fermenter. The richer extractant stream can 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 may 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 fermentation apparatus and an external extractor. The additional volume of fermentation broth present in the external extractor can serve to increase the overall fermenter volume and thus increase the overall production of product alcohol.

  The performance of the external extractor with respect to product alcohol removal is the surface area available for interfacial contact, the physical properties of the fermentation broth and extractant, the two phases present in the external extractor (e.g., the fermentation broth phase and Dependent on the relative amount of extractant phase) and the concentration driving force difference between the fermentation broth phase and extractant phase. Maximizing the efficiency of the external extractor at a given product alcohol concentration driving force can reduce the droplet size of the phase dispersed in the external extractor, eg, nozzle design, internal design, and / or agitation. It can be done by reducing by. In certain embodiments, the design and operation of the external extractor can provide sufficient product alcohol transfer between the fermentation broth phase and the extractant phase to maintain product alcohol productivity requirements. Sufficient mixing can be provided.

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, fermentation broth droplets may adhere to the CO ₂ rising through the extractant phase. In certain embodiments, the extractant phase can 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 flocculation and recovery of the fermentation broth phase, for example, a flocculation pad may be added to the 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 microorganism may be separated from the fermentation broth prior to contacting the fermentation broth with the extractant. In certain embodiments, the microorganism may be separated from the mixture of fermentation broth and extractant prior to separation (or processing) of the mixture. Any separation method capable of separating the microorganisms from the fermentation broth or the mixture of fermentation broth and extractant may be used including, for example, centrifugation. Separating the microorganisms before contacting the fermentation broth with the extractant may allow higher temperatures and / or more stringent extraction conditions such as non-biocompatible extractants to be used. If separation methods are used that are not harmful to microorganisms, 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 is included in the evaporator train feed and can thus be a component of the syrup formed during evaporation and possibly incorporated into animal feed. In certain embodiments, the extractant can be separated from the syrup using any separation means including, for example, centrifugation. A low boiling biocompatible extractant (eg, equivalent to water) may not require such separation because the extractant and water can be reused for use in the production process.

  In a typical product alcohol production plant from corn, by recycling the water in the production plant while distilling the recycled water in the evaporator train to remove salt and other dissolved solids of beer The water balance of the entire production process can be maintained. The syrup obtained from the evaporator train may be mixed with undissolved solids and the mixture can be dried and sold as animal feed. Methods and systems for processing undissolved solids for animal feed are described, for example, in US Patent Application Publication No. 2012/0164302; US Patent Application Publication No. 2011/0315541; US Patent Application Publication No. 2013. No. / 0164795; and PCT International Patent Application No. PCT / US2013 / 51571, the entire contents of each of which are hereby incorporated by reference.

  As described herein, undissolved solids can be removed from the feed (or feed slurry) prior to the addition of feed to the fermentation. If undissolved solids are not removed upstream of the fermentation, centrifuging beer to remove undissolved solids may be necessary to avoid evaporator fouling. For example, in a product alcohol production plant from commercial dry ground 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. An upstream process that removes enough solids to keep the percentage of total suspended solids below these percentage values, for example, the need for centrifugation before sending the beer to the evaporator (or evaporation train). It can disappear. Eliminating this centrifugation will save the capital needed to retrofit the production plant for product alcohol from dry ground corn.

  By removing at least a portion of the undissolved solids present in the raw slurry prior to fermentation, the interfacial surface area between the fermentation broth phase and the extractant phase in the external extractor is reduced to 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. The clear phase separation can also save capital expenditures because it eliminates the need for additional separation steps (eg, centrifugation).

  Separation of the fermentation broth and extractant leaving the external extractor determines the particle size distribution of the solid content and the solid content in the fermentation broth, the gas content and bubble size distribution in the fermentation broth, viscosity, density, and surface tension. It can be influenced by the physical properties of the fermentation broth and extractant, including but not limited to, the design and operation of the external extractor and the design and operation of the fermentation apparatus. These characteristics can determine the need for a separation device (eg, a centrifuge) to separate the fermentation broth and extractant exiting the external extractor or fermenter. By operating under conditions that eliminate the need for a separation device, capital expenditures for performing liquid-liquid extraction ISPR can be minimized. In addition, fermentation can be reduced by minimizing the size of the extractor by maximizing the interfacial area between the fermentation broth phase and the extractant phase that have the physical properties of a given set of fermentation broth and extractant. The ability to phase separate broth and extractant at a low cost can be maintained. By eliminating the capital and operating costs of a separation device such as a centrifuge, the net present value of the product alcohol production plant from dry ground corn using a liquid-liquid extraction ISPR process can be improved.

  In another embodiment of the methods and systems described herein, the extractor design, including phase separation capabilities, can be adjusted to accommodate the physical characteristics 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, there should be enough product alcohol to maintain factory productivity using an extractor that does not include a phase separator. Sometimes it cannot be removed. Accordingly, the present invention provides a method and system that includes solids removal and product alcohol recovery using an external extractor, where the extractor improves phase separation capability for maximum product alcohol recovery. Designed to let you.

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

  As described herein with reference to FIGS. 1-7, feedstock 12 can be processed and solids can be separated (100). Briefly, raw material 12 can be liquefied to produce a raw slurry that includes oil depending on the undissolved solids, fermentable sugar (or fermentable carbon source), and raw material. The raw slurry is then subjected to a separation process to remove suspended solids to produce a wet cake, an aqueous solution 22 (or centrifuge) containing dissolved fermentable sugar, and optionally an oil stream. obtain. Solid separation is 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 separation, grid, porous grid, flotation method, hydrocyclone, filter press, screw press, gravity settler, vortex separator, or combinations thereof may be performed by a number of means.

  The aqueous solution 22 and the microorganism 32 may be added to the fermentation 30, where fermentable sugars are fermented by the microorganism 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 extract 120), where stream 105 is contacted with extractant 124. In certain embodiments, the extractant can be stored in an extractant storage tank or unit. In certain embodiments, stream 105 can 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 fermentation is initiated. In certain embodiments, stream 105 may be removed from fermentation 30 to minimize the effect of product alcohol on microorganism 32. In certain embodiments, the fermentation 30 may include one, two, three, four, five, six, seven, eight, or more fermenters.

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

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

  There may be some loss of extractant or extractant during extraction. In certain embodiments, the extractant 124 may be replenished by addition of the extractant to the extractor 120 or extractant storage unit. In certain embodiments, for example, where the extractant is obtained from a raw material or raw slurry, the extractant 124 can be replenished by converting the oil in the raw material or raw slurry to the extractant. For example, a catalyst can 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. A stream 122 containing the product alcohol-enriched extractant, fatty acid, and / or fatty ester may be sent to separation 130 to produce a stream 125 containing a leaner extractant, fatty acid, and / or fatty ester. In certain embodiments, stream 125 may be further processed before being returned to extractor 120. For example, fatty esters present in stream 125 can be subjected to hydrolysis to produce a stream comprising product alcohol and fatty acids. This stream containing product alcohol and fatty acids may be sent to extractor 120, or this 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 acid may be sent to separation 130 or this stream is 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 the extractor 120 and the product alcohol stream may be combined with the stream 135 and further processed for product alcohol recovery.

  In certain embodiments, the phase separation of the fermentation broth and extractant after passing through the extractor may be insufficient, so that unacceptable levels of dispersed extractant remain in the fermentation broth that returns to the fermentor, And / or unacceptable levels of fermentation broth droplets remain in the extractant proceeding to distillation. In certain embodiments, phase separation of the fermentation broth and extractant can 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 extractant into contact with each other, and the heterogeneous mixture that is formed becomes a water (eg, fermentation broth) phase and organic It may be pumped through one or more hydrocyclones or similar vortex devices to separate the (eg extractant) phase. 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 can be from a fermenter. In certain embodiments, the gas stream can be from a degassing device.

  In a batch or semi-batch fermentation process, when a portion of the fermentable sugar is metabolized by the microorganism 32, the stream 103 containing beer can be sent downstream to the separation 140 to separate the product alcohol from the beer. Stream 145 containing product alcohol can be sent downstream for further processing (eg, distillation) including recovery of product alcohol. In a continuous fermentation process, a stream 103 containing beer may be sent downstream to separation 140 to separate product alcohol from the beer. Stream 142 containing total distillate waste can be sent downstream for further processing including solids removal and production of dilute distillate waste.

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

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

  In certain embodiments of the methods and systems described herein, a decanter can be used for phase separation. In certain embodiments, the decanter can 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 can be added to stream 125 and / or stream 127. In certain embodiments, the nutrition can be soluble in the extractant. In certain embodiments, the concentration of oxygen may be measured in various streams and may be used as part of a control loop to change the oxygen flow into the process. In certain embodiments, mash can be added to the extractor 120 to allow for higher effective titers. In certain 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 can be injected with a solute that facilitates 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 of equilibrating the concentration of product alcohol in the extractant prior to separation (eg, distillation).

In certain embodiments, the extractor 120 may be designed to use the CO ₂ produced during fermentation to mix the fermentation broth and extractant. In certain embodiments, the extractor 120 can be designed to allow easy removal of CO _{2 in} the fermentation broth. This design will facilitate control of the level of mixing due to the CO ₂ bubbles rising through the extractor 120. In certain embodiments, the fermentation broth can be removed from the fermentation 30 to minimize the concentration of CO _{2 in} the stream 105. In certain embodiments, the design of the extractor withdrawal region 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 release region to improve phase separation. In certain embodiments, the external extractor may include an outlet port for the interior or CO _2. For example, an agglomeration pad can be added to an external extractor.

In certain embodiments for minimizing CO ₂ mixing, the extractor can be designed to have a small diameter at the bottom of the extractor and gradually increase in diameter toward the top of the extractor (eg, a cone). form). In certain embodiments, the extractor can 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 incremental increases in diameter. In certain embodiments, the extractor may include one or more regions of constant diameter.

During fermentation, the gas content (eg, CO ₂ ) of the fermentation broth 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 changing the flow rate through the gas stripper and / or the gas stripper pressure. In certain embodiments, the amount of CO ₂ in the fermentation broth can be reduced before transferring the fermentation broth to the extractor. For example, CO ₂ can 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 can be performed at ambient pressure or lower. In certain embodiments, the fermentation may continue in the extractor and CO ₂ may be produced by the microorganism. In order to minimize CO ₂ mixing in the extractor, in certain embodiments, the residence time of the fermentation broth in the extractor can be reduced. In certain embodiments, the residence time can be reduced by adjusting the height of the extractor. In certain 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, the extractor can be replaced with two or more extractors of reduced height to maintain a theoretical number of extraction stages. In certain embodiments, two or more extractors may be continuous. In certain embodiments, two or more extractors may be linked. In certain embodiments, two or more extractors can be coupled to maintain countercurrent. In certain embodiments, a degassing stage can be added to one or more extraction stages.

  Referring to FIG. 8, in certain embodiments, 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 particle size analysis such as focused beam reflectometry (FBRM®) or particle microscopic imaging (PVM®) technology (Mettler-Toledo, LLC, Columbia 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, solids present in the fermentation broth may interact less with the extractant. Absent. In certain embodiments, the conditions of separation 130 can be controlled to minimize the effects of oxidative and thermal instabilities on the extractant.

  In certain embodiments, the quality of the extractant can be monitored and replenished with the extractant as often as necessary for good production of product alcohol. In certain embodiments, the extractant may be incorporated into the total distillation waste solid. The total distillation effluent may be separated into a liquid (eg, dilute distillation effluent) stream and a solid stream, and the solid may be washed to recover the extractant. In certain embodiments, the temperature of the extractor 120 can be adjusted to improve overall process efficiency. In certain embodiments, the fermentation broth and extractant streams to the extractor 120 may be cocurrent or countercurrent. 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, polymer beads or inorganic beads may preferentially absorb product alcohol.

  In certain embodiments, measurements such as in-line, online, at-line, or real-time measurements can 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 change 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. Several processes and flows in FIG. 9 have been identified using the same names and numbers as used in FIGS. 1-8, and the same or similar processes as described in FIGS. And represents the flow.

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

  Aqueous solution 22 and microorganism 32 may be added to fermentation 30 where fermentable sugars are metabolized by microorganism 32 to produce stream 105 containing product alcohol. In certain embodiments, enzymes can be added to fermentation 30. Stream 105 may be sent to extractor 120 and may be contacted with extractant 124. Stream 127 containing the fermentation broth in which the product alcohol is more dilute may be returned to fermentation 30 and stream 122 containing the extractant in which the product alcohol is richer may be sent to separation 130. In certain embodiments, the extractor 120 can be operated such that the stream 122 contains minimal cell mass and minimal substrate. Separation 130 can damage microorganisms 32 or the substrate, which can reduce the fermentation rate. By operating the extractor 120 with minimal cell mass and substrate, the potential for damage due to the separation 130 can be minimized. A stream 125 containing a leaner extractant can be returned to the 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 can be added to fermentation 30. In certain embodiments, a portion of the fermentation broth that includes the extractant may be transferred to the extractor 120, and in certain embodiments, the extractant may be recovered from the fermentation broth that includes the extractant. In certain embodiments, the fermentation broth and extractant flow rates to the extractor can be adjusted to improve phase separation. For example, lowering the overall flow rate entering the extractor early or late in the fermentation can improve the phase separation of the fermentation broth and extractant.

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

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

  In certain embodiments where coarse solids are removed from the liquefied mash, the total distillate waste produced may contain fine solids and insoluble microbial fragments, and these dispersed solids are turbocharged. Can be removed using formula filtration. Turbofiltration can include subjecting the raw material suspension to a centrifugal motion through a filter that can 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, can be used to recover the extractant from the total distillation waste liquid wet cake. In certain embodiments, the organic phase can be formed in a decanter. In certain embodiments, the wet cake washed with product alcohol may then be washed with water to recover the 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 can be circulated 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 circulated to the extractor and / or fermenter. In certain embodiments, the extractant stream can be circulated between the extractant reservoir, the extractor, and the fermentation apparatus. In certain embodiments, upon completion of the fermentation, the extractant reservoir, extractor, and / or fermenter contents may be further processed to recover product alcohol.

  Separation or extraction of the product alcohol from the extractant includes methods known in the art, including but not limited to siphoning, decanting, centrifuging, gravity settling, membrane phase separation, etc. Can be used. In certain embodiments, the extraction can be performed using, for example, a mixer settler. A mixer settler is a stage-wise extractor, pump, stirrer, static mixer, mixing tee, impingement device, circulating screen, or It can be used with various elements for mixing, such as a laying bucket. An example of a mixer settler is shown in FIGS. For example, FIG. 10A shows a mixer settler that uses a pump as the source of mixing. FIG. 10B shows a mixer settler that uses a mixer as the source of mixing. FIG. 10C shows a mixer settler that uses a static mixer as the source of mixing. FIG. 10D shows a mixer settler that uses a mixing tee as a source of mixing. FIG. 10E shows a mixer settler that uses a collision mixer as the source of mixing. FIG. 10F shows a mixer settler that uses a laying bucket or mesh screen as the source of mixing. FIG. 10G shows a mixer settler that uses a centrifuge as the settler. FIG. 10H shows a mixer settler that uses 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, a pitched blade turbine, or a curved propeller. The droplet size produced by the agitator mixer can be controlled by agitator design, tank design, agitator speed, and operating mode. In the case of a static mixer, the droplet size can be controlled by the mixer diameter and flow rate. For example, droplet size can be controlled by changing the flow rate through the mixer during fermentation. In certain embodiments, gases and mixers can be used in the mixing process.

  In certain embodiments, one or more mixer settlers can 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 precipitation areas. In certain embodiments, the settler may include a hydrophilic or hydrophobic surface to facilitate phase separation.

  In another embodiment, a column extractor 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 categorized as non-mechanical, pulse agitation, and rotary agitation. Centrifugal extractors are another type of differential extractor equipped with one such type, Podbielniak (R) 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 interior of the column. The number of nozzles and nozzle diameter can be used to determine the droplet size. In certain embodiments, the spray tower can have an interior. In certain embodiments, the spray tower may include helical tubing. Spiral piping may allow for drop rise and further mixing of the 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 agitation extractor may be used in the methods and systems described herein. Pulse agitation extractors also have a variety of designs, including a reciprocating tray or diaphragm where the tray moves vertically. The overall packed tower and / or sieve tray column can also vibrate vertically to facilitate smaller dispersed phase droplets and more mass transfer. Examples of these extractors are shown in FIG. 10L. In certain embodiments, a rotating agitation 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 stirrer 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, stirred columns can be used in the methods and systems described herein. For example, a stirred column with an interior can provide a high mass transfer rate.

  One aspect of the liquid-liquid extraction process determines good operating conditions for the extractor during constantly changing fermentations. For example, a batch fermentation of a typical corn to product alcohol may involve a first inoculation of microorganisms (or cell clumps) that are added to a specific volume of fermentation broth in the fermentor, followed by a predetermined volume. A further filling of the fermenter is used. Fermentation is continued until a predetermined amount of fermentable carbon source (eg, sugar) is consumed. During batch fermentation, the concentration of cell masses, reaction intermediates, reaction by-products, and substrate components change over time, as well as the physical properties of the fermentation broth including viscosity, density, and surface tension. To improve the performance parameters of fermentation, for example, production speed, titer, and yield parameters and factory economics such as sales, return on investment, and profit, the extractor supplements the changing fermentation broth Can be driven in various ways. Furthermore, the characteristics of dynamic fermentation can affect the size limit of the extractor. Appropriate integration of extractor and fermenter operations can benefit from the use of a mechanical model of the process (see, eg, Daugulis and Kollerup, Biotechnology and Bioengineering 27: 1345-1356, 1985). It is also beneficial to augment the mechanical model, for example to set key model parameters with experimental data. Differential extractor design parameters to account for improved speed, titer, and fermentation process yield include the maximum total flow to the extractor per cross section of the extractor column and the given fermentation broth pair. 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 clear phase separation exiting the extractor. In a staged extractor, the mixing intensity required for efficient mass transfer, the corresponding time required for precipitation, and / or the energy required to separate the phases should be considered. It is a further element. In any type of extractor, staged or differential extractor, the ratio of fermentation broth to extractant fed 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 allowed flow that avoids flooding to the extractor is reduced during fermentation due to changes in the physical properties and concentration of the fermentation broth. When changing from low to high (for example, maximum 1/3 to 2/3 for a given extractor design), the flow to the extractor is maximized while the fermentation is completed in a reasonable amount of time. Can be changed without exceeding. In certain embodiments, when the extractor is agitated, the rate of agitation can be changed during fermentation to compensate for changes in the fermentation broth. The droplet size may be measured in the extractor and the rate to maintain a fixed droplet size can be controlled throughout the fermentation to make up for changes in the fermentation broth. The amount of mass transfer that occurs at any given time measures the concentration of product alcohol in the inlet and outlet streams and adjusts the conditions (eg flow rate, flow ratio, agitation) to control mass transfer during fermentation Can be evaluated.

  In certain embodiments, multiple extractors of various sizes may be used such that the conditions (eg, flow rate, flow ratio, 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 extraction efficiency, increases the concentration of product alcohol in the extractant (corresponding to increased efficiency), and reduces the required flow rate through the extractor. Can be adjusted.

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

  In certain embodiments, product alcohol may be transferred from the fermentation broth to the second water stream or extractant across a selective barrier to product alcohol transport. 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 nature, network size, and the like. In certain embodiments, the hydrogel may comprise a polymer network or polymer blend. Examples of polymer formulations include, but are not limited to, the following: acrylic acid, sodium acrylate, hydroxyethyl acrylate, methacrylate, hydroxybutyl acrylate, butyl acrylate, vinylated polyethylene oxide, vinylated propylene oxide, vinylated One or more of polytetramethylene oxide, polyglycol acrylates and diacrylates, polyvinyl alcohol and hydrocarbon derivatized polyvinyl alcohol, and styrene and styrene derivatives. In certain embodiments, the hydrogel may comprise hydroxyethyl acrylate and methacrylate, hydroxybutyl acrylate and methacrylate, or butyl acrylate and methacrylate.

In other embodiments of the methods and systems described herein, the fermentation broth is removed from the bottom of the fermenter at a pressure above atmospheric pressure and passed through a first flash tank operating at atmospheric pressure, A dissolved gas such as CO ₂ can be released. This first flash tank may be a degassing cyclone, and the steam from this first flash tank may be combined with the steam from the fermenter and sent to the 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 vapor from this second flash tank is recompressed to atmospheric pressure, cooled, partially condensed and then from the fermenter. It can be combined with steam and sent to a scrubber. The fermentation broth exiting this second flash tank is injected into an extraction tower operating at a pressure above atmospheric pressure so that any remaining or newly formed dissolved gas does not form a gas phase in the extraction tower. 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 fermenter. In certain 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 combinations thereof. In certain embodiments, the extractant is not limited to beef tallow, corn oil, canola oil, capric / caprylic triglycerides, castor oil, coconut oil, cottonseed oil, fish oil, jojoba oil, lard, flaxseed oil, cow It can be an oil, such as a leg oil, deer oil, palm oil, peanut oil, rapeseed oil, rice oil, safflower oil, soybean oil, sunflower oil, kiri 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 can be used for these methods and systems. The fermentation broth may be removed from the fermenter and may be cooled using a cooler (eg, an existing cooler in a fermentation production facility) before entering the automatic self-cleaning filter. Some particulate can be retained on the filter screen media as the purified mash passes through the filter. An additional filter may perform backflush at the same time, where a portion of the purified mash can be backflowed through a screen containing particulates and a concentrated solids stream can be discharged. In certain embodiments, a portion of the purified mash can enter the top of the extractor while the extractant is fed to the bottom of the extractor. Purified mash and extractant can be contacted either passively by density differences or using mechanical motion (eg, a Karr® column) by means commonly used in the art. In certain embodiments, an aqueous liquid stream of extractant containing product alcohol exits the top of the extractor and is at least partially reduced in product alcohol compared to purified mash. A stream exits from the bottom of the extractor. The aqueous liquid stream and the concentrated solid stream can be combined and returned to the fermenter. The product alcohol-rich extractant stream may be heated in a heat exchanger that 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 can include a path through the heat transfer device and mass transfer device to allow removal of heat and product alcohol as it passes through the external cooling loop. Further, in certain embodiments, the rate of heat and product alcohol removal can adjust the circulation flow rate through the external cooling loop, adjust the flow rate of the cooling fluid in the heat exchanger, and / or the flow rate of the extractant. By adjusting, the heat during fermentation and the rate of product alcohol production can be balanced.

  In certain embodiments of the methods and systems described herein, phase separation of the extractant from the fermentation broth can be facilitated by adjusting the temperature and / or pH of the process. For example, the process can be operated at a temperature and / or pH different from the temperature and / or pH of the fermenter. In certain embodiments, the process can be operated at a reduced pH compared to the fermenter. In certain embodiments, the process may be operated at a higher temperature compared to the fermenter. In certain embodiments, the process may be operated at a reduced pH and higher temperature compared to the fermenter. Higher temperatures can improve the mass transfer kinetics of product alcohol between the aqueous and organic phases, with extractant droplets dispersed in the aqueous phase and water droplets dispersed in the organic phase. It is possible to improve the dynamic characteristics of the 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 steam or steam injection or indirectly through a heat exchanger. In certain embodiments, the extractant fed to the extractor can result from distillation whose temperature can already be increased. In certain embodiments, the extractant can 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 associated pKa value. In certain embodiments, the pH of the fermentation broth can be lowered below the pKa of the extractant so that the carboxylic acid group of the fatty acid is substantially protonated. In some embodiments, pH may be lowered by injecting a small amount of liquid acid such or sulfuric acid or any other organic or inorganic acids are introduced into the fermentation broth of CO ₂ gas in the fermentation broth. 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 can be distributed 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, a drop of fermentation broth may be passed through an extractant and the product alcohol is transferred to the extractant. Fermentation broth droplets can aggregate at the bottom of the extractor and be returned to the fermenter. The extractant containing the product alcohol can be further processed for recovery of the product alcohol described herein. In addition, product alcohol remaining in the fermenter upon completion of the fermentation can be further processed for product alcohol recovery. In certain embodiments, the extractant phase can 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 improved using electrostatic spraying to disperse the aqueous phase in the extractant phase. In certain 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 certain embodiments, the one or more spray nozzles can be a cathode.

  In certain embodiments, the extractor effluent can be used to facilitate phase separation. For example, a portion of the rich extractant from the top of the extractor (ie, the extractant rich in product alcohol) may be returned to the top of the extractor as reflux, with the remaining rich extractant being It can be further processed for product alcohol recovery. 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 fermenter. In another embodiment, the concentrated extractant can exit the top of the extractor and enter the decanter and 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 flow rates can be used for product alcohol recovery. For example, multiple fermenters and extractors can be used where the fermentation cycle of each fermenter is at a different point. Referring to FIG. 11A as an example, the fermenter 300 is at an earlier point in time compared to the fermenter 400, and the fermenter 400 is at an earlier point compared to the fermenter 500. The fermentation broth containing the product alcohol 302 from the fermenter 300 may be contacted with the extractant 307 in the extractor 305 and the product alcohol is transferred to the extractant to produce the product alcohol enriched extractant 309. Can be generated. The product alcohol enriched extractant 309 from the extractor 305 can be sent to the extractor 405. Fermentation broth containing product alcohol 402 from fermenter 400 may be sent to extractor 405 to produce product alcohol enriched extractant 409. The product alcohol enriched extractant 409 can be sent to the extractor 505. Fermentation broth containing product alcohol 502 from fermenter 500 may be sent to extractor 505. Product alcohol enriched extractant 509 from extractor 505 can be processed for recovery of product alcohol. The fermentation broth (304, 404, 504) that is lean in product alcohol can be returned to the fermentation apparatus 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 reduction in extractor size.

  In another embodiment of this example, there may be an additional fermentor F 'and an additional extractor E' (FIG. 11B). In this embodiment, when the fermenter 500 (at a later point in time compared to fermenters 300 and 400) completes the fermentation, the fermenter 500 may be deactivated (taken off-line), In certain embodiments, the fermentation apparatus 500 may perform hygiene and / or sterilization procedures, such as clean-in-place (CIP) and sterilization-in-place (SIP) procedures. When the fermenter 500 is deactivated, the fermentor F 'can be activated (bront on-line). In this embodiment, the fermenter F ′ is at an early point compared to the fermenter 300, and the fermenter 300 is at an earlier point compared to the fermenter 400. Similar to the description for FIG. 11A, the fermentation broth containing the product alcohol F′-02 from the fermenter F ′ may be contacted with the extractant in the extractor E ′, where the product alcohol is the extractant. To produce the product alcohol-enriched extractant E′-09. Product alcohol enriched extractant E'-09 from extractor E 'can be sent to extractor 305. Fermentation broth containing product alcohol 302 from fermenter 300 may be sent to extractor 305 to produce product alcohol enriched extract 309. The product alcohol enriched extractant 309 can be sent to the extractor 405. Fermentation broth containing product alcohol 402 from fermenter 400 may be sent to extractor 405. Product alcohol enriched extractant 409 from extractor 405 may be processed for product alcohol recovery. The fermentation broth (F'-04, 304, 404) that is lean in product alcohol can be returned to the fermenters F ', 300, and 400, respectively. In certain embodiments, the process comprises multiple cycles, e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, or more Can be repeated over a cycle. In certain embodiments, the process of deactivating a fermenter and operating a further fermenter may be manual or automated. The advantage of this process is reduced extractor flow to product recovery (eg, distillation).

  In certain embodiments, the extractant may reduce the flash point (ie, flammability) of the product alcohol. The flash point refers to the lowest temperature at which flame propagation occurs across the surface of the 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"). By reducing the flash point of the product alcohol, the safety conditions of the alcohol production plant can be improved, for example, by minimizing the risk of fire of potentially flammable product alcohol. Improving safety conditions minimizes the risk of injury as well as the risk of property damage and loss of revenue. In certain embodiments where microbial inactivation is required, the extractant may improve microbial inactivation.

  In certain embodiments, the processes described herein are integrated using, for example, 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 to adjust and control fermentation conditions and / or extractor conditions, for example, in a feedback loop. 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 measurement. obtain. In certain embodiments, the measurement device is: Fourier transform infrared spectrometer (FTIR), near infrared spectrometer (NIR), Raman spectrometer, high pressure liquid chromatography (HPLC), viscometer, density meter, tensiometer. , A droplet size analyzer, a pH meter, a dissolved oxygen (DO) probe, and the like. In certain embodiments, off-gas released from the fermenter can be analyzed, for example, by an in-line mass spectrometer. Measurement of off-gas released from the fermenter can be used as a means to identify chemical species present during the fermentation reaction. The concentration of 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 control system where the conditions in the fermentation device (temperature, pH, nutrients, enzyme and / or substrate concentration) are the concentration, concentration profile. And / or can be modified to maintain conditions in the extractor (fermentation broth flow rate, fermentation broth to extractant flow rate, agitation speed, droplet size, temperature, pH, DO content). Similarly, the conditions in the extractor can be changed to maintain the concentration and / or concentration profile in the fermenter. With such a control system, process parameters can be maintained to improve overall factory productivity and economic goals. In certain embodiments, real-time control of fermentation can be performed by varying concentrations of components (eg, sugars, enzymes, nutrients, etc.) in the fermentation apparatus, varying conditions within 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 concentration of starch, sugars and isobutanol in the fermentation broth changes. In order to maximize the efficiency of the extractor, it may be advantageous to alter 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 the process control method, real-time measurement of isobutanol in the fermentation broth (eg, column feed) can be combined with real-time measurement of isobutanol in the extractant and dilute fermentation broth. These measurements can be used to adjust the ratio (flow rate) of fermentation broth to extractant to the extractor. With the flexibility to match the rate of isobutanol extraction with the rate of isobutanol production, the extractor can be operated efficiently throughout the extraction. Furthermore, by maintaining a high concentration of isobutanol in the extractant, the volumetric 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 the composition of the fermentation broth. In certain embodiments, phase separation can be monitored by conductivity measurements, permittivity 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 can, for example, adjust the flow rate of fermentation broth and extractant to and from the extractor and / or adjust the droplet size of the extractant after mixing the fermentation broth and extractant. Can be used. 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, the droplet size is measured by a process particle analyzer (JM Canty, Inc., Buffalo, NY), a focused beam reflectometry (FBRM®), or a particle microscope imaging method. It can be measured using particle size analysis such as (PVM®) technology (Mettler-Toledo, LLC, Columbia OH). In certain embodiments, these measurements can be real-time in situ particle characterization. By monitoring droplet size in real time, changes in droplet shape and size can be detected, and process steps can be adjusted to adjust droplet size and promote mass transfer rates. For example, the droplet size can be used to monitor the amount of extractant in the 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 fermentor, droplet size monitoring is performed in the fermentation broth back to the fermentor. Will provide a means to minimize the amount of extractant. If the amount of extractant in the fermentation broth is too high, phase separation, for example, adjusts the drop size of the extractant in the extractor and / or adjusts the flow rate of the fermentation broth and extractant to the extractor Can be improved. These adjustments during the process steps not only minimize the amount of extractant in the distillate waste and DDGS, but can also minimize the amount of extractant in the fermentation broth.

  In one embodiment of this control method, the isobutanol in the fermentation broth will not exceed a concentration or set point where the concentration of isobutanol is harmful to the microorganism. The isobutanol fermentation broth set point can be adjusted higher or lower as the fermentation proceeds based on the path of fermentation. For example, a continuous comparison of the concentration of isobutanol in the fermentation broth and the set point concentration of isobutanol can be used to adjust the ratio of fermentation broth to extractant or the flow of fermentation broth and extractant to the extractor. obtain. In order to adjust the isobutanol concentration in the fermentation broth, in-situ measurement of the fermentation broth is performed using Fourier transform infrared spectroscopy (FTIR), near infrared spectroscopy (NIR), and / or Raman spectroscopy. Can be broken. In addition, fermentation headspace measurements can be made using FTIR, Raman spectroscopy, and / or mass spectrometry.

  In certain embodiments, efficient extractor operation can be performed just before the flooding of the extractor. The use of real-time process control with concentration data from the inlet and outlet streams allows the extractor to be easily operated just before flooding. In certain embodiments, real-time extractant monitoring can be used to detect partitioning of by-products or contaminants from the fermentation broth into the extractant. By-products such as alcohol, 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). Can be applied to measurement. The analytical technique selected 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. Real-time data can be used to initiate a remediation of the contaminated extractant or removal of the contaminated extractant from the process. These techniques as well as gas chromatography (GC) and supercritical fluid chromatography (SFC) can also be used to monitor the pyrolysis of the extractant.

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

  As an example, on-line measurements of the 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 the water stream 22, and process imaging may be used to monitor the concentration in the water stream 22 and the size of the oil droplets. In certain embodiments, online measurements of fermentation 30 may be used to monitor product alcohol removal rate. Measurements of fermentable carbon source, dissolved oxygen, product alcohol, and by-products are used to adjust the rate of product alcohol removal to maintain the concentration of product alcohol in 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 measurement of stream 105 and stream 122 can be used to operate the 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; the concentration of product alcohol in stream 122 is this stream to separation 130. And can be used to set the ratio of fermentation broth to extractant. Further, on-line 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 separation 130 can be used to monitor the quality of the phase separation of the extractant and fermentation broth. For example, an on-line measurement device may be used to detect the balance of separation of extractant and fermentation broth, and therefore the feed rate of extractant and fermentation broth can be adjusted to improve phase separation. Online devices such as optical devices may be used to detect, for example, in extractor 120 in the presence of a lag layer (eg, a mixture of oil, aqueous solution, and solid) The agent ratio can be adjusted to minimize the formation of the lug layer. Online measurements 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 insufficient 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 can be performed to improve phase separation. Furthermore, 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. Further, stream 127 can be monitored for the presence of extractant as a means of minimizing the amount of extractant returning to fermentation 30. For example, spectroscopic and process imaging techniques can be used to monitor the presence of extractant in stream 127. Furthermore, a specific concentration of extractant in stream 127 can 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 measurements can be used to monitor pollutant and degradation product streams 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 can 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 of the aqueous and organic phases, and the lag layer composed of solid and extractant (eg, extractant droplets) accumulates and possibly phase. May prevent separation. To mitigate the formation of the lag layer, stirring of the aqueous and organic phases can be used. For example, vanes can be used to disperse the lag layer at the aqueous-organic phase interface. Also, fluid flow or vibrations / oscillations such as recirculation loops can be used to disrupt the formation of the lugs. 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 an agitation unit downstream of the settler or decanter for the treatment of the lag layer, and FIG. 13B upstream of the settler or decanter for the treatment of the lag layer. Illustrates the use of a static mixer in combination with a stirring unit. In certain embodiments, other devices such as coalescers or ultrasonic agitation can be used to disperse the lag layer. In certain embodiments, these devices can be integrated into a settler or decanter.

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

Fed-batch fermentation is a form of batch fermentation where a substrate (eg, fermentable sugar) is added gradually during the fermentation process. Where 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 substrate and / or nutrient addition 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, for example, maintains the level or amount of fermentation broth at the start of the fermentation process. Can be added to the fermenter. In certain embodiments of fed-batch fermentation, an extractant can be added to the fermenter.

  Continuous fermentation is an open system in which fermentation broth is continuously added to the fermenter and an amount 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 can be added and removed from the fermenter. In certain embodiments of continuous fermentation, an extractant can be added to the fermenter. In certain embodiments, the volume of the extractant can be about 3% to about 50% of the fermenter effective volume. In certain embodiments, the volume of the extractant can be about 3% to about 20% of the fermenter effective volume. In certain embodiments, the volume of the extractant can be about 3% to about 10% of the fermenter effective volume.

  In certain embodiments of the methods and systems described herein, gas stripping can be used to remove product alcohol from the fermentation broth. Gas stripping can 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 sprinkling one or more gases through the fermentation broth. In certain embodiments, the gas can be provided by a fermentation reaction. As an example, carbon dioxide can be provided as a byproduct of the metabolism of fermentable carbon sources by microorganisms. In certain embodiments, gas stripping can be used simultaneously with the extractant to remove product alcohol from the fermentation broth. The product alcohol is obtained using methods known in the art, such as using a cooled water trap to condense the product alcohol, or washing the gas phase with a solvent. The product alcohol-containing gas phase can be recovered.

Recombinant microorganisms and biosynthetic pathways While not wishing to be bound by theory, the methods described herein are intended to produce alcohol-producing microorganisms, particularly recombinant microorganisms that produce alcohols with potency above their resistance level. Are considered useful in connection with any microorganism capable of producing a fermentation product comprising

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

  The term “acetyl-CoA acetyltransferase” refers to a polypeptide having an enzymatic activity that catalyzes the conversion of two molecules, acetyl-CoA to acetoacetyl-CoA and coenzyme A (CoA). An example of acetyl-CoA acetyltransferase is acetyl-CoA acetyltransferase with substrate selectivity (forward reaction) for short chain acyl-CoA and acetyl-CoA. C. 2.3.1.9 [Enzyme Nomenclature 1992, Academic Press, San Diego], but enzymes with a wider substrate range (EC 2.3.1.16) function similarly. Acetyl-CoA acetyltransferase is available from several sources, for example, Escherichia coli (GenBank numbers: NP_416728, NC_000913; NCBI amino acid sequence, NCBI nucleotide sequence), Clostridium acetobutylicum (Clostridium acetobutylicum GenBank numbers: NP_349476.1, NC_003030; NP_149242, NC_001988, Bacillus subtilis (GenBank numbers: NP_390297, NC_000964), and Saccharomyces cerevisiae (Saccharice Enbanku (GenBank) number: NP_015297, it is available from NC_001148).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

  The following non-limiting examples further illustrate the present invention. While the following examples include corn as a raw material, it should be understood that other biomass sources such as sugar cane can be used for the raw material without departing from the invention. In addition, the following examples include ethanol and butanol, while other alcohols or fermentation products can 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. “Gpl” means grams / liter, “gpm” means gallons / minute, “h” or “hr” means time, “HPLC” means high performance liquid chromatography, “Kg” means kilograms, “L” means liters, “min” means minutes, “mL” means milliliters, “ppm” means parts per million, “psig” "" Means pounds per square inch (gauge pressure) and "wt%" means weight percent.

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 the description). For example, the commercially available modeling software Aspen Plus® (Aspen Technology, Inc., Burlington, Mass.) Is used by the American Institute to create Aspen models for integrated butanol fermentation, purification, and water management processes. of Chemical Engineers, Inc. (New York, NY) can be used with a physical property database such as DIPPR available. This process modeling can perform many basic engineering calculations such as mass and energy balance, gas / liquid equilibrium, and reaction rate calculations. To generate an Aspen model, information input includes, for example, experimental data, moisture content and ingredient composition, temperature for cooking and flushing mash, saccharification conditions (eg, enzyme ingredient, starch conversion, temperature, pressure) , Fermentation conditions (eg, microbial raw materials, glucose conversion, temperature, pressure), deaeration conditions, solvent towers, preflash towers, condensers, evaporators, centrifuges, and the like.

  The Aspen model is produced with a strict mass and energy balance where 53400 kg / hr of corn is mashed and fermented to produce isobutanol, and the majority of isobutanol is extracted and distilled during fermentation. It 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 wt% suspended solids is injected at 170.7 tons / hour and 85 ° C. via a heat exchanger and water cooler, 32 Feed to fermentation 600 at 0C. A steam stream 602 having an average continuous molar composition of 95.8% carbon dioxide, 3.4% water, and 0.8% isobutanol is passed from fermentation 600 at 17.2 tons / hour at atmospheric pressure. Released to scrubber. An average beer stream 603 containing 12.6 gpl of isobutanol was continuously discharged from fermentation 600 and preheated via a heat exchanger with mash 601 prior to distillation 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 for partial removal of isobutanol. Circulate through. The emerging aqueous broth 605 containing 7.9 gpl isobutanol is cooled to 30 ° C. by heat exchange with cooling tower water (CTW) before re-entering fermentation 600. Solvent containing diisopropylbenzene enters extractor 610 and exits as stream 606 containing 30.1 gpl isobutanol. The extractor 610 effectively provides five theoretical liquid-liquid equilibration stages for contacting the fermentation broth with the solvent. Stream 606 passes through the heat exchanger at 340 tons / hour and enters the middle of the 12 theoretical stages of distillation column 620. The reboiler is operating at 0.6 atmospheres (atm) and 183 ° C. using 150 psig of steam to produce a solvent stream 607 containing diisopropylbenzene and substantially free of isobutanol. 607 exchanges heat with the solvent stream 606 via a heat exchanger and is further cooled by cooling water CTW before entering the extractor 610 again. The top vapor of distillation column 620 is cooled (CTW) and condensed (630) to 23.1 ton / hour reflux, 0.2 ton / hour residual vapor off-gas 608, and 99.2% isoform. A 13.2 ton / hour distillation product 609 is formed containing butanol, 0.6% water, and 0.2% diisopropylbenzene.

Example 2
Method for 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 downward along the length of the column and are attached to a central axis. The shaft is attached to a drive that can move a perforated plate (1/4 inch diameter perforation) up and down in a reciprocating motion. The frequency of movement was variable during the test, but the stroke length of the vibration (0.75 inches) and the tray spacing (2 inches) were both fixed. The column used had a plate stack height of 3000 mm.

  An aqueous feedstock consisting of fermentation broth was provided at the top of the column, while a corn oil fatty acid (COFA) feedstock as an extractant was provided at the bottom of the column. The two feeds flowed counter-current to each other through the column and were collected as products on the opposite side of the column.

Fermentation broth was obtained using a fermentation protocol for the production of ethanol from liquefied and saccharified corn mash, where some of the solids were removed by centrifugation. In some cases, the extraction test was conducted over several days, with one part of the test performed while maximizing or nearly maximizing CO ₂ gas emissions while another part was performed when gas emissions were effectively stopped. . The COFA used for this work was a distillation grade made by 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. In addition, experiments were performed with or without the inside of a predetermined position. Two types of interiors were tested: stainless steel and polytetrafluoroethylene (PTFE). In order to determine the flow regime in which the column can be operated without flooding, the flow range was examined.

Effect of dynamic feed from fermentation During testing, it was found that in some cases the column performance changed as the fermentation progressed. Early in the fermentation, the fermentation broth containing the raw material is high in sugar and at an intermediate time a significant amount of CO ₂ (which can affect the fluid flow) 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 raw material over time was reflected in changes in the capacity of the extraction tower.

Conditions using a PTFE plate and a continuous COFA phase (no agitation) are when the fermentation is at about peak gas evolution (“intermediate broth”) and near the end of the fermentation (“final broth ( end broth))), the performance difference was shown when using the fermentation broth collected. With the final broth, a liquid throughput rate of 14 gpm / ft ² (Sample 3E) was achieved without column flooding. 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 in the final broth (with the formation of droplets) compared to the intermediate broth.

Continuous phase Maximum column throughput was also affected by the nature of the continuous phase. Around the end of the fermentation conditions, operating within a continuous aqueous phase and stainless steel (S. Steel), a total liquid volume of approximately 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 internal material, and Nom. AQ refers to the nominal aqueous flow rate. ORG refers to the nominal organic flow rate, total flow rate (ccm) refers to the total flow rate of aqueous and organic raw materials, and total flow rate (gpm / ft ² ) refers to the total flow rate per unit cross-sectional area.

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

Example 3
Effect of fermentation conditions on the capacity of the extraction tower The properties of the fermentation broth are not unchanged and change as the fermentation process proceeds. During fermentation, the carbohydrate concentration decreases as the carbohydrate is metabolized by the microorganism. This change in composition of the fermentation broth will change physical parameters such as the viscosity and surface tension of the fermentation broth that affect the extraction process. In addition to the concentration change, a significant amount of CO ₂ is generated at an intermediate point; 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 PTFE interior. Treatment was carried out at several points during the fermentation. Organic extractant (COFA) is the continuous phase in the column and the fermentation broth passes through the column as droplets. Prior to the introduction of the fermentation broth into the column, the fermentation broth was passed through a Tee in the line where the CO ₂ bubbles present in the feed were removed through the vents.

In a static interior (no agitation) when using fermentation broth taken at peak gas evolution ("intermediate broth") compared to broth taken at the end of fermentation ("final broth"), A difference in performance was shown. With an intermediate broth, a liquid throughput rate of 14 gpm / ft ² was obtained. The final broth maximum throughput (before column flooding) was less than 9 gpm / ft ² . There were significant differences in water droplet size and appearance. The water phase droplet size was clearly larger in the final broth compared to the intermediate broth.

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

To demonstrate the effect of isobutanol concentration, using a 1 inch diameter glass Karr® extraction tower (Koch Modular Process Systems, Paramus, NJ) equipped with a stainless steel interior, approximately 3 g / The fermentation broth from the fermentation containing L isobutanol was processed. The fermentation broth was formed in the continuous phase in the extractor, while 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 conduit where any CO ₂ bubbles present in the feed were removed before the feed entered the extraction tower. .

  Samples of the feed and the emerging stream 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 equilibria transfer stage (HETS) was calculated using the Kremser equation. For the two data points of the raw fermentation broth, the HETS values were 10 and 13 feet.

  Next, isobutanol was added to the fermentation broth to a concentration of 20 g / L. An extraction test was performed and the data showed that the HETS was 18 feet. This value is about 50% higher than that obtained with neat broth and is consistent with the data obtained with dilute distillation waste liquor containing about 20 g / L of 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 tabletop Karr® column. 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. Parameters such as pO ₂ and pH were monitored for both fermentations. The measured pO ₂ was lower with the implementation using the Karr® column compared to the control performed without using the Karr® column. The absolute pH values were similar for the Karr® column and the control, but the pH profile was different for the two runs. For one gentle peak of the control, the pH in the Karr® column reached the peak quickly, flattened, and then peaked again.

  Two aliquots of 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 fermentation using a Karr® column was comparable to the amount of isobutanol produced during control fermentation. Fermentation using a Karr® column produced a total of 82.4 grams of isobutanol: about 34 grams were in 3.6 liters of organic phase and 48 grams were in the aqueous phase. . The control (30 vol% organic phase added to the fermentor) produced 90 g / L, 60 grams were in 3 liters of 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 fermentor from time zero versus non-zero start of extraction during the Karr® column operation. At Karr® at 22 hours, isobutanol was extracted from the fermentor faster than it was produced. Glucose profiles were generated for the control and Karr® columns. The profiles were similar, indicating that cell growth and metabolism were comparable. Results are shown in FIGS. 16A and 16B. In parentheses are the time points (4-7 hours and 22-33 hours) when the Karr® column was in operation.

Example 6
ISPR using a mixer settler
An external mixer-settler system was used to continuously remove isobutanol 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 fermenter contents were recycled from the fermentor through a mixer-settler extraction system. An extractant containing distilled COFA that did not contain isobutanol was used in an once-through basis.

  Two static mixers were tested. For most of the test, a Kenics® stainless steel static mixer (1/2 inch in diameter and having 36 mixing elements) was used. A plastic mixer was used during the 12-24 hour run (StaMixCo HT-11- 12.6-24, StaMixCo LLC, Brooklyn, NY). Fermentation broth and COFA were fed to the opposite side of the T-tube from which the mixture flowed through a static mixer. The material exiting the static mixer was fed to the settler. The settler was made from a 5 liter glass tank. A dip tube passed over the top of the settler, near the periphery, and extended downward over almost half of 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 to provide gentle mixing at the aqueous-organic phase interface to assist in the separation of the two liquid phases and thereby minimize solids accumulation at the interface. . Data collected during operation is presented in Table 5, and FIG. 17 shows the isobutanol removal rate achieved during the fermentation. As can be seen from the data, the isobutanol level in the aqueous broth remained relatively constant, indicating that isobutanol was removed from the fermentation broth at approximately the same rate that it was produced. . Referring to Table 5, the elapsed time is the time from the start of fermentation, the AQ flow rate is an aqueous feed stream, the ORG flow rate is an organic feed stream, and iB in the AQ feed is in the aqueous feed stream. Isobutanol, iB in the ORG product is isobutanol in the concentrated 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 generate 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 maximize the amount of starch / sugar extracted from the wet cake. Real-time measurements are used to control the backflow, cooking water, or water addition to the slurry tank, for example, to maintain a starch / sugar concentration set point. A 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.

  Corn mash samples were analyzed using Fourier Transform Infrared Spectroscopy (FTIR) with a diamond attenuated total reflection (ATR) probe that allows measurement of the presence of solids. The FTIR was calibrated by collecting a spectrum of the standard sample and the complete starch / sugar measurement using HPLC was completed. Using the HPLC data, a multivariate partial least squares (PLS) model for FTIR was created. FTIR spectra were collected to produce a 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 for two samples: 80 g / L and 70 g / L. The slurry was then separated using a three-phase centrifuge and the wet cake was reslurried. The starch concentration of this slurry was measured and found to be 28.9 g / L. The result is shown in FIG. These measurements were used to measure the exact amount of water and reslurry the wet cake at each stage. By optimizing the addition of water, the starch concentration was maximized and the hydraulic load on the separation process was minimized. The moisture content of the wet cake was measured using near infrared spectroscopy (NIR).

  Corn oil quality is monitored in real time and the data is used to control the variables of the three-phase centrifuge (eg, feed rate, gravity, inlet flow rate, scroll rate). 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 of water using the diamond ATR probe technique was about 500 ppm. A lower detection limit was achieved by using a flow cell with a longer effective path length.

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

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

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

Example 8
Droplet size analysis 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. Approximately 24 hours after the process stream exited the static mixer, a PVM® probe (Mettler-Toledo, LLC, Columbia OH) was inserted into the process stream. Images were collected every 2 minutes during the fermentation run using a PVM® probe. The image showed the presence of both COFA droplets with a size ranging from 50-80 μm in diameter and CO ₂ bubbles ranging in size from 200-400 μm in diameter. Using droplet size monitoring 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, Ensure that it is kept below the average diameter.

  A PVM® probe was also used to image COFA droplets in a dilute broth stream before returning flow to the fermenter. Detection of COFA droplets in this stream indicates the amount of COFA returning to the fermenter. Flow images were collected every 2 minutes during fermentation using a PVM® probe. Unlike the stream exiting the static mixer, the dilute 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 fermenter.

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

  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 fermentation proceeds, 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 scale extraction is used to estimate the size of the large extractor unit. The effects of flow rate, agitation rate, and internal presence or absence on the phase separation of the extractor unit flow from the pilot scale extraction are determined. The total flow rate and the ratio of fermentation broth flow rate to extractant flow rate are changed at a fixed temperature during fermentation, and conditions are observed where phase separation is interrupted. Record the maximum achievable flow rate to the extractor unit per square foot of extractor unit flow surface area. Use the following formula to determine the flow rate per unit area:

U = flow rate per unit area (gallon / min / square foot)
F = total flow of fermentation broth and extractant to extractor unit (gallons / min)
A = cross-sectional area in the direction of flow (square feet)
In the case of an extraction column, this is shown 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 of fermentation broth and extractant to the extractor unit using the following formula:

F _{large scale} = total flow of fermentation broth and extractant to large extractor (gallons / 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 the interior, 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 (gallons / min)
F _extractant = flow rate of extractant to the extractor unit (gallons / 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 material (g / L)
Xn = concentration of product alcohol in the fermentation broth exiting the extractor unit (g / L)
Ys = product alcohol concentration in the extractant entering the extractor unit (g / L)
n = number of theoretical plates obtained by the height of the extractor unit Equation 3 is valid only if 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 height of the theoretical stage of the extractor unit is shown by dividing the height of the extraction column used for pilot scale extraction by the number of theoretical stages realized in a given experiment. The number of theoretical plates required to achieve large scale separation is given by Equation 4:

(Where 'indicates the condition of the large extractor unit) is estimated using the operating conditions predicted on a large scale.

  The product of the number of theoretical plates and the height of the theoretical plates measured for similar flow conditions estimates the total height of the large extractor unit. The predicted flow rate and concentration in large scale extractor units are estimated using a dynamic fermentation model (eg, Daugulis, et al., Biotech. Bioeng. 27: 1345-1356, 1985).

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

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

Claims (39)

Providing a fermentation broth comprising a microorganism that produces a product alcohol;
Contacting the fermentation broth with at least one extractant;
Recovering the product alcohol from the fermentation broth comprising recovering the product alcohol.   The method of claim 1, wherein the step of contacting the fermentation broth with at least one extractant is performed in the fermentation apparatus, an external unit, or both.   The method of claim 2, wherein the external unit is an extractor.   The extractor is selected from a siphon, decanter, centrifuge, gravity settler, phase separator, mixer settler, column extractor, centrifugal extractor, stirred extractor, hydrocyclone, spray tower, or combinations thereof The method according to claim 3. 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, 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 The method of claim 1, selected from: 2-ethyl-1-hexanol, 2-hexyl-1-decanol, 2-octyl-1-dodecanol, and mixtures thereof.   The method of claim 6, wherein a hydrophilic solute is added to the fermentation broth.   8. The method of claim 7, wherein the hydrophilic solute is selected from the group consisting of polyhydroxylated compounds, polycarboxylic acids, polyol compounds, ionic salts, or mixtures thereof.   The method of 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 of claim 1, wherein the step of contacting the fermentation broth with at least one extractant is performed in two or more fermentation apparatuses.   The method of claim 10, wherein the fermenter comprises 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 coalescers, baffles, perforated plates, wells, lamellar separators, cones, or combinations thereof.   The method of claim 1, wherein real time measurements are used to monitor product alcohol extraction.   14. The method of claim 13, wherein product alcohol extraction is monitored by real-time measurement of phase separation.   15. The method of claim 14, wherein phase separation is monitored by measuring phase separation rate, extractant droplet size, and / or fermentation broth composition.   16. The method of claim 15, wherein phase separation is monitored by conductivity measurement, dielectric constant measurement, viscoelasticity measurement, or ultrasonic measurement.   The method of claim 1, wherein the step of providing a fermentation broth comprising microorganisms is performed in two or more fermentation apparatuses.   The method of claim 1, wherein the product alcohol is selected from ethanol, propanol, butanol, pentanol, hexanol, and fusel alcohol.   The method of claim 1, wherein the microorganism comprises a butanol biosynthetic pathway.   20. The method of claim 19, wherein the butanol biosynthetic pathway is a 1-butanol biosynthetic pathway, a 2-butanol biosynthetic pathway, or an isobutanol biosynthetic pathway.   The method according to claim 19, wherein the microorganism is a recombinant microorganism. Providing a raw slurry comprising a fermentable carbon source, an undissolved solid, oil, and water;
Separating the raw slurry, thereby forming (i) an aqueous solution containing a fermentable carbon source, (ii) a wet cake containing a solid, and (iii) an oil;
Adding the aqueous solution to the fermentation broth.   23. The method of claim 22, wherein the oil is hydrolyzed to form a fatty acid.   24. The method of claim 23, wherein the fermentation broth is contacted with the fatty acid.   24. The method of claim 23, wherein the oil is hydrolyzed enzymatically.   26. The method of claim 25, wherein the enzyme is one or more lipases or phospholipases.   23. The method of claim 22, wherein the raw slurry is produced by raw material hydrolysis.   28. The method of claim 27, wherein the raw material is selected from rye, wheat, corn, sugar cane, barley, cellulosic or lignocellulosic materials, or combinations thereof.   The raw slurry is decanter bowl centrifuge, three-phase centrifuge, disc stack centrifuge, filtration centrifuge, decanter centrifuge, filtration, vacuum filtration, belt filter, pressure filtration, sieving filtration, sieving separation, lattice 24. The method of claim 22, wherein the separation is by a porous grid, flotation method, hydrocyclone, filter press, screw press, gravity settler, vortex separator, or combinations thereof.   24. The method of claim 22, wherein the step of separating the feedstock is a single step process.   24. The method of claim 22, wherein the wet cake is combined with the aqueous solution.   23. The method of claim 22, further comprising contacting the aqueous solution with a catalyst to convert the oil in the aqueous solution to a fatty acid.   33. The method of claim 32, wherein the aqueous solution and fatty acid are added to the fermentation broth.   The method of claim 32, wherein the catalyst is deactivated. One or more fermentation equipments:
One or more fermenters including an inlet for receiving the raw slurry; and an outlet for discharging the fermentation broth containing the product alcohol;
One or more extractors:
A first inlet for receiving the fermentation broth;
A second inlet for receiving the extractant;
One or more extractors including a first outlet for discharging the lean fermentation broth; and a second outlet for discharging the rich extractant. One or more liquefaction units;
One or more separation means;
36. The system of claim 35, optionally further comprising one or more cleaning systems.   The separation means is decanter centrifuge, three-phase centrifuge, disc stack centrifuge, filtration centrifuge, decanter centrifuge, filtration, vacuum filtration, belt filter, pressure filtration, membrane filtration, microfiltration, filtration using a sieve 37. The system of claim 36, selected from: sieving separation, grid, porous grid, flotation method, hydrocyclone, filter press, screw press, gravity settler, vortex separator, and combinations thereof.   36. The system of claim 35, further comprising an online measurement device.   The online measuring device is a particle size analyzer, Fourier transform infrared spectrometer, near infrared spectrometer, Raman spectrometer, high pressure liquid chromatography, viscometer, density meter, tension meter, droplet size analyzer, pH meter, dissolved 40. The system of claim 38, wherein the system is selected from an oxygen probe, or a combination thereof.

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