OF FERMENTATION PRODUCTS

[0001] This application claims the benefit of U.S. Provisional Application No. 61/699,976, filed on September 12, 2012; U.S. Provisional Application No. 61/712,385, filed on October 1 1, 2012; U.S. Patent Application No. 13/828,353, filed on March 14, 2013; and U.S. Patent Application No. 13/836, 115, filed on March 15, 2013; the entire contents of each are herein incorporated by reference.

[0002] The Sequence Listing associated with this application is filed in electronic form via EFS-Web and hereby incorporated by reference into the specification in its entirety.

FIELD OF THE INVENTION

[0003] The present invention relates to the production of fermentation products such as alcohols including ethanol and butanol, and processes employing in situ product removal methods.

BACKGROUND OF THE INVENTION

[0004] A number of chemicals and consumer products may be produced utilizing fermentation as the manufacturing process. For example, alcohols such as ethanol and butanol have a variety of industrial and scientific applications such as fuels, reagents, and solvents. Butanol is an important industrial chemical with a variety of applications including use as a fuel additive, as a feedstock chemical in the plastics industry, and as a food-grade extractant in the food and flavor industry. The production of butanol or butanol isomers from materials such as plant-derived materials could minimize the use of petrochemicals and would represent an advance in the art. Furthermore, production of chemicals and fuels using plant-derived materials or other biomass sources would provide eco-friendly and sustainable alternatives to petrochemical processes.

[0005] Techniques such as genetic engineering and metabolic engineering may be utilized to modify a microorganism to produce a certain product from plant-derived materials or other sources of biomass. However, in the production of butanol, for example, some microorganisms that produce butanol in high yields also have low butanol toxicity thresholds. Removal of butanol from the fermentation as it is being produced is a means to manage these low butanol toxicity thresholds. Thus, there is a continuing need to develop efficient methods and systems for producing butanol in high yields despite the low butanol toxicity thresholds of the butanol-producing microorganisms.

[0006] In situ product removal (ISPR) (also referred to as extractive fermentation) can be used to remove butanol or other fermentation products from the fermentation as it is produced, thereby allowing the microorganism to produce butanol at high yields. One ISPR method for removing fermentative alcohol that has been described in the art is liquid-liquid extraction (see, e.g., U.S. Patent Application Publication No. 2009/0305370). In general, with regard to butanol fermentation, the fermentation broth which includes the microorganism is contacted with an extractant at a time before the butanol concentration reaches, for example, a toxic level. Butanol partitions into the extractant decreasing the concentration of butanol in the fermentation broth containing the microorganism, thereby limiting the exposure of the microorganism to the inhibitory butanol.

[0007] In order to be technically and economically viable, liquid-liquid extraction requires contact between the extractant and the fermentation broth for efficient mass transfer of the alcohol into the extractant; phase separation of the extractant from the fermentation broth (during and/or after fermentation); efficient recovery and recycle of the extractant; and minimal decrease of the partition coefficient of the extractant over long-term operation. Extractant can become contaminated over time with each recycle, for example, by the buildup of lipids present in the biomass used as feedstock for fermentation, and this contamination can lead to a concomitant reduction in the partition coefficient of the extractant.

[0008] In addition, the presence of undissolved solids during extractive fermentation can negatively affect the efficiency of alcohol production. For example, the presence of the undissolved solids may lower the mass transfer coefficient, impede phase separation, result in the accumulation of oil from the undissolved solids in the extractant leading to reduced extraction efficiency over time, slow the disengagement of extractant drops from the fermentation broth, result in a lower fermentation vessel volume efficiency, and increase the loss of extractant because it becomes trapped in the solids and ultimately removed as Dried Distillers' Grains with Solubles (DDGS). [0009] Thus, there is a continuing need for alternative extractive fermentation processes that reduce the toxic effect of the fermentative alcohol such as butanol on the microorganism, and which can also reduce the degradation of the partition coefficient of an extractant. The present invention satisfies the needs described herein and provides methods, processes, and systems for the fermentative production of alcohols such as ethanol and butanol.

SUMMARY OF THE INVENTION

[0010] The present invention is directed to a method for recovering a fermentation product from a fermentation broth comprising providing a fermentation broth comprising a microorganism, wherein the microorganism produces fermentation product in a fermentor; contacting the fermentation broth with at least one extractant; and recovering the fermentation product. In some embodiments, the contacting of the fermentation broth with at least one extractant occurs in the fermentor, an external unit, or both. In some embodiments, the external unit is an extractor. In some embodiments, the extractor is selected from siphon, decanter, centrifuge, gravity settler, phase splitter, mixer-settler, column extractor, centrifugal extractor, agitated extractor, hydrocyclone, spray tower, and combinations thereof. In some embodiments, the extractant is selected from C ₇ to C22 fatty alcohols, C7 to C22 fatty acids, esters of C7 to C22 fatty acids, C7 to C22 fatty aldehydes, C7 to C22 fatty amides, and mixtures thereof. In some embodiments, the extractant is selected from 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, lauric aldehyde, 2-methylundecanal, oleamide, linoleamide, palmitamide, stearylamide, 2-ethyl- l -hexanol, 2-hexyl- 1 -decanol, 2- octyl- l -dodecanol, and mixtures thereof. In some embodiments, a hydrophilic solute is added to the fermentation broth. In some embodiments, the hydrophilic solute is selected from polyhydroxlated compounds, polycarboxylic acids, polyol compounds, ionic salts, and mixtures thereof. In some embodiments, the contacting of the fermentation broth with at least one extractant occurs in two or more external units. In some embodiments, the contacting of the fermentation broth with at least one extractant occurs in two or more fermentors. In some embodiments, the fermentors comprise internals or devices to improve phase separation. In some embodiments, the internals or devices are selected from coalescers, baffles, perforated plates, wells, lamella separators, cones, and combinations thereof. In some embodiments, real-time measurements are used to monitor extraction of the fermentation product. In some embodiments, extraction of the fermentation product is monitored by real-time measurements of phase separation. In some embodiments, phase separation is monitored by measuring rate of phase separation, extractant droplet size, and/or composition of fermentation broth. In some embodiments, phase separation is monitored by conductivity measurements, dielectric measurements, viscoelastic measurements, and/or ultrasonic measurements. In some embodiments, providing a fermentation broth comprising a microorganism occurs in two or more fermentors. In some embodiments, the fermentation product may be a product alcohol. In some embodiments, the product alcohol is selected from ethanol, propanol, butanol, pentanol, hexanol, and fusel alcohols. In some embodiments, the microorganism comprises a butanol biosynthetic pathway. In some embodiments, the butanol biosynthetic pathway is a 1 -butanol biosynthetic pathway, a 2- butanol biosynthetic pathway, an isobutanol biosynthetic pathway, or a 2-butanone pathway. In some embodiments, the microorganism is a recombinant microorganism. In some embodiments, the method further comprises the steps of providing a feedstock slurry comprising fermentable carbon source, undissolved solids, oil, and water; separating the feedstock slurry forming three streams: (i) an aqueous solution comprising fermentable carbon source, (ii) a wet cake comprising solids, and (iii) oil; and adding the aqueous solution to the fermentation broth. In some embodiments, the oil is hydrolyzed to form fatty acids. In some embodiments, the fermentation broth is contacted with the fatty acids. In some embodiments, the oil is hydrolyzed by an enzyme. In some embodiments, the enzyme is one or more lipases or phospholipases. In some embodiments, the feedstock slurry is generated by hydrolysis of feedstock. In some embodiments, feedstock is selected from rye, wheat, corn, cane, barley, cellulosic or lignocellulosic material, and combinations thereof. In some embodiments, the feedstock slurry is separated by decanter bowl centrifugation, three- phase centrifugation, disk stack centrifugation, filtering centrifugation, decanter centrifugation, filtration, membrane filtration, microfiltration, vacuum filtration, beltfilter, pressure filtration, filtration using a screen, screen separation, grating, porous grating, flotation, hydrocyclone, filter press, screwpress, gravity settler, vortex separator, or combination thereof. In some embodiments, separating the feedstock is a single step process. In some embodiments, the wet cake is combined with the aqueous solution. In some embodiments, the method further comprises contacting the aqueous solution with a catalyst converting oil in the aqueous solution to fatty acids. In some embodiments, the aqueous solution and fatty acids are added to the fermentation broth. In some embodiments, the catalyst is deactivated.

[0011] The present invention is also directed to a system comprising one or more fermentors comprising: an inlet for receiving feedstock slurry; and an outlet for discharging fermentation broth comprising fermentation product; and one or more extractors comprising: a first inlet for receiving the fermentation broth; a second inlet for receiving extractant; a first outlet for discharging a lean fermentation broth; and a second outlet for discharging a rich extractant. In some embodiments, the system further comprises one or more liquefaction units; one or more separation means; and optionally one or more wash systems. In some embodiments, the separation means is selected from decanter bowl centrifugation, three- phase centrifugation, disk stack centrifugation, filtering centrifugation, decanter centrifugation, filtration, vacuum filtration, belt filter, pressure filtration, membrane filtration, microfiltration, filtration using a screen, screen separation, grating, porous grating, flotation, hydrocyclone, filter press, screwpress, gravity settler, vortex separator, and combinations thereof. In some embodiments, the system also comprises on-line measurement devices. In some embodiments, the on-line measurement devices are selected from particle size analyzers, Fourier transform infrared spectroscopes, near-infrared spectroscopes, Raman spectroscopes, high pressure liquid chromatography, viscometers, densitometers, tensiometers, droplet size analyzers, pH meters, dissolved oxygen probes, and combinations thereof.

DESCRIPTION OF THE DRAWINGS

[0012] The accompanying drawings, which are incorporated herein and form a part of the specification, illustrate the present invention and, together with the description, further serve to explain the principles of the invention and to enable a person skilled in the pertinent art to make and use the invention.

[0013] Figure 1 schematically illustrates an exemplary process and system of the present invention, in which undissolved solids are removed via separation after liquefaction and before fermentation. [0014] Figure 2 schematically illustrates an exemplary process and system of the present invention, in which ISPR is conducted downstream of fermentation.

[0015] Figure 3 schematically illustrates another exemplary alternative process and system of the present invention, in which an oil stream is discharged.

[0016] Figure 4 schematically illustrates another exemplary alternative process and system of the present invention, in which the wet cake is subjected to wash cycles.

[0017] Figure 5 schematically illustrates another exemplary alternative process and system of the present invention, in which an oil stream is discharged and wet cake is subjected to wash cycles.

[0018] Figures 6A and 6B schematically illustrates another exemplary alternative process and system of the present invention, in which the aqueous solution and wet cake are combined and conducted to fermentation (Figure 6A) and aqueous solution, oil, and wet cake are combined and conducted to fermentation (Figure 6B).

[0019] Figures 7A-7D schematically illustrates exemplary alternative processes and systems of the present invention, in which the aqueous solution is subjected to conversion (e.g., hydrolysis, transesterification) and/or deactivation.

[0020] Figure 8 schematically illustrates an exemplary fermentation process of the present invention including downstream processing.

[0021] Figure 9 schematically illustrates an exemplary fermentation process of the present invention including downstream processing.

[0022] Figures 10A-10M illustrated various systems that may be used in the processes described herein.

[0023] Figures 11 A and 1 IB schematically illustrate multiple pass extractant flow systems.

[0024] Figure 12 schematically illustrates an exemplary fermentation process of the present invention utilizing on-line, in-line, at-line, and/or real-time measurements for monitoring fermentation processes.

[0025] Figures 13A and 13B schematically illustrates exemplary processes of the present invention for mitigating formation of a rag layer.

[0026] Figure 14 schematically illustrates an exemplary process of the present invention including fermentation, extraction, and distillation processes.

[0027] Figure 15 shows the effects of the fermentation broth to extractant ratios (aq/org) on extraction column efficiency. [0028] Figures 16A and 16B show the effects of ISPR using an external extraction column on isobutanol concentrations and glucose profiles.

[0029] Figure 17 show the effects of ISPR using a mixer-settler on isobutanol removal rates.

[0030] Figure 18 shows FTIR spectra of the range of starch concentrations using in-line measurements.

[0031] Figure 19 shows FTIR spectra of the starch concentration of wet cake during processing of corn mash.

[0032] Figure 20 shows FTIR spectra of corn oil during processing of corn mash.

[0033] Figure 21 demonstrates a real-time measurement of isobutanol in COFA.

DESCRIPTION OF THE INVENTION

[0034] 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 the 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 incorporated by reference in their entireties for all purposes.

[0035] In order to further define this invention, the following terms and definitions are herein provided.

[0036] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains," or "containing," or any other variation thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. For example, a composition, a mixture, a process, a method, an article, or an apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, "or" refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present). [0037] Also, the indefinite articles "a" and "an" preceding an element or component of the invention are intended to be nonrestrictive regarding the number of instances, that is, occurrences of the element or component. Therefore, "a" or "an" should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.

[0038] The term "invention" or "present invention" as used herein is a non-limiting term and is not intended to refer to any single embodiment of the invention but encompasses all possible embodiments as described in the application.

[0039] As used herein, the term "about" modifying the quantity of an ingredient or reactant of the invention employed refers to variation in the numerical quantity that can occur, for example, through typical measuring and liquid handling procedures used for making concentrates or solutions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make the compositions or to carry out the methods; and the like. The term "about" also encompasses amounts that differ due to different equilibrium conditions for a composition resulting 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, alternatively within 5% of the reported numerical value.

[0040] "Biomass" as used herein refers to a natural product containing hydrolyzable polysaccharides that provide fermentable sugars and/or starches including any sugar and starch derived from natural resources such as corn, sugar cane, wheat, cellulosic or lignocellulosic material, and materials comprising cellulose, hemicellulose, lignin, starch, oligosaccharides, disaccharides, and/or monosaccharides, and mixtures thereof. Biomass may also comprise additional components such as protein and/or lipids. Biomass may be derived from a single source or biomass may comprise a mixture derived from more than one source. For example, 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, sludge from paper manufacture, yard waste, wood and forestry waste (e.g., forest thinnings). Examples of biomass include, but are not limited to, corn, corn cobs, crop residues such as corn husks, corn stover, grasses, wheat, rye, wheat straw, spelt, triticale, barley, barley straw, oats, hay, rice, rice straw, switchgrass, potato, sweet potato, cassava, Jerusalem artichoke, sugar cane bagasse, sorghum, sugar cane, sugar beet, fodder beet, soy, palm, coconut, rapeseed, safflower, sunflower, millet, eucalyptus, miscanthus, components obtained from milling of grains, trees (e.g., branches, roots, leaves), wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, manure, and mixtures thereof. For example, mash, juice, molasses, or hydrolysate may be formed from biomass by any processing known in the art for processing biomass for purposes of fermentation such as milling and liquefaction. For example, cellulosic and/or lignocellulosic biomass may be processed to obtain a hydrolysate containing fermentable sugars by any method known to one skilled in the art, such as low ammonia pretreatment disclosed in U.S. Patent Application Publication No. 2007/0031918, which is herein incorporated by reference. Enzymatic saccharification of cellulosic and/or lignocellulosic biomass typically makes use of an enzyme consortium (e.g., cellulases, xylanases, glucosidases, glucanases, lyases) for breaking down cellulose and hemicellulose to produce a hydrolysate containing sugars including glucose, xylose, and arabinose. Saccharification enzymes suitable for cellulosic and/or lignocellulosic biomass are reviewed in Lynd, et al. (Microbiol. Mol. Biol. Rev. 66:506-577, 2002).

[0041] "Fermentable carbon source" or "fermentable carbon substrate" as used herein refers to a carbon source capable of being metabolized by microorganisms. Suitable fermentable carbon sources include, but are not limited to, monosaccharides such as glucose or fructose; disaccharides such as lactose or sucrose; oligosaccharides; polysaccharides such as starch or cellulose; one carbon substrates; and mixtures thereof.

[0042] "Fermentable sugar" as used herein refers to one or more sugars capable of being metabolized by the microorganisms disclosed herein for the production of fermentation products.

[0043] "Feedstock" as used herein refers to a feed in a fermentation process, the feed containing a fermentable carbon source with or without undissolved solids and oil, and where applicable, the feed containing the fermentable carbon source before or after the fermentable carbon source has been removed from starch or obtained from the breakdown of complex sugars by further processing such as by liquefaction, saccharification, or other process. Feedstock includes or may be derived from biomass. Suitable feedstocks include, but are not limited to, rye, wheat, corn, corn mash, cane, cane mash, barley, cellulosic material, lignocellulosic material, or mixtures thereof. Where reference is made to "feedstock oil," it will be appreciated that the term encompasses the oil produced from a given feedstock.

[0044] "Fermentation broth" as used herein refers to a mixture of water, fermentable carbon sources (e.g., sugars), dissolved solids, optionally microorganisms producing fermentation products (e.g., product alcohol), optionally fermentation products (e.g., product alcohol), and other constituents. In some embodiments, fermentation broth refers to the material held in the fermentor in which the fermentation product (e.g., product alcohol) is being made by the metabolism of fermentable carbon sources by the microorganisms. From time to time as used herein, the term "fermentation broth" may be used synonymously with "fermentation medium" or "fermented mixture." In some embodiments, fermentation broth comprising product alcohol may be referred to as fermentation beer or beer.

[0045] "Fermentor" or "fermentation vessel" as used herein refers to the unit in which the fermentation reaction is carried out whereby fermentation product (e.g., product alcohol such as ethanol or butanol) is produced from fermentable carbon sources. The term "fermentor" may be used synonymously herein with "fermentation vessel."

[0046] "Liquefaction unit" as used herein refers to the unit in which liquefaction is carried out. Liquefaction is the process in which oligosaccharides are released from feedstock. In some embodiments where the feedstock is corn, oligosaccharides are released from the corn starch content during liquefaction.

[0047] "Saccharification unit" as used herein refers to the unit in which saccharification (i.e., the breakdown of oligosaccharides into monosaccharides) is carried out. Where fermentation and saccharification occur simultaneously, the saccharification unit and the fermentor may be the same unit.

[0048] "Sugar" as used herein refers to oligosaccharides, disaccharides, monosaccharides, and/or mixtures thereof. The term "saccharide" also includes carbohydrates including starches, dextrans, glycogens, cellulose, pentosans, as well as sugars.

[0049] As used herein, "saccharification enzyme" refers to one or more enzymes that are capable of hydrolyzing polysaccharides and/or oligosaccharides, for example, alpha- 1,4- glucosidic bonds of glycogen or starch. Saccharification enzymes may include enzymes capable of hydrolyzing cellulosic or lignocellulosic materials as well.

[0050] "Undissolved solids" as used herein refers to non-fermentable portions of feedstock, for example, germ, fiber, gluten, and any additional components that do not dissolve in aqueous media. For example, the non- fermentable portions of feedstock include the portion of feedstock that remains as solids and can absorb liquid from the fermentation broth.

[0051] "Oil" as used herein refers to lipids obtained from plants (e.g., biomass) or animals. Examples of oils include, but are not limited to, tallow, corn, canola, capric/caprylic triglycerides, castor, coconut, cottonseed, fish, jojoba, lard, linseed, neetsfoot, oiticica, palm, peanut, rapeseed, rice, safflower, soya, sunflower, tung, jatropha, and vegetable oil blends.

[0052] "Product alcohol" as used herein refers to any alcohol that can be produced by a microorganism in a fermentation process that utilizes biomass as a source of fermentable carbon substrate. Product alcohols include, but are not limited to, Ci to Cs alkyl alcohols. In some embodiments, the product alcohols are C2 to Cs alkyl alcohols. In other embodiments, the product alcohols are C2 to C5 alkyl alcohols. It will be appreciated that Ci to Cs alkyl alcohols include, but are not limited to, methanol, ethanol, propanol, butanol, pentanol, and hexanol. Likewise C2 to Cs alkyl alcohols include, but are not limited to, ethanol, propanol, butanol, pentanol, and hexanol. In some embodiments, product alcohol may also include fusel alcohols (or fusel oils) and glycerol. "Alcohol" is also used herein with reference to a product alcohol.

[0053] "Butanol" as used herein refers to the butanol isomers 1 -butanol (1-BuOH), 2- butanol (2-BuOH), tert-butanol (i-BuOH), and/or isobutanol (iBuOH, Z-BuOH, I-BUOH, iB also known as 2-methyl-l -propanol), either individually or as mixtures thereof. From time to time, when referring to esters of butanol, the terms "butyl esters" and "butanol esters" may be used interchangeably.

[0054] "Propanol" as used herein refers to the propanol isomers isopropanol or 1 -propanol.

[0055] "Pentanol" as used herein refers to the pentanol isomers 1 -pentanol, 3 -methyl- 1- butanol, 2-methyl-l -butanol, 2,2-dimethyl-l -propanol, 3-pentanol, 2-pentanol, 3-methyl-2- butanol, or 2-methyl-2 -butanol.

[0056] "In Situ Product Removal (ISPR)" as used herein refers to the selective removal of a specific product from a biological process such as fermentation to control the product concentration in the biological process as the product is produced.

[0057] "Extractant" as used herein refers to a solvent used to extract a fermentation product

(e.g., product alcohol). From time to time as used herein, the term "extractant" may be used synonymously with "solvent." [0058] "Water-immiscible" as used herein refers to a chemical component such as an extractant or solvent, which is incapable of mixing with an aqueous solution such as fermentation broth, in such a manner as to form one liquid phase.

[0059] "Carboxylic acid" as used herein refers to any organic compound with the general chemical formula -COOH in which a carbon atom is bonded to an oxygen atom by a double bond to make a carbonyl group (-C=0) and to a hydroxyl group ( ^~ OH) by a single bond. A carboxylic acid may be in the form of the protonated carboxylic acid, in the form of a salt of a carboxylic acid (e.g., an ammonium, sodium, or potassium salt), or as a mixture of protonated carboxylic acid and salt of a carboxylic acid. The term carboxylic acid may describe a single chemical species (e.g., oleic acid) or a mixture of carboxylic acids as can be produced, for example, by the hydrolysis of biomass-derived fatty acid esters or triglycerides, diglycerides, monoglycerides, and phospholipids.

[0060] "Fatty acid" as used herein refers to a carboxylic acid (e.g., aliphatic monocarboxylic acid) having C ₄ to C28 carbon atoms (most commonly C ₁₂ to C2 ₄ carbon atoms), which is either saturated or unsaturated. Fatty acids may also be branched or unbranched. Fatty acids may be derived from, or contained in esterified form, an animal or vegetable fat, oil, or wax. Fatty acids may occur naturally in the form of glycerides in fats and fatty oils or may be obtained by hydrolysis of fats or by synthesis. The term fatty acid may describe a single chemical species or a mixture of fatty acids. In addition, the term fatty acid also encompasses free fatty acids.

[0061] "Fatty alcohol" as used herein refers to an alcohol having an aliphatic chain of C ₄ to C22 carbon atoms, which is either saturated or unsaturated.

[0062] "Fatty aldehyde" as used herein refers to an aldehyde having an aliphatic chain of C ₄ to C22 carbon atoms, which is either saturated or unsaturated.

[0063] "Fatty amide" as used herein refers to an amide having an aliphatic chain of C ₄ to C22 carbon atoms, which is either saturated or unsaturated.

[0064] "Fatty ester" as used herein refers to an ester having an aliphatic chain of C ₄ to C22 carbon atoms, which is either saturated or unsaturated.

[0065] "Aqueous phase" as used herein refers to the aqueous phase of, for example, a biphasic mixture containing, for example, a liquid phase and a vapor phase, to the aqueous phase of a triphasic mixture containing two liquid phases (e.g., an organic phase and an aqueous phase) and a vapor phase, to the aqueous phase of either a biphasic or triphasic mixture where the aqueous phase contains some amount of suspended solids, or to a quartphasic mixture comprising a vapor phase, an organic phase, an aqueous phase and a solid phase. In some embodiments, a triphasic mixture may comprise a vapor phase, a liquid phase, and a solid phase. In some embodiments, an aqueous phase may be obtained by contacting a fermentation broth with a water-immiscible organic extractant. In an embodiment of a process described herein that includes fermentative extraction, the term "fermentation broth" then may refer to the aqueous phase in biphasic fermentative extraction.

[0066] "Organic phase" as used herein refers to the non-aqueous phase of a mixture (e.g., biphasic mixture, triphasic mixture, quartphasic mixture) obtained by contacting a fermentation broth with a water-immiscible organic extractant. From time to time as used herein, the terms "organic phase" may be used synonymously with "extractant phase."

[0067] "Effective titer" as used herein refers to the total amount of a particular fermentation product (e.g., product alcohol) produced by fermentation per liter of fermentation broth.

[0068] "Portion" as used herein with reference to a process stream refers to any fractional part of the stream which retains the composition of the stream, including the entire stream, as well as any component or components of the stream, including all components of the stream.

[0069] The present invention provides processes and methods for producing fermentation products such as product alcohols using fermentation. Other fermentation products that may be produced using the processes and methods described herein include propanediol, butanediol, acetone, acids such as lactic acid, acetic acid, butyric acid, and propionic acid; gases such as hydrogen methane, and carbon dioxide; amino acids; vitamins such as biotin, vitamin B ₂ (riboflavin), vitamin Bi ₂ (e.g., cobalamin), ascorbic acid (e.g., vitamin C), vitamin E (e.g., a-tocopherol), and vitamin K (e.g., menaquinone); antibiotics such as erythromycin, penicillin, streptomycin, and tetracycline; and other products such as citric acid, invertase, sorbitol, pectinase, and xylitol.

[0070] The present invention provides processes and systems for producing a product alcohol by fermentative processes and recovering a product alcohol produced by a fermentative process. As an example of an embodiment of the processes described herein, fermentation may be initiated by introducing feedstock directly into a fermentor. In some embodiments, one or more fermentors may be used in the processes described herein. Suitable feedstocks include, but are not limited to, rye, wheat, corn, corn mash, cane, cane mash, barley, cellulosic material, lignocellulosic material, or mixtures thereof. These feedstocks may be processed using methods such as dry milling or wet milling. In some embodiments, prior to the introduction to the fermentor, the feedstock may be liquefied to create feedstock slurry which may comprise undissolved solids, a fermentable carbon source (e.g., sugar), and oil. Liquefaction of the feedstock may be accomplished by any known liquefying processes including, but not limited to, acid process, enzyme process (e.g., alpha- amylase), acid-enzyme process, or combinations thereof. In some embodiments, liquefaction may take place in a liquefaction unit.

[0071] If the feedstock slurry is fed directly to the fermentor, the undissolved solids and/or oil may interfere with efficient removal and recovery of a product alcohol. In particular, when liquid-liquid extraction is utilized to extract a product alcohol from the fermentation broth, the presence of the undissolved solids (e.g., particulates) may cause system inefficiencies including, but not limited to, decreasing the mass transfer rate of the product alcohol to the extractant by interfering with the contact between the extractant and the fermentation broth; creating or promoting an emulsion in the fermentor and thereby interfering with phase separation of the extractant and the fermentation broth; reducing the efficiency of recovering and recycling the extractant because at least a portion of the extractant and product alcohol becomes "trapped" in the solids which may be removed as Distillers' Dried Grains with Solubles (DDGS); lowering fermentor volume efficiency because there are solids taking up volume in the fermentor and because there is a slower disengagement of the extractant from the fermentation broth; and shortening the life cycle of the extractant by contamination with oil. These effects may result in higher capital and operating costs. In addition, extractant "trapped" in the DDGS may detract from the DDGS value and qualification for sale as animal feed. Thus, in order to avoid and/or minimize these problems, at least a portion of the undissolved solids may be removed from the feedstock slurry prior to the addition of the feedstock slurry to the fermentor. Extraction activity and efficiency of product alcohol production may be increased when extraction is performed on a fermentation broth where the undissolved solids have been removed.

[0072] Processes and systems to process feedstock generating a feedstock slurry and to separate feedstock slurry generating an aqueous phase comprising fermentable carbon source and a solid phase (e.g., wet cake) are described herein with reference to the Figures. As shown in Figure 1, in some embodiments, the system includes liquefaction 10 configured to liquefy feedstock to create a feedstock slurry. For example, feedstock 12 may be introduced to liquefaction 10 (e.g., via an inlet in the liquefaction unit). Feedstock 12 can be any suitable biomass material known in the industry including, but not limited to, barley, oat, rye, sorghum, wheat, triticale, spelt, millet, cane, corn, or combinations thereof that contains a fermentable carbon source such as sugar and/or starch. Water may also be introduced to liquefaction 10.

[0073] The process of liquefying feedstock 12 involves hydrolysis of starch in feedstock 12 to water-soluble sugars. Any known liquefying processes, as well as liquefaction unit, utilized by the industry can be used including, but not limited to, an acid process, an enzyme process, or an acid-enzyme process. Such processes can be used alone or in combination. In some embodiments, the enzyme process may be utilized and an appropriate enzyme 14, for example, alpha-amylase, is introduced to liquefaction 10. Examples of alpha-amylases that may be used in the systems and processes of the present invention are described in U.S. Patent No. 7,541,026; U.S. Patent Application Publication No. 2009/0209026; U.S. Patent Application Publication No. 2009/0238923; U.S. Patent Application Publication No. 2009/0252828; U.S. Patent Application Publication No. 2009/0314286; U.S. Patent Application Publication No. 2010/02278970; U.S. Patent Application Publication No. 2010/0048446; U.S. Patent Application Publication No. 2010/0021587, the entire contents of each are herein incorporated by reference.

[0074] In some embodiments, the enzymes for liquefaction and/or saccharification may be produced by the microorganism. Examples of microorganisms producing such enzymes are described in U.S. Patent No. 7,498, 159; U.S. Patent Application Publication No. 2012/0003701; U.S. Patent Application Publication No. 2012/0129229; PCT International Publication No. WO 2010/096562; and PCT International Publication No. WO 2011/153516, the entire contents of each are herein incorporated by reference. In some embodiments, enzymes for liquefaction and/or saccharification may be expressed by a microorganism that also produces a product alcohol. In some embodiments, enzymes for liquefaction and/or saccharification may be expressed by a microorganism that also expresses a butanol biosynthetic pathway. In some embodiments, the butanol biosynthetic pathway may be 1- butanol biosynthetic pathway, 2-butanol biosynthetic pathway, isobutanol biosynthetic pathway, or 2-butanone biosynthetic pathway.

[0075] The process of liquefying feedstock 12 creates feedstock slurry 16 (also referred to as mash or thick mash) that includes fermentable carbon source (e.g., sugar) and undissolved solids. In some embodiments, feedstock slurry 16 may include fermentable carbon source (e.g., sugar), oil, and undissolved solids. The undissolved solids may be non-fermentable portions of feedstock 12. In some embodiments, feedstock 12 may be corn, such as dry milled, unfractionated corn kernels, and feedstock slurry 16 is corn mash slurry. Feedstock slurry 16 may be discharged from an outlet of liquefaction 10, and may be conducted to separation 20.

[0076] Separation 20 has an inlet for receiving feedstock slurry 16, and may be configured to remove undissolved solids from feedstock slurry 16. Separation 20 may also be configured to remove oil, and/or oil and undissolved solids. Separation 20 may agitate or spin feedstock slurry 16 to create a liquid phase or aqueous solution 22 and a solid phase or wet cake 24.

[0077] Aqueous solution 22 may include sugar, for example, in the form of oligosaccharides, and water. Aqueous solution 22 may comprise at least about 10% by weight oligosaccharides, at least about 20% by weight of oligosaccharides, or at least about 30% by weight of oligosaccharides. Aqueous solution 22 may be discharged from separation 20 via an outlet. In some embodiments, the outlet may be located near the top of separation 20.

[0078] Wet cake 24 may include undissolved solids. Wet cake 24 may be discharged from separation 20 via an outlet. In some embodiments, the outlet may be located near the bottom of separation 20. Wet cake 24 may also include a portion of sugar and water. Wet cake 24 may be washed with additional water in separation 20 after aqueous solution 22 has been discharged from separation 20. Alternatively, wet cake 24 may be washed with additional water by additional separation devices. Washing wet cake 24 will recover the sugar (e.g., oligosaccharides) present in the wet cake, and the recovered sugar and water may be recycled to liquefaction 10. After washing, wet cake 24 may be further processed to form Dried Distillers' Grains with Solubles (DDGS) through any suitable known process. The formation of DDGS from wet cake 24 formed in separation 20 has several benefits. Since the undissolved solids do not go to the fermentor, DDGS is not subjected to the conditions of the fermentor. For example, DDGS does not contact the microorganisms present in the fermentor or any other substances that may be present in the fermentor (e.g., extractant and/or product alcohol) and therefore, the microorganism and/or other substances are not trapped in the DDGS. These effects provide benefits to subsequent processing and selling of DDGS, for example, as animal feed. [0079] Separation 20 may be any conventional separation device utilized in the industry, including, for example, centrifuges such as a decanter bowl centrifuge, three-phase centrifuge, disk stack centrifuge, filtering centrifuge, or decanter centrifuge. In some embodiments, removal of the undissolved solids from feedstock slurry 16 may be accomplished by filtration, vacuum filtration, belt filter, pressure filtration, membrane filtration, microfiltration, filtration using a screen, screen separation, grates or grating, porous grating, flotation, hydrocyclone, filter press, screwpress, gravity settler, vortex separator, or any method or device that may be used to separate solids from liquids. In some embodiments, separation 20 is a single step process. In one embodiment, undissolved solids may be removed from feedstock slurry 16 to form two product streams, for example, an aqueous solution of oligosaccharides which contains a lower concentration of solids as compared to feedstock slurry 16 and a wet cake which contains a higher concentration of solids as compared to feedstock slurry 16. In addition, a third stream containing oil may be generated, for example, if three-phase centrifugation is utilized for solids removal from feedstock slurry 16. As such, a number of product streams may be generated by using different separation techniques or combinations thereof.

[0080] A three-phase centrifuge may be used for three-phase separation of feedstock slurry such as separation of the feedstock slurry to generate two liquid phases (e.g., aqueous stream and oil stream) and a solid phase (e.g., solids or wet cake) (see, e.g., Flottweg Tricanter®, Flottweg AG, Vilsibiburg, Germany). The two liquid phases may be separated and decanted, for example, from the bowl of the centrifuge via two discharge systems to prevent cross contamination and the solids phase may be removed via a separate discharge system.

[0081] In some embodiments using corn as feedstock, a three-phase centrifuge may be used to remove solids and corn oil simultaneously from liquefied corn mash. The solids may be undissolved solids remaining after starch is hydrolyzed to soluble oligosaccharides during liquefaction. The corn oil may be released from the germ of the corn kernel during grinding and/or liquefaction. In some embodiments, the three-phase centrifuge may have one feed stream and three outlet streams. The feed stream may consist of liquefied corn mash produced during liquefaction. The mash may consist of an aqueous solution of oligosaccharides (e.g., liquefied starch); undissolved solids which consist of insoluble, non- starch components from the corn; and corn oil which consists of glycerides and free fatty acids. The three outlet streams from the three-phase centrifuge may be a wet cake which contains most of the undissolved solids from the mash; a heavy centrate stream which contains most of the liquefied starch from the mash; and a light centrate stream which contains most of the corn oil from the mash. The heavy centrate stream may be fed to fermentation. The wet cake may be washed with process recycle water, such as evaporator condensate and/or backset as described herein, to recover soluble starch from the wet cake. The light centrate stream may be sold as a co-product, converted to another co-product, or used in processing such as converting the corn oil to corn oil fatty acids (COFA). In some embodiments, COFA may be used as an extractant.

[0082] Referring to Figure 1, fermentation 30 (or fermentor 30), configured to ferment aqueous solution 22 to produce a product alcohol, has an inlet for receiving aqueous solution 22. Fermentation 30 may be any suitable fermentor known in the art. Fermentation 30 may include fermentation broth. In some embodiments, simultaneous saccharification and fermentation (SSF) may occur inside fermentation 30. Any known saccharification process utilized by the industry may be used including, but not limited to, an acid process, an enzyme process, or an acid-enzyme process. In some embodiments, enzyme 38 (e.g., such as glucoamylase) may be introduced to an inlet in fermentation 30 in order to hydrolyze oligosaccharides in aqueous solution 22 forming monosaccharides. Examples of glucoamylases that may be used in the systems and processes of the present invention are described in U.S. Patent No. 7,413,887; U.S. Patent No. 7,723,079; U.S. 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, the entire contents of each are herein incorporated by reference. In some embodiments, glucoamylase may be expressed by the microorganism. In some embodiments, glucoamylase may be expressed by a microorganism that also produces a product alcohol. In some embodiments, glucoamylase may be expressed by a microorganism that also expresses a butanol biosynthetic pathway. In some embodiments, the butanol biosynthetic pathway may be 1 -butanol biosynthetic pathway, 2- butanol biosynthetic pathway, isobutanol biosynthetic pathway, or 2-butanone biosynthetic pathway.

[0083] In some embodiments, enzymes such as glucoamylases may be added to liquefaction. The addition of enzymes such as glucoamylases to liquefaction may reduce the viscosity of the feedstock slurry or liquefied mash and may improve separation efficiency. In some embodiments, any enzyme capable of reducing the viscosity of the feedstock slurry may be used (e.g., Viscozyme®, Sigma-Aldrich, St. Louis, MO). Viscosity of the feedstock may be measured by any method known in the art (e.g., viscometers, rheometers).

[0084] Microorganism 32 may be introduced to fermentation 30. In some embodiments, microorganism 32 may be included in the fermentation broth. In some embodiments, microorganism 32 may be propagated in a separate vessel or tank (e.g., propagation tank). In some embodiments, microorganisms from the propagation tank may be used to inoculate one or more fermentors. In some embodiments, one or more propagation tanks may be used in the processes and systems described herein. In some embodiments, the propagation tank may be about 2% to about 5% the size of the fermentor. In some embodiments, the propagation tank may comprise one or more of the following mash, water, enzymes, nutrients, extractant, and microorganisms. In some embodiments, product alcohol may be produced in the propagation tank.

[0085] In some embodiments, microorganism 32 may be bacteria, cyanobacteria, filamentous fungi, or yeast. In some embodiments, microorganism 32 metabolizes the sugar in aqueous solution 22 and produces product alcohol. In some embodiments, microorganism 32 may be a recombinant microorganism. In some embodiments, microorganism 32 may be immobilized, such as by adsorption, covalent bonding, crosslinking, entrapment, and encapsulation. Methods for encapsulating cells are known in the art, for example, as described in U.S. Patent Application Publication No. 2011/0306116, which is incorporated herein by reference.

[0086] In some embodiments, in situ product removal (ISPR) may be utilized to remove product alcohol from fermentation 30 as the product alcohol is produced by microorganism 32. In some embodiments, liquid-liquid extraction may be utilized for ISPR. In some embodiments, fermentation 30 may have an inlet for receiving extractant 34. In some embodiments, extractant 34 may be added to the fermentation broth downstream of fermentation 30. Alternative means of additions of extractant 34 to fermentation 30 or downstream of fermentation 30 are represented by the dotted lines. In some embodiments, ISPR may be conducted in a propagation tank. In some embodiments, ISPR may be conducted in the fermentor and the propagation tank. In some embodiments, ISPR may be performed at the initiation (e.g., time 0) of fermentation and/or propagation. By initiating ISPR at the beginning of fermentation and/or propagation, the concentration of product alcohol in the fermentor and propagation tank may be maintained at low levels, and thereby minimize the effects of product alcohol on the microorganism and allowing the microorganism to achieve increased cell mass. In some embodiments, extractant may be added to the propagation tank. In some embodiments, extractant may be added prior to inoculation of the propagation tank. In some embodiments, extractant may be added after inoculation of the propagation tank. In some embodiments, extractant may be added at various time points after inoculation of the propagation tank. In some embodiments, extractant may be added to the fermentor. In some embodiments, extractant may be added prior to inoculation of the fermentor. In some embodiments, extractant may be added after inoculation of the fermentor. In some embodiments, extractant may be added at various time points after inoculation of the fermentor. In some embodiments, extractant may be added to the fermentor and the propagation tank. Examples of liquid-liquid extraction are described herein. Processes for producing and recovering alcohols from fermentation broth using extractive fermentation are described in U.S. Patent Application Publication No. 2009/0305370; U.S. Patent Application Publication No. 2010/0221802; U.S. Patent Application Publication No. 2011/0097773; U.S. Patent Application Publication No. 201 1/0312044; U.S. Patent Application Publication No. 2011/0312043; and PCT International Publication No. WO 201 1/159998; the entire contents of each are herein incorporated by reference.

[0087] Extractant 34 contacts the fermentation broth forming stream 36 comprising, for example, a biphasic mixture (e.g., extractant-rich phase with product alcohol and aqueous phase depleted of product alcohol). In some embodiments, stream 36 may be a quartphasic mixture comprising, for example, a vapor phase, an organic phase, an aqueous phase, and a solid phase. Product alcohol, or a portion thereof, in the fermentation broth is transferred to extractant 34. In some embodiments, stream 36 may be discharged through an outlet in fermentation 30. Product alcohol may be separated from the extractant in stream 36 using conventional techniques.

[0088] In some embodiments, fermentor internals or devices may be used to improve phase separation between fermentation broth and extractant. For example, the internal or device may serve as a coalescer to promote phase separation between fermentation broth and extractant and/or act as a physical barrier to improve phase separation. These fermentor internals or devices may also prevent solids from settling in the extractant phase (or layer), promote coalescensce of aqueous droplets that may be entrained in the extractant layer, and promote removal of off-gases (e.g., CO ₂ , air), and thereby minimize disturbance of the extractant phase and/or liquid-liquid interface. Examples of internals or devices that may be used in the processes and systems described herein include, but are not limited to, baffles, perforated plates, deep wells, lamella separators, cones, and the like. In some embodiments, the perforated plate may be a flat horizontal perforated plate. In some embodiments, the cone may be an inverted cone or concentric cone(s). In some embodiments, the internals may be rotating. In some embodiments, the internals or devices may be located at or about the level of the liquid-liquid interface of fermentation broth and extractant. In some embodiments, a coalescing pad may be added and/or exit ports may be relocated to improve coalescence and recovery of the aqueous phase.

[0089] In some embodiments prior to ISPR and/or completion of fermentation, stream 35 may be discharged from an outlet in fermentation 30. Discharged stream 35 may include microorganism 32. Microorganism 32 may be separated from stream 35, for example, by centrifugation or membrane filtration. In some embodiments, by removing the microorganism prior to addition of extractant to the fermentation broth, the microorganism is not exposed to the extractant and therefore, not exposed to any negative impact that the extractant may have on the microorganism. In addition, by removing the microorganism upstream of the extraction process, a more aggressive extraction process (e.g., heating or cooling the mixture to enhance separation, using a higher ¾ and/or higher selectivity extractant, or an extractant with improved properties but lower biocompatibility) may be employed to recover the product alcohol. In some embodiments, microorganism 32 may be recycled to fermentation 30 which can increase the production rate of product alcohol, thereby resulting in an increase in the efficiency of product alcohol production.

[0090] Referring to Figure 2, in some embodiments, ISPR may be conducted downstream of fermentation 30. In some embodiments, stream 33 including product alcohol and microorganism 32 may be discharged from an outlet in fermentation 30 and conducted downstream, for example, to an extraction column for recovery of product alcohol. In some embodiments, stream 33 may be processed by separating microorganism 32 prior to ISPR. For example, removal of microorganism 32 from stream 33 may be accomplished by centrifugation, filtration, vacuum filtration, belt filter, pressure filtration, membrane filtration, microfiltration, filtration using a screen, screen separation, grates or grating, porous grating, flotation, hydrocyclone, filter press, screwpress, gravity settler, vortex separator, or any method or separation device that may be used to separate solids (e.g., microorganisms) from liquids. Following removal of microorganism 32, stream 33 may be conducted to an extraction column for recovery of product alcohol.

[0091] Additional embodiments of the processes and systems described herein are illustrated in Figures 3 to 6. Figures 3 to 6, including the options for the addition of extractant to the fermentor (e.g., generating stream 36) or extraction conducted downstream of the fermentor (e.g., generating stream 33), are similar to Figures 1 and 2, respectively, and therefore will not be described in detail again.

[0092] Referring to Figure 3, the systems and processes of the present invention may include discharging oil 26 from an outlet of separation 20. Feedstock slurry 16 may be separated into a first liquid phase or aqueous solution 22 comprising a fermentable sugar, a solid phase or wet cake 24 comprising undissolved solids, and a second liquid phase comprising oil 26 which may exit separation 20. In some embodiments, separation of feedstock slurry 16 into a first liquid phase, a second liquid phase, and a solid phase may occur in a single step. In some embodiments, feedstock 12 is corn and oil 26 is corn oil. In some embodiments, oil 26 may be conducted to a storage tank or any unit that is suitable for oil storage. Any suitable separation device may be used to discharge aqueous solution 22, wet cake 24, and oil 26, for example, a three-phase centrifuge. In some embodiments, a portion of the oil from feedstock 12 such as corn oil when the feedstock is corn, remains in wet cake 24. In some embodiments, when oil 26 is removed via separation 20 from feedstock 12 (e.g., corn), the fermentation broth in fermentation 30 includes a reduced amount of corn oil.

[0093] As described herein, in some embodiments, oil may be separated from the feedstock or feedstock slurry and may be stored in an oil storage unit. For example, oil may be separated from the feedstock or feedstock slurry using any suitable means for separation including a three-phase centrifuge or mechanical extraction. To improve the removal of oil from the feedstock or feedstock slurry, oil extraction aids such surfactants, anti-emulsifiers, or flocculents as well as enzymes may be utilized. Examples of oil extraction aids include, but are not limited to, non-polymeric, liquid surfactants; talcum powder; microtalcum powder; salts (NaOH); calcium carbonate; and enzymes such as Pectinex® Ultra SP-L, Celluclast®, and Viscozyme® L (Sigma-Aldrich, St. Louis, MO), and NZ 33095 (Novozymes, Franklinton, NC).

[0094] As illustrated in Figure 4, if oil is not discharged separately it may be removed with wet cake 24. When wet cake 24 is removed via separation 20, in some embodiments, a portion of the oil from feedstock 12, such as corn oil when the feedstock is corn, remains in wet cake 24. Wet cake 24 may be conducted to mix 60 and combined with water or other solvents forming wet cake mixture 65. In some embodiments, water may be fresh water, backset, cook water, process water, lutter water, evaporation water, or any water source available in the fermentation processing facility, or any combination thereof. Wet cake mixture 65 may be conducted to separation 70 producing wash centrate 75 comprising fermentable sugars recovered from wet cake 24, and wet cake 74. Wash centrate 75 may be recycled to liquefaction 10.

[0095] In some embodiments, separation 70 may be any separation device capable of separating solids and liquids including, for example, decanter bowl centrifugation, three- phase centrifugation, disk stack centrifugation, filtering centrifugation, decanter centrifugation, filtration, vacuum filtration, belt filter, pressure filtration, membrane filtration, filtration using a screen, screen separation, grating, porous grating, flotation, hydrocyclone, filter press, screwpress, gravity settler, vortex separator, or combinations thereof.

[0096] In some embodiments, wet cake may be subjected to one or more wash cycles or wash systems. For example, wet cake 74 may be further processed by conducting wet cake 74 to a second wash system. In some embodiments, wet cake 74 may be conducted to a second mix 60' forming wet cake mixture 65'. Wet cake mixture 65' may be conducted to a second separation 70' producing wash centrate 75' and wet cake 74'. Wash centrate 75' may be recycled to liquefaction 10. In some embodiments, wash centrate 75' may be combined with wash centrate 75, and recycled to liquefaction 10. In some embodiments, wet cake 74' may be combined with wet cake 74 for further processing as described herein. In some embodiments, separation 70' may be any separation device capable of separating solids and liquids including, for example, decanter bowl centrifugation, three-phase centrifugation, disk stack centrifugation, filtering centrifugation, decanter centrifugation, filtration, vacuum filtration, belt filter, pressure filtration, membrane filtration, microfiltration, filtration using a screen, screen separation, grating, porous grating, flotation, hydrocyclone, filter press, screwpress, gravity settler, vortex separator, or combination thereof. In some embodiments, the wet cake may be subjected to one, two, three, four, five, or more wash cycles or wash systems.

[0097] Wet cake 74 may be combined with syrup and then dried to form DDGS through any suitable known process. The formation of the DDGS from wet cake 74 has several benefits. Since the undissolved solids do not go to the fermentor, the DDGS does not have trapped extractant and/or product alcohol, it is not subjected to the conditions of the fermentor, and it does not contact the microorganisms present in the fermentor. These benefits make it easier to process DDGS, for example, as animal feed.

[0098] In some embodiments, a portion of undissolved solids may be conducted to fermentation 30. In some embodiments, this portion of undissolved solids may have smaller particle sizes (e.g., fines). In some embodiments, this portion of undissolved solids may form whole stillage. In some embodiments, this whole stillage may be processed to form thin stillage and a wet cake. In some embodiments, the wet cake formed from whole stillage and wet cake 74 and/or 74' may be combined and further processed to produce DDGS.

[0099] As shown in Figure 4, oil is not discharged separately from the wet cake, but rather oil is included as part of the wet cake and is ultimately present in the DDGS. If corn is utilized as feedstock, corn oil contains triglycerides, diglycerides, monoglycerides, fatty acids, and phospholipids, which provide a source of metabolizable energy for animals. The presence of oil (e.g., corn oil) in the wet cake and ultimately DDGS may provide a desirable animal feed, for example, a high fat content animal feed.

[00100] In some embodiments, oil may be separated from wet cake and DDGS and converted to an ISPR extractant for subsequent use in the same or different alcohol fermentation processes. Methods for deriving extractants from biomass are described in U.S. Patent Application Publication No. 201 1/0312044; U.S. Patent Application Publication No. 201 1/0312043; and U.S. Patent Application Publication No. 2012/0156738; the entire contents of each are herein incorporated by reference. Oil may be separated from wet cake and DDGS using any suitable process including, for example, a solvent extraction process. In one embodiment of the invention, wet cake or DDGS may be added to an extraction unit and washed with a solvent such as hexane to remove oil. Other solvents that may be utilized include, for example, butanol, isohexane, ethanol, petroleum distillates such as petroleum ether, or mixtures thereof. Following oil extraction, wet cake or DDGS may be treated to remove any residual solvent. For example, wet cake or DDGS may be heated to vaporize any residual solvent using any method known in the art. Following solvent removal, wet cake or DDGS may be subjected to a drying process to remove any residual water. The processed wet cake may be used to generate DDGS. The processed DDGS may be used as a feed supplement for animals such as dairy and beef cattle, poultry, swine, livestock, equine, aquaculture, and domestic pets.

[00101] In some embodiments, extractant may be used as a means to modify the color of the wet cake. For example, feedstocks such as corn contain pigments (e.g., xanthophylls) which may be used as a coloring agent in food products including animal feeds (e.g., poultry feed). Exposure to extractants can modify these pigments resulting in a wet cake that is, for example, lighter in color. A lighter color wet cake may produce DDGS with a lighter color, which may be a desirable quality for certain animal feeds.

[00102] In some embodiments, where corn is used as the feedstock, xanthophylls may be isolated from corn and/or undissolved solids and used as a pigment ingredient in DDGS or animal feed, or as a supplement for pharmaceutical and nutraceutical applications. Methods for isolating xanthophylls include, but are not limited to, chromatography such as size exclusion chromatography, solvent extraction such as ethanol extraction, and enzyme treatment such as alcalase hydrolysis (see, e.g., Tsui, et al, J. Food Eng. 83 :590-595, 2007; Li, et al, Food Science 31 : 72-77, 2010: U.S. Patent No. 5,648,564; U.S. Patent No. 6, 169,217; U.S. Patent No. 6,329,557; U.S. Patent No. 8,236,929; the entire contents of each are herein incorporated by reference). In some embodiments, xanthophylls may be isolated from corn and/or undissolved solids and added to COFA. In some embodiments, COFA and/or xanthophylls may be used for food, pharmaceutical, and nutraceutical applications.

[00103] After extraction from wet cake or DDGS, the resulting oil and solvent mixture may be collected for separation of oil and solvent. In one embodiment, the oil/solvent mixture may be processed by evaporation whereby the solvent is evaporated and may be collected and recycled. The recovered oil may be converted to an ISPR extractant for subsequent use in the same or different alcohol fermentation processes.

[00104] Removal of the oil component of the feedstock is advantageous to product alcohol production because oil present in the fermentor can break down into fatty acids and glycerin. Glycerin can accumulate in water and reduce the amount of water that is available for recycling throughout the system. Thus, removal of the oil component of feedstock can increase the efficiency of product alcohol production by increasing the amount of water that can be recycled through the system.

[00105] Referring to Figure 5, oil may be removed at various points during the processes described herein. Feedstock slurry 16 may be separated, for example, using a three-phase centrifuge, into a first liquid phase or aqueous solution 22, a second liquid phase comprising oil 26, and a solid phase or wet cake 24. Wet cake 24 may be further processed to recover fermentable sugars and oil. Wet cake 24 may be conducted to mix 60 and combined with water or other solvents forming wet cake mixture 65. In some embodiments, water may be backset, cook water, process water, lutter water, water collected from evaporation, or any water source available in the fermentation processing facility, or any combination thereof. Wet cake mixture 65 may be conducted to separation 70 (e.g., three-phase centrifuge) producing wash centrate 75 comprising fermentable sugars, oil stream 76, and wet cake 74. Wash centrate 75 may be recycled to liquefaction 10.

[00106] As described herein, wet cake may be subjected to one or more wash cycles or wash systems. In some embodiments, wet cake 74 may be conducted to a second mix 60' forming wet cake mixture 65'. Wet cake mixture 65' may be conducted to a second separation 70' producing wash centrate 75', oil stream 76' and wet cake 74'. Wash centrate 75' may be recycled to liquefaction 10. In some embodiments, wash centrate 75' may be combined with wash centrate 75, and recycled to liquefaction 10. In some embodiments, wet cake 74' may be combined with wet cake 74 for further processing as described below. In some embodiments, oil stream 76' and oil 26 may be combined and further processed for the generation of extractant that may be used in the fermentation process or oil stream 76' and oil 26 may be combined and further processed for the manufacture of consumer products.

[00107] Wet cake 74 may be combined with syrup and then dried to form DDGS utilizing any suitable process. The formation of DDGS from wet cake 74 has several benefits. Since the undissolved solids do not go to the fermentor, the DDGS does not contain extractant and/or product alcohol, it is not subjected to the conditions of the fermentor, and it does not contact the microorganisms present in the fermentor. These benefits make it easier to process DDGS, for example, as animal feed. As described herein, in some embodiments, wet cake 74, 74', and wet cake formed from whole stillage may be combined and further processed to produce DDGS. [00108] As illustrated in Figure 6A, aqueous solution 22 and wet cake 24 may be combined, cooled, and conducted to fermentation 30. Feedstock slurry 16 may be separated, for example, using a three-phase centrifuge, into a first liquid phase or aqueous solution 22, a second liquid phase comprising oil 26, and a solid phase or wet cake 24. In some embodiments, oil 26 may be conducted to a storage tank or any unit that is suitable for oil storage. Aqueous solution 22 and wet cake 24 may be conducted to mix 80 and re-slurried forming aqueous solution/wet cake mixture 82. Mixture 82 may be conducted to cooler 90 producing cooled mixture 92 which may be conducted to fermentation 30. In some embodiments, when oil 26 is removed via separation 20 from feedstock slurry 16, mixtures 82 and 92 include a reduced amount of oil.

[00109] In another embodiment, as illustrated in Figure 6B, feedstock slurry 16 may be separated using a separation device (e.g., a three-phase centrifuge) to generate a first liquid phase or aqueous solution 22, a second liquid phase comprising oil 26, and a solid phase or wet cake 24. Aqueous solution 22, wet cake 24, and oil 26, or portions thereof, may be conducted to fermentation 30. In some embodiments, aqueous solution 22, wet cake 24, and oil 26, or portions thereof, may be combined, for example, by mixing, forming an aqueous solution, wet cake, and oil mixture, and the mixture may be conducted to fermentation 30. In some embodiments, aqueous solution 22 and wet cake 24 may be combined forming an aqueous solution and wet cake mixture, then oil 26 may be added to the mixture forming an aqueous solution, wet cake, and oil mixture and this mixture may be conducted to fermentation 30. In some embodiments, aqueous solution 22 and wet cake 24 may be combined forming an aqueous solution and wet cake mixture, and this mixture and oil 26, or a portion thereof, may be conducted to fermentation 30 as separate streams.

[00110] In additional embodiments of the processes and systems described herein, saccharification may occur in a separate saccharification system. In some embodiments, a saccharification system may be located between liquefaction 10 and separation 20 or between separation 20 and fermentation 30. In some embodiments, liquefaction and/or saccharification may be conducted utilizing raw starch enzymes or low temperature hydrolysis enzymes such as Stargen™ (Genencor International, Palo Alto, CA) and BPX™ (Novozymes, Franklinton, NC). In some embodiments, feedstock slurry may be subjected to raw starch hydrolysis (also known as cold cooking or cold hydrolysis). [00111] In some embodiments, the systems and processes of the present invention may include a series of two or more separation devices (e.g., centrifuges) for the removal of undissolved solids and/or oil. For example, aqueous solution discharged from a first separation unit may be conducted to an inlet of a second separation unit. The first separation unit and second separation unit may be identical (e.g., two three-phase centrifuges) or may be different (e.g., a three-phase centrifuge and a decanter centrifuge). Separation may be accomplished by a number of means including, but not limited to, decanter bowl centrifugation, three-phase centrifugation, disk stack centrifugation, filtering centrifugation, decanter centrifugation, filtration, vacuum filtration, belt filter, pressure filtration, membrane filtration, filtration using a screen, screen separation, grating, porous grating, flotation, hydrocyclone, filter press, screwpress, gravity settler, vortex separator, or combinations thereof.

[00112] The absence or minimization of undissolved solids in the fermentation broth has several benefits. For example, the need for units of operation in the downstream processing may be eliminated, thereby resulting in an increased efficiency for product alcohol production. Also, some or all of the centrifuges used to process whole stillage may be eliminated as a result of less undissolved solids in the fermentation broth exiting the fermentor. Removal of undissolved solids from feedstock slurry may improve the processing productivity of biomass and cost effectiveness. Improved productivity may include increased efficiency of product alcohol production and/or increased extraction activity relative to processes and systems that do not remove undissolved solids prior to fermentation. For additional description of processes and systems for separating undissolved solids from feedstock slurry see, for example, U.S. Patent Application Publication No. 2012/0164302 and PCT International Patent Application No. PCT/US2013/51571 , the entire contents of each are herein incorporated by reference.

[00113] As described herein, product alcohol may be recovered from fermentation broth using a number of methods including liquid-liquid extraction. In some embodiments of the processes and systems described herein, an extractant may be used to recover product alcohol from fermentation broth. Extractants used herein may have, for example, one or more of the following properties and/or characteristics: (i) biocompatible with the microorganisms, (ii) immiscible with the fermentation broth, (iii) a high partition coefficient (Kp) for the extraction of product alcohol, (iv) a low partition coefficient for the extraction of nutrients and/or water, (v) low viscosity (μ), (vi) high selectivity for product alcohol as compared to, for example, water, (vii) low density (p) relative to the fermentation broth or a density that is different as compared to the density of the fermentation broth, (viii) a boiling point suitable for downstream processing of the extractant and product alcohol, (ix) a melting point lower than ambient temperature, (x) minimal absorbency in solids, (xi) a low tendency to form emulsions with the fermentation broth, (xii) stability throughout the fermentation process, (xiii) low cost, and (xiv) nonhazardous.

[00114] In some embodiments, the extractant may be selected based upon certain properties and/or characteristics as described herein. For example, viscosity of the extractant can influence the mass transfer properties of the system, that is, the efficiency with which the product alcohol may be extracted from the aqueous phase to the extractant phase (i.e., organic phase). The density of the extractant can affect phase separation. In some embodiments, selectivity refers to the relative amounts of product alcohol to water taken up by the extractant. The boiling point can affect the cost and method of product alcohol recovery. For example, in the case where butanol is recovered from the extractant phase by distillation, the boiling point of the extractant should be sufficiently low as to enable separation of butanol while minimizing any thermal degradation or side reactions of the extractant, or the need for deep vacuum in the distillation process.

[00115] The extractant may be biocompatible with the microorganism, that is, nontoxic to the microorganism or toxic only to such an extent that the microorganism is impaired to an acceptable level. In some embodiments, biocompatible refers to the measure of the ability of a microorganism to utilize fermentable carbon sources in the presence of an extractant. The extent of biocompatibility of an extractant may be determined, for example, by the glucose utilization rate of the microorganism in the presence of the extractant and product alcohol. In some embodiments, a non-biocompatible extractant refers to an extractant that interferes with the ability of a microorganism to utilize fermentable carbon sources. For example, a non- biocompatible extractant does not permit the microorganism to utilize glucose at a rate greater than about 25%, greater than about 30%, greater than about 35%, greater than about 40%, greater than about 45%, or greater than about 50% of the rate when the extractant is not present.

[00116] One skilled in the art may select an extractant to maximize the desired properties and/or characteristics as described herein and to optimize recovery of a product alcohol. One of skill in the art can also appreciate that it may be advantageous to use a mixture of extractants. For example, extractant mixtures may be used to increase the partition coefficient for the product alcohol. Additionally, extractant mixtures may be used to adjust and optimize physical characteristics of the extractant, such as the density, boiling point, and viscosity. For example, the appropriate combination may provide an extractant which has a sufficient partition coefficient for the product alcohol and sufficient biocompatibility to enable its economical use for removing product alcohol from fermentative broth.

0117] In some embodiments, extractants useful in the processes and systems described herein may be organic solvents. In some embodiments, extractants useful in the processes and systems described herein may be water-immiscible organic solvents. In some embodiments, the extractant may be an organic extractant selected from the group consisting of saturated, mono-unsaturated, poly-unsaturated C ₁₂ to C22 fatty alcohols, C ₁₂ to C22 fatty acids, esters of C ₁₂ to C22 fatty acids, C ₁₂ to C22 fatty aldehydes, C ₁₂ to C22 fatty amides, and mixtures thereof. In some embodiments, the extractant may also be an organic extractant selected from the group consisting of saturated, mono-unsaturated, poly-unsaturated C ₄ to C22 fatty alcohols, C ₄ to C28 fatty acids, esters of C ₄ to C28 fatty acids, C ₄ to C22 fatty aldehydes, C ₄ to C22 fatty amides, and mixtures thereof. In some embodiments, the fatty acid may be a C ₄ to C2 ₄ fatty acid and/or the ester may be an ester of a C ₄ to C2 ₄ fatty acid. In some embodiments, the extractant may be an organic extractant selected from the group consisting of saturated, mono-unsaturated, poly-unsaturated C ₁₂ to C ₁₈ fatty alcohols, C ₁₂ to Ci ₈ fatty acids, esters of C ₁₂ to C ₁₈ fatty acids, C ₁₂ to C ₁₈ fatty aldehydes, C ₁₂ to C ₁₈ fatty amides, and mixtures thereof. In some embodiments, the extractant may be an organic extractant selected from the group consisting of saturated, mono-unsaturated, polyunsaturated C ₁₄ to Ci ₈ fatty alcohols, C ₁₄ to C ₁₈ fatty acids, esters of C ₁₄ to C ₁₈ fatty acids, C ₁₄ to Ci ₈ fatty aldehydes, C ₁₄ to C ₁₈ fatty amides, and mixtures thereof. In some embodiments, the extractant may be an organic extractant selected from the group consisting of saturated, mono-unsaturated, poly-unsaturated Ci ₆ to C ₁₈ fatty alcohols, Ci ₆ to C ₁₈ fatty acids, esters of C _½ to C ₁₈ fatty acids, Ci ₆ to C ₁₈ fatty aldehydes, C _½ to C ₁₈ fatty amides, and mixtures thereof. In some embodiments, the extractant may comprise carboxylic acids. In some embodiments, the ester of a fatty acid may be the combination of a fatty acid with an alcohol (e.g., fatty ester). In some embodiments, the alcohol may be a product alcohol. In some embodiments, the ester may be methyl ester, ethyl ester, propyl ester, butyl ester, pentyl ester, hexyl ester, or glycerides.

[00118] In some embodiments, the extractant may include a first extractant selected from C to C22 fatty alcohols, C ₁₂ to C22 fatty acids, esters of C ₁₂ to C22 fatty acids, C ₁₂ to C22 fatty aldehydes, C ₁₂ to C22 fatty amides, and mixtures thereof; and a second extractant selected from C12 to C22 fatty alcohols, C ₁₂ to C22 fatty acids, esters of C ₁₂ to C22 fatty acids, C ₁₂ to C22 fatty aldehydes, C ₁₂ to C22 fatty amides, and mixtures thereof. In some embodiments, the extractant may include a first extractant selected from C ₁₂ to C22 fatty alcohols, C ₁₂ to C22 fatty acids, esters of C ₁₂ to C22 fatty acids, and mixtures thereof; and a second extractant selected from C ₁₂ to C22 fatty alcohols, C ₁₂ to C22 fatty acids, esters of C ₁₂ to C22 fatty acids, and mixtures thereof. In some embodiments, the extractant may include a first extractant selected from C ₁₂ to C ₁₈ fatty alcohols, C ₁₂ to C ₁₈ fatty acids, esters of C ₁₂ to C ₁₈ fatty acids, and mixtures thereof; and a second extractant selected from C ₁₂ to C ₁₈ fatty alcohols, C ₁₂ to Ci ₈ fatty acids, esters of C ₁₂ to C ₁₈ fatty acids, and mixtures thereof. In some embodiments, the extractant may include a first extractant selected from C ₁₄ to C ₁₈ fatty alcohols, C ₁₄ to C ₁₈ fatty acids, esters of C ₁₄ to C ₁₈ fatty acids, and mixtures thereof; and a second extractant selected from C ₁₄ to C ₁₈ fatty alcohols, C ₁₄ to C ₁₈ fatty acids, esters of C14 to C ₁₈ fatty acids, and mixtures thereof. In some embodiments, the extractant may include a first extractant selected from C ₁₂ to C22 fatty alcohols, C ₁₂ to C22 fatty acids, esters of C ₁₂ to C22 fatty acids, C12 to C22 fatty aldehydes, C ₁₂ to C22 fatty amides, and mixtures thereof; and a second extractant selected from C7 to Cn fatty alcohols, C7 to Cn fatty acids, esters of C7 to Cn fatty acids, C7 to Cn fatty aldehydes, and mixtures thereof.

[00119] In some embodiments, the extractant may be an organic extractant such as oleyl alcohol, behenyl alcohol, cetyl alcohol, lauryl alcohol (also referred to as 1-dodecanol), myristyl alcohol, stearyl alcohol, oleic acid, lauric acid, linoleic acid, linolenic acid, myristic acid, palmitic acid, stearic acid, octanoic acid, decanoic acid, undecanoic acid, methyl myristate, methyl oleate, 1-nonanol, 1-decanol, 2-undecanol, 1-nonanal, 1-undecanol, undecanal, lauric aldehyde, 2-methylundecanal, oleamide, linoleamide, palmitamide, stearylamide, 2-ethyl-l-hexanol, 2 -hexyl- 1-decanol, 2-octyl- 1-dodecanol, and mixtures thereof. In some embodiments, the extractant may comprise 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 some embodiments, the extractant may comprise one or more of the following oleic acid, linoleic acid, linolenic acid, myristic acid, palmitic acid, and stearic acid. In some embodiments, the extractant may comprise one or more of the following oleic acid, linoleic acid, palmitic acid, and stearic acid. In some embodiments, the extractant may comprise 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 one or more esters of oleic acid, lauric acid, linoleic acid, linolenic acid, myristic acid, palmitic acid, stearic acid, octanoic acid, decanoic acid, and undecanoic acid. In some embodiments, the extractant may comprise one or more of the following oleic acid, linoleic acid, linolenic acid, myristic acid, palmitic acid, and stearic acid, and one or more esters of oleic acid, linoleic acid, linolenic acid, myristic acid, palmitic acid, and stearic acid. In some embodiments, the extractant may comprise 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. In some embodiments, the extractant may comprise one or more of the following oleyl alcohol, behenyl alcohol, cetyl alcohol, lauryl alcohol, myristyl alcohol, stearyl alcohol. In some embodiments, the extractant may comprise one or more of the following 1-nonanol, 1-decanol, 2-undecanol, 1-nonanal, 1- undecanol, undecanal, 2-ethyl-l-hexanol, 2-hexyl- 1 -decanol, 2-octyl-l-dodecanol.

[00120] In some embodiments, the extractant may be a mixture of biocompatible and non- biocompatible extractants. Examples of mixtures of biocompatible and non-biocompatible extractants include, but are not limited to, oleyl alcohol and nonanol, oleyl alcohol and 1- undecanol, oleyl alcohol and 2-undecanol, oleyl alcohol and 1-nonanal, oleyl alcohol and decanol, and oleyl alcohol and dodecanol. Additional examples of biocompatible and non- biocompatible extractants are described in U.S. Patent Application Publication No. 2009/0305370 and U.S. Patent Application Publication No. 201 1/0097773; the entire contents of each herein incorporated by reference. In some embodiments, biocompatible extractants may have high atmospheric boiling points. For example, biocompatible extractants may have atmospheric boiling points greater than the atmospheric boiling point of water.

[00121] In some embodiments, a hydrophilic solute may be added to fermentation broth that is contacted with an extractant. The presence of a hydrophilic solute in the aqueous phase may improve phase separation and may increase the fraction of product alcohol that partitions into the organic phase. Examples of a hydrophilic solute may include, but are not limited to, polyhydroxylated compounds, polycarboxylic compounds, polyol compounds, and dissociating ionic salts. Sugars such as glucose, fructose, sucrose, maltose, and oligosaccharides may serve as a hydrophilic solute. Other polyhydroxylated compounds may include glycerol, ethylene glycol, propanediol, polyglycerol, and hydroxylated fullerene. Polycarboxylic compounds may include citric acid, tartaric acid, maleic acid, succinic acid, polyacrylic acid, and sodium, potassium, or ammonium salts thereof. Ionic salts that may be used as a hydrophilic solute in fermentation broth comprise cations that include sodium, potassium, ammonium, magnesium, calcium, and zinc; and anions that include sulfate, phosphate, chloride, and nitrate. The amount of hydrophilic solute in the fermentation broth may be selected by one skilled in the art to maximize the transfer of product alcohol from the aqueous phase (e.g., fermentation broth) to the organic phase (e.g., extractant) while not having a negative impact on the growth and/or productivity of the product alcohol-producing microorganism. High levels of hydrophilic solute may impose osmotic stress and/or toxicity on the microorganism. One skilled in the art may use any number of known methods to determine an optimal amount of hydrophilic solute to minimize the effects of osmotic stress and/or toxicity on the microorganism.

0122] In some embodiments where the product alcohol is butanol, the extractant may be selected for attracting the alkyl portion of butanol and for providing little or no affinity to water. An extractant that offers no hydrogen bonding, for example, to water will absorb the alcohol selectively. In some embodiments, the extractant may comprise an aromatic compound. In some embodiments, the extractant may comprise alkyl substituted benzenes including, but not limited to, cumene, para-cymene (also known as l-methyl-4-(l- methylethyl)benzene), meta-cymene (also known as l-methyl-3-(l-methylethyl)benzene), meta-diisopropylbenzene, para-diisopropylbenzene, triethylbenzene, ethyl butyl benzene, and tert-butylstyrene. An advantage of using an alkyl-substituted benzene is the comparatively higher butanol affinity relative to other hydrocarbons. In addition, isopropyl-substituted or isobutyl-substituted benzenes may offer a particular advantage in butanol affinity over other substituted benzenes. Another advantage is the lower viscosity, lower surface tension, lower density, higher thermal stability, and higher chemical stability that aids in phase separability and long-term reuse. In some embodiments, an extractant that attracts the alkyl portion of butanol may be combined with another extractant that offers affinity in the form of hydrogen bonding, for example, to the hydroxyl portion of butanol such that the mixture provides an optimal balance between selectivity and partitioning over water. In some embodiments, an extractant containing butanol may be phase separated from fermentation broth and distilled in a column operating under vacuum. This distillation may operate with reflux in order to maintain a distillate of high purity butanol that contains very little extractant. The bottoms may comprise a portion of the butanol contained in the distillation feed such that the reboiling temperature under vacuum is suitable for delivering heat indirectly from available steam. Distillation may be carried out with a partial condenser where only reflux liquid is condensed, and a vapor distillate of substantially butanol composition may be directed into the bottom of a rectification column that is simultaneously fed a butanol stream decanted from condensed beer column overhead vapor. An advantage of this type of distillation is that the need for a reboiler to purify the decanted butanol stream is eliminated by heat integrating the vapor generated from stripping butanol out of the extractant.

[00123] In some embodiments, extractant may be generated from feedstock. For example, oils such as corn oil present in feedstock may be used for the generation of extractant for extractive fermentation. The glycerides in oil may be chemically or enzymatically converted into a reaction product, such as fatty acids and/or fatty esters (e.g., ethyl esters, butyl esters, fusel esters) which may be used as an extractant for the recovery of the product alcohol. Using corn oil as an example, corn oil triglycerides may be reacted with a base such as ammonia hydroxide to obtain fatty amides and glycerol. In some embodiments, oil in the feedstock may be hydrolyzed by a catalyst to generate fatty acids. In some embodiments, at least a portion of the acyl glycerides in oil may be hydrolyzed to carboxylic acid by contacting the oil with catalyst. In some embodiments, the resulting acid/oil composition includes monoglycerides and/or diglycerides from the partial hydrolysis of the acyl glycerides in the oil. In some embodiments, the resulting acid/oil composition includes glycerol, a by-product of acyl glyceride hydrolysis. In some embodiments, the resulting acid/oil composition includes lysophospholipids from the partial hydrolysis of phospholipids in the oil. Methods for deriving extractants from biomass are described in U.S. Patent Application Publication No. 2011/0312044; U.S. Patent Application Publication No. 201 1/0312043; and U.S. Patent Application Publication No. 2012/0156738, the entire contents of each are all herein incorporated by reference.

[00124] In some embodiments, the conversion of oil (e.g., hydrolysis, transesterification) in the feedstock or feedstock slurry may occur in the fermentor by the addition of a catalyst to the fermentor. For example, a catalyst such as lipase may be added to the fermentor, converting the oil present in the feedstock or feedstock slurry to fatty acids and/or fatty esters. In some embodiments, the conversion of oil in the feedstock or feedstock slurry may occur in a separate unit. For example, the feedstock or feedstock slurry may be conducted to a unit, and a catalyst such as lipase may be added to the unit, converting the oil present in the feedstock or feedstock slurry to fatty acids. As another example, the feedstock or feedstock slurry may be conducted to a unit, and a catalyst such as lipase and an alcohol (e.g., ethanol, butanol, fusel alcohols) may be added to the unit, converting the oil present in the feedstock or feedstock slurry to fatty esters. In some embodiments, the fatty acids and/or fatty esters may be added to the fermentor and may be used as an extractant for the recovery of the product alcohol. In some embodiments, the fatty acids and/or fatty esters may be added to an external extractor or extractant column and may be used as an extractant for the recovery of the product alcohol.

[00125] In some embodiments, oil may be separated from feedstock slurry and the oil may be conducted to a unit, and a catalyst such as lipase may be added to the unit, generating a fatty acid stream. The fatty acid stream may be heated to deactivate the lipase and then the fatty acid stream may be conducted to an external extractor or a storage tank. Fatty acids from the storage tank may be conducted to an external extractor for extraction of product alcohol from fermentation broth. In some embodiments, oil separated from feedstock slurry may be stored in a storage tank. A catalyst such as lipase may be added to the storage tank, generating a fatty acid stream. The fatty acid stream may be heated to deactivate the lipase, cooled, and then conducted to an external extractor for extraction of product alcohol from fermentation broth. In some embodiments, oil separated from feedstock slurry may be conducted to a unit, and a catalyst such as lipase may be added to the unit, generating a fatty acid stream. The fatty acid stream may be heated to deactivate the lipase, cooled, and then the fatty acid stream may be conducted to a fermentor.

[00126] In some embodiments, the one or more catalysts may be one or more enzymes, for example, hydrolase enzymes. In some embodiments, the one or more catalysts may be one or more enzymes, for example, lipase enzymes. Lipase enzymes may be derived from any source including, for example, Absidia, Achromobacter, Aeromonas, Alcaligenes, Alternaria, Aspergillus, Achromobacter, Aureobasidium, Bacillus, Beauveria, Brochothrix, Candida, Chromobacter, Coprinus, Fusarium, Geotricum, Hansenula, Humicola, Hyphozyma, Lactobacillus, Metarhizium, Mucor, Nectria, Neurospora, Paecilomyces, Penicillium, Pseudomonas, Rhizoctonia, Rhizomucor, Rhizopus, Rhodosporidium, Rhodotorula, Saccharomyces, Sus, Sporobolomyces, Thermomyces, Thiarosporella, Trichoderma, Verticillium, and/or Yarrowia. In some embodiments, the source of the lipase may be selected from the group consisting of Absidia blakesleena, Absidia corymbifera, Achromobacter iophagus, Alcaligenes sp., Alternaria brassiciola, Aspergillus flavus, Aspergillus niger, Aspergillus tubingensis, Ameobasidium pullulans, Bacillus coagulans, Bacillus pumilus, Bacillus strearothermophilus , Bacillus subtilis, Brochothrix thermosohata, Burkholderia cepacia, Candida cylindracea (Candida rugosa), Candida paralipolytica, Candida antarctica lipase A, Candida antarctica lipase B, Candida ernobii, Candida deformans, Candida rugosa, Candida parapsilosis, Chromobacter viscosum, Coprinus cinerius, Fusarium heterosporum, Fusarium oxysporum, Fusarium solani, Fusarium solani pisi, Fusarium roseum culmorum, Geotrichum candidum, Geotricum penicillatum, Hansenula anomala, Humicola brevispora, Humicola brevis var. thermoidea, Humicola insolens, Lactobacillus curvatus, Rhizopus oryzae, Mucor javanicus, Neurospora crassa, Nectria haematococca, Penicillium cyclopium, Penicillium crustosum, Penicillium expansum, Penicillium roqueforti, Penicillium camembertii, Penicillium sp. I, Penicillium sp. II, Pseudomonas aeruginosa, Pseudomonas alcaligenes, Pseudomonas cepacia (syn. Burkholderia cepacia), Pseudomonas fluorescens, Pseudomonas fragi, Pseudomonas maltophilia, Pseudomonas mendocina, Pseudomonas mephitica lipolytica, Pseudomonas alcaligenes, Pseudomonas plantari, Pseudomonas pseudoalcaligenes, Pseudomonas putida, Pseudomonas stutzeri, and Pseudomonas wisconsinensis, Rhizoctonia solani, Rhizomucor miehei, Rhizopus arrhizus, Rhizopus delemar, Rhizopus japonicus, Rhizopus microsporus, Rhizopus nodosus, Rhizopus oryzae, Rhodosporidium toruloides, Rhodotorula glutinis, Saccharomyces cerevisiae, Sporobolomyces shibatanus, Sus scrofa, Thermomyces lanuginosus (formerly Humicola lanuginose), Thiarosporella phaseolina, Trichoderma harzianum, Trichoderma reesei, and Yarrowia lipolytica.

[00127] In some embodiments, hydrolase and/or lipase may be expressed by the microorganism. In some embodiments, the microorganism may be engineered to express homologous or heterologous hydrolase and/or lipase. In some embodiments, hydrolase and/or lipase may be expressed by a microorganism that also produces a product alcohol. In some embodiments, hydrolase and/or lipase may be expressed by a microorganism that also expresses a butanol biosynthetic pathway. In some embodiments, the butanol biosynthetic pathway may be a 1 -butanol biosynthetic pathway, 2-butanol biosynthetic pathway, isobutanol biosynthetic pathway, or 2-butanone biosynthetic pathway.

[00128] Commercial lipase preparations suitable as a catalyst include, but are not limited to, Lipolase® 100 L, Lipex® 100L, Lipoclean® 2000T, Lipozyme® CALB L, Novozyme® CALA L, and Palatase 20000L, available from Novozymes (Franklinton, NC), or lipases from Pseudomonas fluorescens, Pseudomonas cepacia, Mucor miehei, hog pancreas, Candida cylindracea, Candida rugosa, Rhizopus niveus, Candida antarctica, Rhizopus arrhizus or Aspergillus available from Sigma Aldrich (St. Louis, MO). In some embodiments, the lipase may be thermostable and/or thermotolerant, and/or solvent tolerant.

[00129] In some embodiments, the one or more catalysts may be phospholipases. A phospholipase useful in the present invention may be obtained from a variety of biological sources, for example, but not limited to, filamentous fungal species within the genus Fusarium, such as a strain of Fusarium culmorum, Fusarium heterosporum, Fusarium solani, or Fusarium oxysporum; or a filamentous fungal species within the genus Aspergillus, such as a strain of Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus niger or Aspergillus oryzae. Also useful in the present invention are Thermomyces lanuginosus phospholipase variants such as the commercial product Lecitase® Ultra (Novozymes A'S, Denmark). One or more phospholipases may be applied as lyophilized powder, immobilized, or in aqueous solution.

[00130] In some embodiments, phospholipase may be expressed by the microorganism. In some embodiments, the microorganism may be engineered to express homologous or heterologous phospholipases. In some embodiments, phospholipase may be expressed by a microorganism that also produces a product alcohol. In some embodiments, phospholipase may be expressed by a microorganism that also expresses a butanol biosynthetic pathway. In some embodiments, the butanol biosynthetic pathway may be a 1 -butanol biosynthetic pathway, 2-butanol biosynthetic pathway, isobutanol biosynthetic pathway, or 2-butanone biosynthetic pathway.

[00131] By-products of fermentation such as isobutyric acid, phenylethanol, 3-methyl-l- butanol, 2-methyl-l -butanol, isobutyraldehyde, acetic acid, ketoisovaleric acid, pyruvic acid, and dihydroxyisovaleric acid may have an inhibitory effect on the microorganism. In some embodiments, these by-products may be modified by esterification. For example, the by- products may be esterified with carboxylic acids, alcohols, fatty acids, or other by-products. In some embodiments, these esterification reactions may be catalyzed by lipases or phospholipases. As an example, lipase present in the fermentation broth may catalyze the esterification of by-products generated during fermentation. Esterification of these byproducts may minimize their inhibitory effects on the microorganism.

[00132] Referring to Figure 7A, feedstock 12 may be processed as described in Figures 1 to 6, and therefore will not be described in detail. Aqueous solution 22 may then be further treated to remove any residual oil. In some embodiments, aqueous solution 22 may be subjected to centrifugation, decantation, or any other method that may be used for oil removal. In some embodiments, aqueous solution 22 may be conducted to unit 25 (or vessel) and catalyst 23 (e.g., lipase) may be added to unit 25, converting the oil present in aqueous solution 22 to fatty acids, generating stream 27. Stream 27 may then be conducted to fermentation 30 and microorganism 32 may also be added to fermentation 30 for the production of product alcohol. Following fermentation 30, stream 31 comprising product alcohol and fatty acids may be conducted to an external unit, for example, an external extractor or external extraction loop for the recovery of product alcohol.

[00133] Referring to Figure 7B, in some embodiments, catalyst 23 may be deactivated, for example, by heating. In some embodiments, stream 27 comprising catalyst 23 may be heated (q) to deactivate catalyst 23 prior to addition to fermentation 30. Referring to Figure 7C, in some embodiments, deactivation may be conducted in a separate unit, for example, a deactivation unit. In some embodiments, stream 27 may be conducted to deactivation 28. Following deactivation, stream 27' may be conducted to fermentation 30 and microorganism 32 may also be added to fermentation 30 for production of product alcohol.

[00134] Removing oil from aqueous solution 22 by converting the oil to fatty acids can result in energy savings for the production plant due to more efficient fermentation, less fouling of the equipment due to the removal of the oil, decreased energy requirements, for example, the energy needed to dry distillers grains, and improved operation of evaporators or evaporation train. In addition, removal of the oil component of the feedstock is advantageous to product alcohol production because oil present in the fermentor can break down into fatty acids and glycerin. The glycerin can accumulate in the water and reduce the amount of water that is available for recycling throughout the system. Thus, removal of the oil component of the feedstock increases the efficiency of the product alcohol production by increasing the amount of water that can be recycled through the system. Also, stable emulsions are less likely to occur by removal of oil. In some embodiments of the present invention, in the event that an emulsion forms, emulsions may be readily broken by mechanical processing, addition of protic solvents, or by other conventional means.

[00135] In another embodiment, referring to Figure 7D, aqueous solution 22 may be conducted to fermentation 30 and catalyst 23 (e.g., lipase) may be added to fermentation 30, converting oil present in aqueous solution 22 to fatty acids and/or fatty esters. In some embodiments, fatty esters may be derived from the combination of fatty acids with an alcohol. In some embodiments, the alcohol may be any alcohol in fermentation 30 including a product alcohol. In some embodiments, the amount of oil in aqueous solution 22 converted to fatty acids and/or fatty esters may be at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%. In some embodiments, the ratio of fatty esters and fatty acids generated by the conversion of oil may be about 75:25. In some embodiments, the ratio of fatty esters and fatty acids may be about 80:20. In some embodiments, catalyst 23 may be added to fermentation 30 in an amount to maintain a certain oil conversion rate.

[00136] Following fermentation 30, stream 31 comprising product alcohol, fatty acids, and fatty esters may be further processed for recovery of product alcohol. For example, stream 31 may be conducted to an external unit, for example, an external extractor or external extraction loop for the recovery of product alcohol. In some embodiments, the fatty acids and fatty esters in stream 31 may be used as an extractant. In some embodiments, the external unit may comprise extractant. In some embodiments, the extractant may comprise fatty acids and/or esters of fatty acids.

[00137] The present invention also provides processes and systems for recovering a product alcohol produced by a fermentative process. One such process for product alcohol recovery is liquid-liquid extraction. Using liquid-liquid extraction as an ISPR technique is best served by a liquid-liquid extraction process that maximizes the net present value of the capital investment required to practice the technology. An aspect of maximizing the net present value of a liquid-liquid extraction process is to avoid large capital and operating cost expenditures associated with separating extractant from fermentation broth.

[00138] In one embodiment of a liquid-liquid extraction process, extractant may be added directly to the fermentor, and fermentation broth and extractant may be mixed together in a way that effects mass transfer (e.g., transfer of product alcohol from fermentation broth to extractant) and allows the fermentation to proceed to high effective product alcohol titer. In such a process, if mixing is too intense or vigorous, the fermentation broth and extractant may have to be separated using a separation device such as a centrifuge. If mixing is not too intense, phase separation may be achieved through gravity settling brought on by the density difference between the extractant and the fermentation broth. In either case, additional fermentors may be required to overcome the loss of fermentor volume taken up by extractant added to the fermentor. Adding extractant directly to the fermentor may be carried out in batch, semi-batch, or continuous modes irrespective of phase separation within the fermentor. If continuous mode is employed and gravity separation of fermentation broth and extractant is not possible, then a separation device such as a centrifuge may be required for the separation of product alcohol from extractant. If the separation process employed to remove product alcohol from extractant is such that the microorganism present in the fermentation broth is viable the separation process, then separation of fermentation broth from product alcohol/extractant may not be required.

[00139] Another embodiment of a liquid-liquid extraction process may include an external extractor or extraction column. For example, fermentation broth from the fermentor may be conducted to an external extractor where the fermentation broth is mixed with extractant. The mixture of fermentation broth and extractant may then be separated, generating a fermentation broth stream leaner in product alcohol and an extractant stream richer in product alcohol. The leaner fermentation broth stream may be returned to the fermentor. The richer extractant stream may be processed further to separate at least a portion of product alcohol from the extractant for product alcohol recovery. In some embodiments, the rate of product alcohol recovery from the extractant stream may be set at a rate to maintain plant production. In some embodiments, the liquid-liquid extraction process may comprise one or more external liquid-liquid extractors.

[00140] In some embodiments, fermentation may occur in the fermentor and the external extractor. The additional volume of fermentation broth present in the external extractor may serve to increase the overall fermentor volume and therefore, may increase the overall production of product alcohol.

[00141] The performance of the external extractor with regard to removing product alcohol may depend on the surface area available for interfacial contact, the physical nature of the fermentation broth and extractant, the relative amounts of the two phases (e.g., fermentation broth phase and extractant phase) present in the external extractor, and the concentration driving force difference between the fermentation broth and extractant phases. Maximizing the efficiency of the external extractor for a given product alcohol concentration driving force may be accomplished by reducing the droplet size of the dispersed phase in the external extractor, for example, via nozzle design, internals design, and/or agitation. In some embodiments, the design and operation of the external extractor may provide enough mixing to effect adequate product alcohol transfer between the fermentation broth and extractant phases to maintain product alcohol productivity requirements.

[00142] In some instances, CO ₂ from fermentation may be generated in the external extractor, leading to the formation of droplets which may interfere with phase separation. For example, droplets of fermentation broth may attach to CO ₂ rising through the extractant phase. In some embodiments, the extractant phase may be maintained as the continuous phase to improve the coalescence of droplets. In some embodiments, the external extractor may include internals or exit ports for CO ₂ . For example, a coalescing pad may be added to the external extractor and/or the exit ports may be located to improve coalescence and recovery of the fermentation broth phase.

[00143] Conditions to separate product alcohol from fermentation broth may be deleterious to the microorganism present in the fermentation broth. In some embodiments, the microorganism may be separated from fermentation broth prior to contacting the fermentation broth with the extractant. In some embodiments, the microorganism may be separated from a mixture of fermentation broth and extractant prior to the separation (or processing) of this mixture. Any separation method capable of separating the microorganism from fermentation broth or mixture of fermentation broth and extractant may be used including, for example, centrifugation. By separating the microorganism prior to contacting the fermentation broth with extractant, it may be possible to use more rigorous extraction conditions such as higher temperatures and/or non-biocompatible extractants. If a separation method was used that was not deleterious to the microorganism, then separating the fermentation broth and extractant prior to product alcohol removal may not be required.

[00144] If extractant and fermentation broth are not separated, then the extractant may be included in the evaporator train feed and therefore, become a component of the syrup formed during evaporation, and possibly incorporated in animal feed. In some embodiments, extractant may be separated from the syrup using any separation means including, for example, centrifugation. A low boiling point (e.g., comparable to water) biocompatible extractant may not require such separation because the extractant and water may be recycled for use in the production process.

[00145] In a typical corn-to-product alcohol production plant, the water balance of the overall production process may be maintained by recycling water of the production plant with recycled water distilled in an evaporator train to remove salts and other dissolved solids of the beer. The resulting syrup from the evaporator train may be mixed with undissolved solids, and the mixture may be dried and sold as animal feed. Processes and systems for processing undissolved solids for animal feed are described, for example, in U.S. Patent Application Publication No. 2012/0164302; U.S. Patent Application Publication No. 201 1/0315541; U.S. Patent Application Publication No. 2013/0164795; and PCT International Patent Application No. PCT/US2013/51571, the entire contents of each are herein incorporated by reference.

[00146] As described herein, undissolved solids may be removed from feedstock (or feedstock slurry) prior to the addition of the feedstock to fermentation. If undissolved solids are not removed upstream of fermentation, then centrifugation of the beer to remove undissolved solids may be necessary to avoid fouling of the evaporators. For example, in a commercial corn-to-product alcohol dry-grind production plant, undissolved solids content in an evaporator train feed may operate at about 3% total suspended solids, and may be as high as 3.5-4% total suspended solids. An upstream process that removes enough solids to maintain the percentage of total suspended solids at or below these percentage values may eliminate the need for centrifugation, for example, prior to conducting the beer to the evaporators (or evaporation train). The elimination of this centrifugation would result in a savings on the capital required to retrofit a dry-grind corn-to-product alcohol production plant.

[00147] By removing at least a portion of the undissolved solids present in the feedstock slurry prior to fermentation, the interfacial surface area between the fermentation broth and extractant phases in an external extractor may be increased by reducing the amount of undissolved solids at the interface, enhancing product alcohol transfer between the fermentation broth and the extractant and providing for a clean phase separation between the fermentation broth and extractant. A clean phase separation may also eliminate the need for additional separation steps (e.g., centrifugation) and therefore, a savings on capital expenses.

[00148] The separation of fermentation broth and extractant leaving the external extractor may be influenced by the solids content and particle size distribution of the solids content in the fermentation broth, the gas content and gas bubble size distribution in the fermentation broth, the physical properties of the fermentation broth and extractant including, but not limited to, viscosity, density, and surface tension as well as the design and operation of the external extractor and the design and operation of the fermentor. These properties may determine the need for separation devices (e.g., centrifuges) to separate the fermentation broth and extractant leaving the external extractor or the fermentor. Operating under conditions that eliminate the need for separation devices may minimize the capital expenditure to practice liquid-liquid extraction ISPR. In addition, minimizing the size of the extractors by maximizing the interfacial area between fermentation broth and extractant phases for a given set of fermentation broth and extractant physical properties can maintain the ability to inexpensively phase separate fermentation broth and extractant. By eliminating the capital and operating cost of separation devices such as centrifuges, the net present value of a dry grind corn-to-product alcohol production plant employing a liquid-liquid extraction ISPR process may be improved.

[00149] In another embodiment of the processes and systems described herein, the extractor design including phase separation capacity may be tailored to accommodate the physical properties of the fermentation broth and extractant. If undissolved solids are not removed from feedstock slurry or if the concentration of product alcohol in the fermentation broth is too low, it may not be possible to remove enough product alcohol to maintain plant productivity employing an extractor that does not include phase separation equipment. Therefore, the present invention provides for processes and systems that include solids removal as well as recovery of product alcohol utilizing an external extractor wherein the extractor has been designed to improve phase separation capacity for maximum product alcohol recovery.

[00150] An exemplary process of the present invention is described in Figure 8. Some processes and streams in Figure 8 have been identified using the same name and numbering as used in Figures 1-7 and represent the same or similar processes and streams as described in Figures 1-7. [00151] Feedstock 12 may be processed and solids separated (100) as described herein with reference to Figures 1-7. Briefly, feedstock 12 may be liquefied to generate feedstock slurry comprising undissolved solids, fermentable sugars (or fermentable carbon source), and depending on the feedstock, oil. The feedstock slurry may then be subjected to separation methods to remove suspended solids, generating a wet cake, an aqueous solution 22 (or centrate) comprising dissolved fermentable sugars, and optionally an oil stream. Solids separation may be accomplished by a number of means including, but not limited to, decanter bowl centrifugation, three-phase centrifugation, disk stack centrifugation, filtering centrifugation, decanter centrifugation, filtration, vacuum filtration, belt filter, pressure filtration, membrane filtration, microfiltration, filtration using a screen, screen separation, grating, porous grating, flotation, hydrocyclone, filter press, screwpress, gravity settler, vortex separator, or combination thereof.

[00152] Aqueous solution 22 and microorganism 32 may be added to fermentation 30 where the fermentable sugars are fermented by microorganism 32 to produce stream 105 comprising product alcohol. In some embodiments, during fermentation, a portion of stream 105 may be transferred to extractor 120 (or extraction 120) where stream 105 is contacted with extractant 124. In some embodiments, extractant may be stored in an extractant storage tank or unit. In some embodiments, stream 105 may be removed from fermentation 30 when the concentration of product alcohol and/or other metabolic products reach a predetermined concentration. In some embodiments, the predetermined concentration may be a concentration of product alcohol and/or other metabolic products which negatively impact the metabolism of the microorganism. In some embodiments, stream 105 may be removed from fermentation 30 when fermentation is initiated. In some embodiments, stream 105 may be removed from fermentation 30 to minimize the effects of product alcohol on microorganism 32. In some embodiments, fermentation 30 may comprise one, two, three, four, five, six, seven, eight, or more fermentors.

[00153] In some embodiments, extractant may be added to fermentation 30. In some embodiments, a portion of fermentation broth comprising extractant may be transferred to extractor 120, and in some embodiments, extractant may be recovered from the fermentation broth comprising extractant. By adding extractant to fermentation 30, ISPR may be initiated in fermentation 30. [00154] Product alcohol, or a portion thereof, transfers from stream 105 to extractant 124, and stream 122 comprising extractant richer in product alcohol may be conducted to separation 130. Stream 127 comprising fermentation broth leaner in product alcohol may be returned to fermentation 30. Separation 130 removes a portion of product alcohol from stream 122, and stream 125 comprising leaner extractant may be returned to extractor 120. In some embodiments, extractor 120 may be external to fermentation 30. In some embodiments, fermentation 30 may comprise an extractor. In some embodiments, extractant, fermentation broth, or both may be at least partially immiscible. Stream 135 may be conducted downstream for further processing (e.g., distillation) including recovery of product alcohol.

[00155] Over the course of extraction, there may be a loss of extractant or a portion of extractant. In some embodiments, extractant 124 may be replenished by the addition of extractant to extractor 120 or an extractant storage unit. In some embodiments, for example, where extractant may be derived from feedstock or feedstock slurry, extractant 124 may be replenished by converting oil in the feedstock or feedstock slurry to extractant. For example, a catalyst may be added to fermentation 30, converting oil in aqueous solution 22 to fatty acids and/or fatty esters (see, e.g., Figure 7D), and a portion of stream 105 comprising product alcohol, fatty acids, and/or fatty esters may be transferred to extractor 120 where stream 105 may be contacted with extractant 124. Stream 122 comprising product alcohol- rich extractant, fatty acids, and/or fatty esters may be conducted to separation 130 generating stream 125 comprising leaner extractant, fatty acids, and/or fatty esters. In some embodiments, stream 125 may be further processed prior to its return to extractor 120. For example, fatty esters present in stream 125 may be subjected to hydrolysis generating a stream comprising product alcohol and fatty acids. This stream comprising product alcohol and fatty acids may be conducted to extractor 120, or this stream may be combined with stream 122 and the combined stream may be conducted to separation 130. In another embodiment, this stream comprising product alcohol and fatty acids may be conducted to separation 130 or this stream may be conducted to another separation unit generating a product alcohol stream and a fatty acid stream. The fatty acid stream may be conducted to extractor 120, and the product alcohol stream may be combined with stream 135 and further processed for product alcohol recovery. [00156] In some embodiments, phase separation of fermentation broth and extractant after passing through an extractor may be insufficient such that an unacceptable level of dispersed extractant remains in the fermentation broth returning to the fermentor and/or an unacceptable level of fermentation broth droplets remain in the extractant advancing to distillation. In some embodiments, the phase separation of fermentation broth and extractant may be enhanced by processing a heterogeneous mixture exiting the top or bottom of an extractor through one or more hydrocyclones or similar vortex device. In some embodiments, a static mixer may be used in place of an extractor to bring fermentation broth and extractant into contact with each other and the heterogeneous mixture that is formed may be pumped through one or more hydrocyclones or similar vortex device to effect a separation of the aqueous (e.g., fermentation broth) and organic (e.g., extractant) phases. In some embodiments, one or more hydrocyclones or similar vortex device may be used to remove liquid or liquid droplets from a gas stream. In some embodiments, the gas stream may be from the fermentor. In some embodiments, the gas stream may be from a degassing device.

[00157] In a batch or semi-batch fermentation process, when a portion of the fermentable sugars has been metabolized by microorganism 32, stream 103 comprising beer may be conducted downstream to separation 140 to separate product alcohol from the beer. Stream 145 comprising product alcohol may be conducted downstream for further processing (e.g., distillation) including recovery of product alcohol. In a continuous fermentation process, stream 103 comprising beer may be conducted downstream to separation 140 to separate product alcohol from the beer. Stream 142 comprising whole stillage may be conducted downstream for further processing including solids removal and generation of thin stillage.

[00158] In some embodiments, fermentation 30 may comprise two or more fermentors, and stream 105 may comprise combined multiple streams from the two or more fermentors. In some embodiments, the combined multiple streams may be conducted to extractor 120. In some embodiments, stream 127 may be split and portions of stream 127 may be returned to the multiple fermentors. In some embodiments, extractor 120 may be a series of units connected together in parallel or in series.

[00159] In some embodiments, extraction may be conducted for a certain period of time. Extraction may be conducted, for example, until the concentration of product alcohol in fermentation 30 is low enough that separation 140 is not required. In some embodiments, extraction may be conducted for an extended period of time.

[00160] In some embodiments of the processes and systems described herein, a decanter may be used for phase separation. In some embodiments, a decanter may be used in combination with an extractor. In some embodiments, the surfaces of the decanter may be modified to improve phase separation. For example, surfaces of the decanter may be modified by the addition of hydrophilic and/or hydrophobic surfaces.

[00161] In some embodiments, oxygen, air, and/or nutrients may be added to stream 125 and/or stream 127. In some embodiments, the nutrients may be soluble in extractant. In some embodiments, the concentration of oxygen may be measured in the various streams, and may be used as part of a control loop to vary the flow of oxygen into the process. In some embodiments, mash may be added to extractor 120 to allow for higher effective titers. In some embodiments, separation 130 and 140 may be extractors. In some embodiments, these extractors may use water to extract product alcohol from extractant, and product alcohol may be subsequently separated from an aqueous phase. In some embodiments, extractant may be infused with solutes that enhance its capacity to extract product alcohol from fermentation broth. In some embodiments, a surge tank may be located between extractor 120 and separation 130 as a means to equilibrate the concentration of product alcohol in the extractant prior to separation (e.g., distillation).

[00162] In some embodiments, extractor 120 may be designed to utilize CO ₂ generated during fermentation for the purpose of mixing fermentation broth and extractant. In some embodiments, extractor 120 may be designed to allow for ready disengagement of CO ₂ in the fermentation broth. This design would facilitate the control of the level of mixing by CO ₂ bubbles rising through extractor 120. In some embodiments, fermentation broth may be removed from fermentation 30 to minimize the concentration of CO ₂ in stream 105. In some embodiments, the design of extractor disengagement zones may include surfaces to promote phase separation between fermentation broth and extractant. In some embodiments, hydrophilic and/or hydrophobic surfaces may be installed in the disengagement zones to improve phase separation. In some embodiments, the external extractor may include internals or exit ports for CO ₂ . For example, a coalescing pad may be added to the external extractor. [00163] In some embodiments to minimize CO ₂ mixing, the extractor may be designed with a small diameter at the bottom of the extractor, graduating to a large diameter at the top of the extractor (e.g., conical shape). In some embodiments, the extractor may be designed with a stepwise increase in diameter. For example, the extractor may comprise a first region of constant diameter flowed by a stepwise increase of diameter to a second region of constant diameter. In some embodiments, the extractor may further comprise a second stepwise increase of diameter to a third region of constant diameter. In some embodiments, the extractor may comprise one or more stepwise increases of diameter. In some embodiments, the extractor may comprise one or more regions of constant diameter.

[00164] Over the course of fermentation, the gas content (e.g., C0 ₂ ) of the fermentation broth changes, and these gases may be removed from the fermentation broth by utilizing a gas stripper. The amount of gas stripped from the fermentation broth may be adjusted by varying the flow through the gas stripper and/or the pressure of the gas stripper. In some embodiments, the amount of CO ₂ in the fermentation broth may be reduced prior to transferring the fermentation broth to an extractor. For example, CO ₂ may be stripped from the fermentation broth using a gas stripper or any means known to those skilled in the art. In some embodiments, removal of CO ₂ may be performed at or below ambient pressure. In some embodiments, fermentation may continue in the extractor, and CO ₂ may be produced by the microorganism. In some embodiments to minimize CO ₂ mixing in the extractor, the residence time of the fermentation broth in the extractor may be reduced. In some embodiments, residence time may be reduced by modifying the height of the extractor. In some embodiments, the height of the extractor may be reduced. Reducing the height of the extractor may reduce the number of theoretical extraction stages. In some embodiments, to maintain the number of theoretical extraction stages, the extractor may be replaced with two or more extractors of reduced height. In some embodiments, the two or more extractors may be in series. In some embodiments, the two or more extractors may be connected. In some embodiments, the two or more extractors may be connected in such a way to maintain countercurrent flow. In some embodiments, a degassing stage may be added to one or more extraction stages.

[00165] Referring to Figure 8, in some embodiments, the size of dispersed phase droplets in extractor 120 may be measured and adjusted through various means to enhance the rate of mass transfer. For example, droplet size may be measured using particle size analysis such as focused beam reflectance measurement (FBRM®) or particle vision and measurement (PVM®) technologies (Mettler-Toledo, LLC, Columbus OH). In some embodiments, the fermentation broth may be the dispersed phase and extractant may be the continuous phase, and under these conditions, solids present in the fermentation broth may interact to a lesser degree with the extractant. In some embodiments, conditions of separation 130 may be controlled to minimize oxidative and thermal instabilities effects on the extractant.

[00166] In some embodiments, the quality of the extractant may be monitored and extractant replenished at a frequency necessary for successful production of product alcohol. In some embodiments, extractant may be taken up by whole stillage solids. The whole stillage may be separated into liquid (e.g., thin stillage) and solid streams, and the solids may be washed to recover the extractant. In some embodiments, the temperature of extractor 120 may be adjusted to improve the efficiency of the overall process. In some embodiments, the flows of fermentation broth and extractant to extractor 120 may be co-current or countercurrent. In some embodiments, membranes may be used to minimize the mixing of fermentation broth and extractant. In some embodiments, extractant may be polymer beads or inorganic beads that absorb product alcohol. In some embodiments, the polymer beads or inorganic beads may be preferentially absorb product alcohol.

[00167] In some embodiments, measurements such as in-line, on-line, at-line, or real-time measurements may be used to measure the concentration of product alcohol and/or metabolic by-products in the various streams. These measurements may be used as part of a control loop to vary the flow between the various units or vessels (e.g., fermentation 30, extractor 120, separations 130 and 140, etc.) and to improve the overall process.

[00168] Another exemplary process of the present invention is described in Figure 9. Some processes and streams in Figure 9 have been identified using the same name and numbering as used in Figures 1-8 and represent the same or similar processes and streams as described in Figures 1-8.

[00169] Feedstock 12 may be processed and solids separated (100) as described herein with reference to Figures 1-7. In some embodiments, feedstock 12 may be mixed with recycled water (e.g., stream 162) generated by evaporation 160. As described herein, feedstock slurry may be subjected to separation methods to remove suspended solids, generating a wet cake 24, an aqueous solution 22 (or centrate) comprising dissolved fermentable sugars, and depending on the feedstock, oil. Wet cake 24 may be dried in dryer 170 and used to produce DDGS. In some embodiments, wet cake 24 may be re-slurried with water (e.g., recycled water/stream 162) and subjected to separation to remove additional fermentable sugars, generating washed wet cake (e.g., 74, 74' as described in Figures 4 and 5). In some embodiments, wet cake streams 24, 74, and 74' may be combined and the combined wet cake streams may be dried in a dryer 170 and used to produce DDGS.

[00170] Aqueous solution 22 and microorganism 32 may be added to fermentation 30 where the fermentable sugars are metabolized by microorganism 32 to produce stream 105 comprising product alcohol. In some embodiments, enzyme may be added to fermentation 30. Stream 105 may be conducted to extractor 120, and may be contacted with extractant 124. Stream 127 comprising fermentation broth leaner in product alcohol may be returned to the fermentation 30 and stream 122 comprising extractant richer in product alcohol may be conducted to separation 130. In some embodiments, extractor 120 may be operated in such a way that stream 122 contains minimal cell mass and minimal substrate. Separation 130 may damage microorganism 32 or substrate resulting in a decrease in the fermentation rate. Operating extractor 120 with minimal cell mass and substrate may minimize any potential damage by separation 130. Stream 125 comprising leaner extractant may be returned to extractor 120. Stream 135 from separation 130 may be conducted to purification 150 for further processing including recovery of product alcohol. In some embodiments, extractant may be added to fermentation 30. In some embodiments, a portion of fermentation broth comprising extractant may be transferred to extractor 120, and in some embodiments, extractant may be recovered from the fermentation broth comprising extractant. In some embodiments, the flow rates of fermentation broth and extractant to extractor may be modified to improve phase separation. For example, lower overall flow rates entering the extractor in the early or later stages of fermentation can improve the phase separation of fermentation broth and extractant.

[00171] As described herein, after a batch fermentation process or as a steady effluent stream in a continuous fermentation process, stream 103 comprising beer may be conducted downstream to separation 140 to separate product alcohol from the whole stillage 142. Utilizing an upstream solids removal process may lower the undissolved solids content in the thin mash and therefore, it may not be necessary to centrifuge whole stillage 142 to remove solids. Thus, whole stillage 142 may be conducted directly to evaporation 160. Syrup 165 generated by evaporation 160 may be mixed with wet cake 24, 74, 74' in dryer 170 to form DDGS.

[00172] In some embodiments, backset comprising total suspended solids (TSS) from whole stillage may be used (or recycled) for feedstock slurry preparation. In some embodiments, whole stillage or a portion of whole stillage may be processed through a solids separation system including, but not limited to, turbo filtration or ultracentrifugation prior to evaporation, or whole stillage or a portion of whole stillage may be processed for self- cleaning water purification.

[00173] In some embodiments where coarse grain solids are removed from liquefied mash, the whole stillage that is produced may contain fine solids and insoluble microorganism fragments, and these dispersed solids may be removed using turbo filtration. Turbo filtration may include subjecting a feed suspension to centrifugal motion through a strainer 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 of and inside the pores of fine grain particles. In some instances, washing the wet cake with water is insufficient for recovering extractant from the wet cake. In some embodiments, a concentrated product alcohol stream such as the organic phase may be used to recover extractant from whole stillage wet cake. In some embodiments, this organic phase may be formed in a decanter. In some embodiments, the wet cake that has been washed with product alcohol may be subsequently washed with water to recover the product alcohol from the wet cake.

[00174] In some embodiments, the processes and systems described herein may include an extractant reservoir (or tank or vessel). Extractant may be added to the extractant reservoir and this extractant may be circulated to an extractor. In some embodiments, extractant may be conducted to an extractor and a stream from the extractor may be returned to the extractant reservoir. In some embodiments, extractant from an extractant reservoir may be circulated to an extractor and/or fermentor. In some embodiments, an extractant stream may be circulated between an extractant reservoir, an extractor, and a fermentor. In some embodiments, at the completion of fermentation, the contents of the extractant reservoir, extractor, and/or the fermentor may be further processed to recover product alcohol.

[00175] Separation or extraction of product alcohol from extractant may be accomplished using methods known in the art, including but not limited to, siphoning, decantation, centrifugation, gravity settler, membrane-assisted phase splitting, and the like. In some embodiments, extraction may be performed using, for example, mixer-settlers. Mixer- settlers are stage-wise extractors and are available with various elements for mixing such as, pumps, agitators, static mixers, mixing tees, impingement devices, circulating screens, or raining buckets. Examples of mixer-settlers are shown in Figures 10A-10H. For example, Figure 10A illustrates a mixer-settler using a pump as the source of mixing. Figure 10B illustrates a mixer-settler using a mixer as the source of mixing. Figure IOC illustrates a mixer-settler using a static mixer as the source of mixing. Figure 10D illustrates a mixer- settler using a mixing tee as the source of mixing. Figure 10E illustrates a mixer-settler using an impingement mixer as the source of mixing. Figure 10F illustrates a mixer-settler using a raining bucket or meshed screen as the source of mixing. Figure 10G illustrates a mixer- settler using a centrifuge as a settler. Figure 10H illustrates a mixer-settler using a hydrocyclone or vortex separator as a settler. In some embodiments, one or more mixing devices may be used in the processes and systems as described herein.

[00176] In some embodiments, mixers may comprise agitators such as, for example, flat blades, pitched blade turbines, or curved propellers. Droplet size produced by agitated mixers may be controlled by agitator design, tank design, agitator speed, and mode of operation. For static mixers, droplet size may be controlled by the diameter of the mixer and flow rate. For example, droplet size may be controlled by varying the flow through the mixer over the course of the fermentation. In some embodiments, gases and mixers may be used for mixing purposes.

[00177] In some embodiments, one or more mixer-settlers may be used in the processes and systems as described herein. In some embodiments, the one or more mixer-settlers may be arranged in series or in countercurrent mode as illustrated in Figures 101 and 10J. In some embodiments, mixer-settlers may be stacked in a column arrangement, providing multiple mixing and settling zones. In some embodiments, the settler may comprise hydrophilic or hydrophobic surfaces to promote phase separation.

[00178] In another embodiment, column extractors or centrifugal extractors may be used in the processes and systems as described herein. Column extractors are differential extractors providing conditions for mass transfer over their length with a steadily changing concentration profile. The different types of differential extractors may be divided into non- mechanical, pulse-agitated, and rotary-agitated. Centrifugal extractors are a separate class of differential extractors with the Podbielniak® centrifugal contactor being one such type. [00179] In some embodiments, non-mechanical spray towers may be used in the processes and systems as described herein. One example of a non-mechanical spray tower includes a non-mechanical spray tower without column internals. The number of nozzles and nozzle diameter may be used to determine droplet size. In some embodiments, the spray tower may have internals. In some embodiments, a spray tower may comprise helical piping. Helical piping may allow for droplet rise and additional mixing of fermentation broth and extractant. In some embodiments, non-mechanical extractors such as packed towers, sieve trays, and baffle trays may be used in the processes and systems as described herein. Examples of these extractors are shown in Figure 10K. In some embodiments, the packing of such extractors may be random or structured.

[00180] In some embodiments, pulsed-agitated extractors may be used in the processes and systems as described herein. Pulsed-agitated extractors have different designs as well including reciprocating trays or vibrating plates where the trays move in vertical fashion. The entire packed and/or sieve tray column can also vibrate in a vertical fashion to promote smaller dispersed phase droplets and more mass transfer. Examples of these extractors are shown in Figure 10L. In some embodiments, rotary-agitated or rotating disc contactors may be used in the processes and systems as described herein. Examples of these extractors are shown in Figure 10M.

[00181] In some embodiments, agitated extractors may be used in the processes and systems as described herein. For example, agitated extractors with centrifuges may provide high mass transfer rates and clean phase separation. In some embodiments, agitated columns may be used in the processes and systems as described herein. For example, agitated columns with internals may provide high mass transfer rates.

[00182] One aspect of a liquid-liquid extraction process is determining successful operating conditions for the extractor over the course of the constantly changing fermentation. For example, a typical corn-to-product alcohol batch fermentation employs an initial inoculum of microorganism (or cell mass) added to a certain volume of fermentation broth in the fermentor, followed by further filling of the fermentor to a specified volume. The fermentation is permitted to proceed until a pre-determined amount of the fermentable carbon source (e.g., sugar) is consumed. Over the course of batch fermentation, the concentrations of cell mass, reaction intermediates, reaction by-products, and substrate components change with time as do the physical properties of the fermentation broth including viscosity, density, and surface tension. To improve performance parameters of the fermentation, for example, rate, titer, and yield parameters of production and plant economics such as sales volume, return on investment, and profit, the extractor may be operated in a variable way to compensate for the changing fermentation broth. In addition, properties of a dynamic fermentation may impact the size limits of the extractor. Proper integration of the operation of the extractor and the fermentor may be benefit by use of mathematical models of the process (see, e.g., Daugulis and Kollerup, Biotechnology and Bioengineering 27: 1345-1356, 1985). Augmenting the mathematical model, for example, setting the key model parameters with experimental data is also valuable. Design parameters for differential extractors to consider for improved rate, titer, and yield of the fermentation process include the maximum total flow to the extractor per cross-sectional area of the extractor column as well as the height of the extractor required to remove enough product alcohol at a given fermentation broth to extractant ratio. It may be necessary to change the maximum flow per unit area and extractor height during a batch fermentation. Another consideration for differential extractors is droplet size of the dispersed phase. Appropriate droplet size may be a balance between small enough to provide adequate mass transfer but large enough to allow for clean phase separation exiting the extractor. In stage-wise extractors, the mixing intensity required for efficient mass transfer, the corresponding time needed to settle, and/or energy needed to separate the phases are additional elements to consider. In either type of extractor, stage-wise or differential, the ratio of fermentation broth to extractant fed to the extractor plays a role in determining the size of the extractor.

0183] In some embodiments, if an extractor of a fixed size were utilized and the maximum allowable flow that avoids flooding to the extractor varied from a low value to a high value (e.g., from 1/3 to 2/3 the maximum for a given extractor design) over the course of the fermentation owing to changes in the physical properties and concentrations of the fermentation broth, then the flows to the extractor may be varied, not exceeding the maximum flow, while still completing the fermentation in a reasonable time. In some embodiments, if an extractor is agitated, the speed of the agitation may be varied over the course of the fermentation to offset changes in the fermentation broth. Droplet size may be measured within the extractor, and the speed to maintain a fixed droplet size may be controlled throughout the fermentation to offset changes in the fermentation broth. The amount of mass transfer occurring at any time point may be assessed by measuring the concentrations of product alcohol in the inlet and outlet streams and adjusting conditions (e.g., flow, flow ratio, agitation) to control the mass transfer over the course of the fermentation.

[00184] In some embodiments, multiple extractors of different sizes may be utilized and conditions (e.g., flow, flow ratio, agitation) in each extractor may be adjusted to provide improved control of the fermentation process. In some embodiments, the ratio of fermentation broth to extractant may be modified to improve extraction efficiency, increase the concentration of product alcohol in the extractant (equivalent to increased efficiency), and reduce the required flows through the extractor.

[00185] In additional embodiments of the processes and systems described herein, there may be two or more fermentation broth or aqueous streams. An extractant phase that has absorbed product alcohol from a first aqueous stream may be brought into contact with a second aqueous stream that contains less product alcohol than the first aqueous stream or fermentation broth, enabling the transfer of product alcohol from the rich extractant phase to the second aqueous phase. In some embodiments, contacting the rich extractant with a dilute aqueous stream may take place in a multi-stage contacting device or in a static mixer followed by a settler. In some embodiments, contacting the rich extractant with a dilute aqueous stream may take place in the same device where lean extractant is contacted with fermentation broth. An extractor with perforated baffles would allow downflow of both fermentation broth and a dilute aqueous stream in separate compartments while an extractant that is lean in product alcohol may form a continuous phase throughout all compartments. An advantage of this configuration is a reduced amount of extractant would be needed in the production plant if the extractant remains confined to the closed volume of an extractor. Another advantage of this configuration is that the extractant is not subjected to potential degradation during distillation and therefore, may exhibit a longer service life. By transferring product alcohol to a homogeneous aqueous stream, the product alcohol may be conducted to more than one stripping column via partitioning of the dilute aqueous stream, taking into consideration column capacities and heat integration. The need to clean equipment that is exposed to an extractant may be reduced when product alcohol is extracted into an aqueous medium during or immediately after the product alcohol is extracted from fermentation broth. [00186] In some embodiments, product alcohol may be transferred from fermentation broth to a second aqueous stream or an extractant across a barrier that is selective for product alcohol transport. In some embodiments, this barrier may be provided by a membrane material. The membrane material may be either organic or inorganic. Examples of membrane material include polymers and ceramics. In some embodiments, product alcohol may be separated from fermentation broth utilizing a hydrogel. In some embodiments, the hydrogel may comprise functional elements that promote interaction with a product alcohol such as, but not limited to, hydroxyl functionality, hydrocarbon character, network size, and the like. In some embodiments, a hydrogel may comprise a polymeric network structure or polymer formulations. Examples of polymer formulations include, but are not limited to, one or more of the following: acrylic acid, sodium acrylate, hydroxyethyl acrylate, methacrylate, hydroxybutyl acrylate, butylacrylate, vinylated polyethylene oxide, vinylated polypropylene oxide, vinylated polytetratmethylene oxide, acrylates and diacrylates of polyglycols, polyvinyl alcohol and hydrocarbon derivatized polyvinyl alcohol, and styrene and styrene derivatives. In some embodiments, the hydrogel may comprise hydroxyethyl acrylate and methacrylate, hydroxybutyl acrylate and methacrylate, or butylacrylate and methacrylate.

[00187] In other embodiments of the processes and systems described herein, fermentation broth may be removed from the bottom of the fermentor at above atmospheric pressure and passed through a first flash tank operating at atmospheric pressure to release dissolved gases such as CO ₂ . This first flash tank may be a degassing cyclone and the vapors from this first flash tank may be combined with vapors from the fermentor and directed to a scrubber. In some embodiments, the fermentation broth from the first flash tank may be passed through a second flash tank operating below atmospheric pressure to release more dissolved gases such as CO ₂ . This second flash tank may be a degassing cyclone and the vapors from this second flash tank may be re-compressed to atmospheric pressure, cooled, and partially condensed prior to being combined with vapors from the fermentor and being directed to a scrubber. The fermentation broth exiting this second flash tank may be pumped to an extraction column operating at above atmospheric pressure so that any remaining or newly formed dissolved gases will not lead to formation of a vapor phase in the extraction column.

[00188] In another embodiment of the processes and systems described herein, fermentation broth may be conducted to an extractor and contacted with extractant generating an aqueous stream and organic stream comprising extractant and product alcohol. This organic stream may be conducted to a flash tank (e.g., vacuum flash) for separation of product alcohol from extractant. In some embodiments, the extractant stream from the flash tank may be recycled to the extractor and/or the fermentor. In some embodiments, the organic stream may be conducted to a second extractor prior to the flash tank. This second extractor may be used to remove, for example, any residual water in the organic stream. The extractors may be siphons, decanters, centrifuges, gravity settlers, mixer-settlers, or combinations thereof. In some embodiments, the extractant may be an oil such as, but are not limited to, tallow, corn, canola, capric/caprylic triglycerides, castor, coconut, cottonseed, fish, jojoba, lard, linseed, neetsfoot, oiticica, palm, peanut, rapeseed, rice, safflower, soya, sunflower, tung, jatropha, and vegetable oil blends, or fatty acids derived therefrom.

0189] In some embodiments of the processes and systems described herein, automatic self-cleaning filtration may be used in these processes and systems. Fermentation broth may be removed from a fermentor and may be cooled using a cooler (e.g., an existing cooler in a fermentation production facility) before entering an automatic self-cleaning filter. Some particulates may be retained on the screen medium of the filter as clarified mash passes through the filter. Additional filters may be simultaneously undergoing backflush where a portion of the clarified mash flows back through the screen carrying the particulates with it, discharging a concentrated solids stream. In some embodiments, a portion of the clarified mash may enter the top of an extractor while an extractant is fed in the bottom of the extractor. The clarified mash and extractant may be brought into contact either passively by density differences or with the aid of mechanical motion (e.g., a Karr® column) by means commonly used in the art. In some embodiments, an organic liquid stream of extractant containing product alcohol emerges from the top of the extractor and an aqueous liquid stream of fermentation broth that has been at least partially depleted of product alcohol relative to clarified mash emerges from the bottom of the extractor. The aqueous liquid stream and concentrated solids stream may be combined and returned to the fermentor. The extractant stream rich in product alcohol may be heated in a heat exchanger that transfers heat from an extractant stream that is lean in product alcohol and that originates from the bottom of the extractor. After releasing some heat, the lean extractant may be further cooled with water in a heat exchanger to reach a temperature that is suitable for fermentation. Circulation of fermentation broth may include a pathway through a heat transfer device and mass transfer device enabling the removal of heat and product alcohol per pass through an external cooling loop. Moreover, in some embodiments, the rate of heat and product alcohol removal may be balanced with the rate of heat and product alcohol production during fermentation by adjusting the circulation flow through the external cooling loop, adjusting the flow of cooling fluid in a heat exchanger, and/or adjusting the flow of extractant.

[00190] In some embodiments of the processes and systems described herein, phase separation of extractant from fermentation broth may be enhanced by modifying the temperature and/or pH of the process. For example, the process may be operated at temperatures and/or pH that are different than the temperature and/or pH of the fermentor. In some embodiments, the process may be operated at a reduced pH as compared to the fermentor. In some embodiments, the process may be operated at a higher temperature as compared to the fermentor. In some embodiments, the process may be operated at a reduced pH and a higher temperature as compared to the fermentor. A higher temperature can increase the kinetics of mass transfer of product alcohol between the aqueous and organic phases and may increase the kinetics of coalescence for extractant droplets dispersed in the aqueous phase and for aqueous droplets dispersed in the organic phase. In some embodiments, the temperature inside an extractor containing fermentation broth and extractant may be increased by heating the fermentation broth and/or extractant entering the extractor. The fermentation broth may be heated either directly with injection of water vapor or steam or indirectly via a heat exchanger. In some embodiments, the extractant feeding the extractor may originate from distillation where its temperature may already be elevated. In some embodiments, the extractant may be cooled to a temperature higher than the fermentation temperature.

[00191] In some embodiments, a reduced pH can minimize the solubility and dispersibility of extractant in the aqueous broth phase. In some embodiments, the extractant may be a fatty acid with a known associated pKa value. In some embodiments, the pH of the fermentation broth may be reduced to below the pKa of the extractant such that the carboxylic acid groups of the fatty acid are substantially protonated. In some embodiments, the pH may be reduced by introducing CO ₂ gas into the fermentation broth or by injecting a small amount of liquid acids such as sulfuric acid or any other organic or inorganic acid into the fermentation broth. In some embodiments, the pH of the fermentation broth after separating from the extractant may be adjusted to the pH of fermentation. [00192] In some embodiments where the extractant phase is the continuous phase, the aqueous phase may be distributed or dispersed in the extractant phase. For example, fermentation broth comprising product alcohol may be conducted to an extractor (e.g., external extractor) via a distributor or dispersal device. In some embodiments, the distributor or dispersal device may be a nozzle such as a spray nozzle. In some embodiments, the distributor or dispersal device may be a spray tower. As an example, droplets of fermentation broth may be passed through extractant, and product alcohol is transferred to the extractant. Droplets of fermentation broth coalesce at the bottom of the extractor and may be returned to the fermentor. Extractant comprising product alcohol may be further processed for recovery of product alcohol as described herein. In addition, at the completion of fermentation, residual product alcohol in the fermentor may also be further processed for recovery of product alcohol. In some embodiments, the extractant phase may be countercurrent.

[00193] In some embodiments where the extractant phase is the continuous phase and the aqueous phase is the dispersed phase, mass transfer rates may be improved by using electrostatic spraying to disperse the aqueous phase in the extractant phase. In some embodiments, one or more spray nozzles may be utilized for electrostatic spraying. In some embodiments, the one or more spray nozzles may be an anode. In some embodiments, the one or more spray nozzles may be a cathode.

[00194] In some embodiments, extractor effluent may be used to enhance phase separation. For example, a portion of rich extractant (i.e., extractant rich in product alcohol) from the top of the extractor may be returned to the top of the extractor as reflux, and the remaining rich extractant may be further processed for recovery of product alcohol. Also, a portion of lean fermentation broth from the bottom of the extractor may be returned to the bottom of the extractor as reflux and the remaining lean fermentation broth may be returned to the fermentor. In another embodiment, rich extractant may exit the top of the extractor into a decanter and separated into a heavy phase and light phase. The heavy phase from the decanter may be conducted to the top of the extractor to enhance phase separation. The light phase from the decanter may be may be further processed for recovery of product alcohol.

[00195] In some embodiments of the processes and systems described herein, multiple pass extractant flow may be utilized for product alcohol recovery. For example, multiple fermentors and extractors may be used, where the fermentation cycle of each fermentor is at a different timepoint. Referring to Figure 1 1A as an example, fermentor 300 is at an earlier timepoint as compared to fermentor 400 which is at an earlier timepoint as compared to fermentor 500. Fermentation broth comprising product alcohol 302 from fermentor 300 may be contacted with extractant 307 in extractor 305, and product alcohol may be transferred to extractant generating product alcohol-rich extractant 309. Product alcohol-rich extractant 309 from extractor 305 may be conducted to extractor 405. Fermentation broth comprising product alcohol 402 from fermentor 400 may be conducted to extractor 405, producing product alcohol-rich extractant 409. Product alcohol-rich extractant 409 may be conducted to extractor 505. Fermentation broth comprising product alcohol 502 from fermentor 500 may be conducted to extractor 505. Product alcohol-rich extractant 509 from extractor 505 may be processed for recovery of product alcohol. Product alcohol-lean fermentation broth (304, 404, 504) may be returned to fermentors 300, 400, and 500, respectively. The number of fermentors and extractors may vary depending on the operational facility. A benefit of this process is, for example, the reduction in total extractant processing and the size of the extractor.

0196] In another embodiment of this example, there may be an additional fermentor F' and an additional extractor E' (Figure 1 IB). In this embodiment, when fermentor 500 (which is at a later timepoint compared to fermentors 300 and 400) has completed fermentation, fermentor 500 may be taken off-line, and in some embodiments, fermentor 500 may undergo sanitation and/or sterilization procedures such as clean-in-place (CIP) and sterilization-in- place (SIP) procedures. When fermentor 500 is taken off-line, fermentor F' may be brought on-line. In this embodiment, fermentor F' is at an earlier timepoint as compared to fermentor 300 which is at an earlier timepoint as compared to fermentor 400. Similar to the description for Figure 1 1A, fermentation broth comprising product alcohol F'-02 from fermentor F' may be contacted with extractant in extractor E', and product alcohol may be transferred to extractant generating product alcohol-rich extractant E'-09. Product alcohol- rich extractant E'-09 from extractor E' may be conducted to extractor 305. Fermentation broth comprising product alcohol 302 from fermentor 300 may be conducted to extractor 305, producing product alcohol-rich extractant 309. Product alcohol-rich extractant 309 may be conducted to extractor 405. Fermentation broth comprising product alcohol 402 from fermentor 400 may be conducted to extractor 405. Product alcohol-rich extractant 409 from extractor 405 may be processed for recovery of product alcohol. Product alcohol-lean fermentation broth (F'-04, 304, 404) may be returned to fermentors F', 300, and 400, respectively. In some embodiments, this process may be repeated for multiple cycles, for example, at least one, at least two, at least three, at least four, at least five, at least ten, at least fifteen, at least twenty, or more cycles. In some embodiments, the process of taking fermentors off-line and putting additional fermentors on-line may be manual or automated. A benefit of this process is reduced extractor flow to product recovery (e.g., distillation).

[00197] In some embodiments, an extractant may reduce the flashpoint (i.e., flammability) of the product alcohol. Flashpoint refers to the lowest temperature at which flame propagation occurs across the surface of a liquid. Flashpoint may be measured, for example, using the ASTM D93-02 method ("Standard Test Methods for Flash Point by Pensky- Martens Closed Tester"). Reduction of the flashpoint of the product alcohol can improve the safety conditions of an alcohol production plant, for example, by minimizing the fire hazard of the potentially flammable product alcohol. By improving safety conditions, the risk of injury is minimized as well as the risk of property damage and revenue loss. In some embodiments where inactivation of the microorganism is required, an extractant may improve the inactivation of the microorganism.

[00198] In some embodiments, the processes described herein may be integrated extraction fermentation processes using on-line, in-line, at-line, and/or real-time measurements, for example, of concentrations and other physical properties of the fermentation broth and extractant. These measurements may be used, for example, in feed-back loops to adjust and control the conditions of the fermentation and/or the conditions of the extractor. In some embodiments, the concentration of product alcohol and/or other metabolites and substrates in the fermentation broth may be measured using any suitable measurement device for on-line, in-line, at-line, and/or real-time measurements. In some embodiments, the measurement device may be one or more of the following: Fourier transform infrared spectroscope (FTIR), near-infrared spectroscope (NIR), Raman spectroscope, high pressure liquid chromatography (HPLC), viscometer, densitometer, tensiometer, droplet size analyzer, pH meter, dissolved oxygen (DO) probe, and the like. In some embodiments, off-gas venting from the fermentor may be analyzed, for example, by an in-line mass spectrometer. Measuring off-gas venting from the fermentor may be used as a means to identify species present in the fermentation reaction. The concentration of product alcohol and other metabolites and substrates dissolved in the extractant may also be measured using the techniques and devices described herein.

[00199] In some embodiments, measured inputs may be sent to a controller and/or control system, and conditions within the fermentor (temperature, pH, nutrients, enzyme and/or substrate concentration) may be varied to maintain a concentration, concentration profile, and/or conditions within the extractor (fermentation broth flow, fermentation broth to extractant flow, agitation rate, droplet size, temperature, pH, DO content). Similarly, conditions within the extractor may be varied to maintain a concentration and/or concentration profile within the fermentor. By utilizing such a control system, process parameters may be maintained in such a way to improve overall plant productivity and economic goals. In some embodiments, real-time control of fermentation may be achieved by variation of concentrations of components (e.g., sugars, enzymes, nutrients, and the like) in the fermentor, variation of conditions within the extractor, or both.

[00200] As an example of an isobutanol fermentation process, the efficiency of isobutanol extraction in a Karr® column is continuously changing as the concentrations of starch, sugars and isobutanol change in the fermentation broth. 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. Isobutanol concentrations in the extractant may be maximized resulting in more energy efficient distillation operations.

[00201] As part of a process control strategy, real-time measurements of isobutanol in the fermentation broth (e.g., column feed) may be coupled with real-time measurements of isobutanol in the extractant and in the lean fermentation broth. These measurements may be used to adjust the fermentation broth to extractant ratio (flows) to the extractor. The flexibility to match the rate of isobutanol extraction with the rate of isobutanol generation may allow the extractor to be operated efficiently throughout the extraction. In addition, by maintaining a high concentration of isobutanol in the extractant, the volumetric flow rate to the distillation columns can be minimized, resulting in an energy savings for distillation operations. Phase separation may also be monitored using real-time measurements, for example, by monitoring the rate of phase separation, extractant droplet size, and/or composition of fermentation broth. In some embodiments, phase separation may be monitored by conductivity measurements, dielectric measurements, viscoelastic measurements, or ultrasonic measurements. In some embodiments, an automated phase separation detection system may be used to monitor phase separation. This automated system may be used to adjust the flow rates of fermentation broth and extractant to and from the extractor and/or adjust the droplet size of extractant, for example, after mixing of fermentation broth and extractant. By using these real-time monitoring systems, clean phase separation of aqueous and organic phases may be accomplished.

[00202] As another example of process control strategy, droplet size may be measured using particle size analysis such as a process particle analyzer (JM Canty, Inc., Buffalo, NY), focused beam reflectance measurement (FBRM®), or particle vision and measurement (PVM®) technologies (Mettler-Toledo, LLC, Columbus OH). In some embodiments, these measurements may be real-time in situ particle system characterizations. By monitoring droplet size in real time, changes in droplet shape and dimensions may be detected and process steps may be adjusted to modify droplet size and enhance the rate of mass transfer. For example, droplet size may be used to monitor the amount of extractant in fermentation broth. Following phase separation, some extractant may be present in the fermentation broth, and in some embodiments where the fermentation broth is recycled to the fermentor, monitoring droplet size would provide a means to minimize the amount of extractant in the fermentation broth returning to the fermentor. If the amount of extractant in the fermentation broth is too high, then phase separation may be improved, for example, by adjusting the droplet size of extractant in the extractor and/or adjusting the flow rates of fermentation broth and extractant to the extractor. These adjustments in the process steps can minimize the amount of extractant in the fermentation broth, as well as minimize the amount of extractant in thin stillage and DDGS.

[00203] In one embodiment of this control strategy, isobutanol in the fermentation broth would not exceed a concentration or setpoint at which the concentration of isobutanol becomes deleterious to the microorganism. The isobutanol fermentation broth setpoint may be adjusted higher or lower as the fermentation progresses based upon the trajectory of the fermentation. For example, continuous comparison of the concentration of isobutanol in the fermentation broth to a setpoint concentration of isobutanol can be utilized to modify fermentation broth to extractant ratios or flow rates of fermentation broth and extractant to an extractor. To monitor isobutanol concentrations in the fermentation broth, in situ measurements of the fermentation broth may be performed using Fourier transform infrared spectroscopy (FTIR), near infrared spectroscopy (NIR), and/or Raman spectroscopy. In addition, measurements of the fermentor headspace may be performed using FTIR, Raman spectroscopy, and/or mass spectrometry.

[00204] In some embodiments, efficient extractor operation may occur close to the point of extractor flooding. The use of real-time process control that utilizes concentration data from inlet and outlet streams may allow the extractor to be operated reliably near the point of flooding. In some embodiments, real-time extractant monitoring may be used to detect the partitioning of by-products from the fermentation broth or contaminants into the extractant. By-products such as alcohols, lipids, oils, and other fermentation components may reduce the extraction efficiency of the extractant. Numerous process monitoring techniques may be applied to this measurement including, but are not limited to, Fourier transform infrared spectroscopy (FTIR), near infrared spectroscopy (NIR), high performance liquid chromatography (HPLC), and nuclear magnetic resonance (NMR). The analytical technique selected to monitor the extractant for the presence of by-products or contamination may be a different technique than employed for real-time alcohol determination. Real-time data may be used to trigger the remediation of contaminated extractant or the purge of contaminated extractant from the process. These techniques as well as gas chromatography (GC) and supercritical fluid chromatography (SFC) may also be utilized to monitor thermal breakdown of extractant.

[00205] Referring to Figure 12, the systems and processes of the present invention may include means for on-line, in-line, at-line, and/or real-time measurements (circles represent measurement devices and dotted lines represent feedback loops). Figure 12 is similar to Figure 9, except for the addition of measurement devices for on-line, in-line, at-line, and/or real-time measurements, and therefore will not be described in detail again.

[00206] As an example, on-line measurements of aqueous stream 22 may be utilized to monitor the concentration of fermentable carbon sources (e.g., polysaccharides), oil, and/or dissolved oxygen. For example, FTIR may be used to monitor the dispersion of oil in aqueous stream 22, and process imaging may be used to monitor the concentration and size of oil droplets in the aqueous stream 22. In some embodiments, on-line measurements of fermentation 30 may be utilized to monitor removal rates of product alcohol. Measurements of fermentable carbon sources, dissolved oxygen, product alcohol, and by-products may be used to adjust the removal rate of product alcohol in order to maintain a concentration of product alcohol in fermentation 30 that is tolerable to microorganisms. By maintaining a setpoint product alcohol concentration, product inhibition and toxicity may be minimized.

[00207] On-line measurements of stream 105 and stream 122 may be used to operate process control feedback loops. For example, the concentration of product alcohol in stream 105 may be used to control the flow rate of this stream to extractor 120; and the concentration of product alcohol in stream 122 may be used to control the flow rate of this stream to separation 130 and to set the ratio of fermentation broth to extractant. In addition, on-line measurements of stream 105 and stream 122 may also be utilized to establish realtime product alcohol mass balance. Process control feedback loops for extractor 120 and separation 130 may be used to monitor the quality of phase separation of extractant and fermentation broth. For example, on-line measurement devices may be used to detect the balance of the separation of extractant and fermentation broth, and feed rates of extractant and fermentation broth may be adjusted accordingly to improve phase separation. On-line devices such as optical devices may be used to detect the presence of a rag layer (e.g., mixture of oil, aqueous solution, and solids) in, for example, extractor 120, and the ratio of fermentation broth to extractant may be adjusted to minimize the formation of a rag layer. On-line measurements of stream 135 from separation 130 may be used to monitor the presence of fermentation broth in this stream, and the presence of fermentation broth in stream 135 may indicate poor phase separation. If the concentration of fermentation broth in stream 135 exceeds a certain setpoint, process changes such as flow rate adjustments or adjustments to the ratio of fermentation broth to extractant may be implemented to improve phase separation. In addition, the concentration of product alcohol in stream 135 may be used as a process control feedback loop to ensure efficient operation of separation 130.

[00208] As another example, on-line measurements of the concentration of product alcohol in stream 127 may be used to monitor extraction efficiency and to maintain a concentration of product alcohol in fermentation 30 that is tolerable to microorganisms. In addition, stream 127 may be monitored for the presence of extractant as a means to minimize the amount of extractant returning to fermentation 30. For example, spectroscopic and process imaging techniques may be used to monitor the presence of extractant in stream 127. Furthermore, a certain concentration of extractant in stream 127 may be maintained to improve extraction efficiency and phase separation. [00209] In another embodiment, stream 135 from separation 130 may be conducted to purification 150 for further processing including recovery of product alcohol and extractant 152. Extractant 152 may be conducted to extractor 120. On-line measurements may be used to monitor stream 152 for contaminants and degradation products. By monitoring stream 152, the potential for contamination of extractor 120 and fermentation 30 is minimized. If there is an increase in contaminants in stream 152, this stream may be further processed to remove these contaminants, for example, by absorption or chemical reaction.

[00210] During the extraction process, a rag layer may form at the interface of the aqueous and organic phases, and the rag layer, composed of solids and extractant (e.g., droplets of extractant), can accumulate and possibly interfere with phase separation. To mitigate the formation of rag layer, agitation of the aqueous and organic phases may be employed. For example, an impeller may be used to disperse the rag layer at the aqueous-organic interface. Also, fluid flow such as a recirculating loop or vibrations/oscillations may be used to disrupt rag formation. Figures 13A and 13B illustrate exemplary processes for mitigating formation of a rag layer. Figure 13A exemplifies the use of a static mixer in combination with an agitation unit downstream of the settler or decanter for the treatment of a rag layer, and Figure 13B exemplifies the use of a static mixer in combination with an agitation unit upstream of the settler or decanter for the treatment of a rag layer. In some embodiments, other devices such as coalescers or sonic agitation may be used to disperse the rag layer. In some embodiments, these devices may be integrated into the settler or decanter.

[00211] The processes and systems described herein may be conducted using batch, fed- batch, or continuous fermentation. Batch fermentation is a closed system in which the composition of the fermentation broth is established at the beginning of the fermentation and is not subjected to artificial alterations during the fermentation process. In some embodiments of batch fermentation, extractant may be added to the fermentor. In some embodiments, the volume of extractant may be about 20% to about 60% of the fermentor working volume.

[00212] Fed-batch fermentation is a variation of batch fermentation, in which substrates (e.g., fermentable sugars) are added in increments during the fermentation process. Fed-batch systems are useful when catabolite repression may inhibit the metabolism of the microorganism and where it is desirable to have limited amounts of substrate in the media. In some embodiments, concentrations of substrate and/or nutrients may be monitored during fermentation. In some embodiments, parameters such as pH, dissolved oxygen, and gases (e.g., CO ₂ ) may be monitored during fermentation. From these measurements, the rate or amount of substrate and/or nutrients addition may be determined. In some embodiments, as the level or amount of fermentation broth decreases during fermentation, additional mash may be added to the fermentor to maintain the level or amount of fermentation broth, for example, maintain the level or amount of fermentation broth at the initiation of the fermentation process. In some embodiments of fed-batch fermentation, extractant may be added to the fermentor.

[00213] Continuous fermentation is an open system where fermentation broth is added continuously to a fermentor and an amount of fermentation broth is removed for further processing (e.g., recovery of product alcohol). In some embodiments, addition and removal of fermentation broth may be simultaneous. In some embodiments, equal amounts of fermentation broth may be added and removed from the fermentor. In some embodiments of continuous fermentation, extractant may be added to the fermentor. In some embodiments, the volume of extractant may be about 3% to about 50% of the fermentor working volume. In some embodiments, the volume of extractant may be about 3% to about 20% of the fermentor working volume. In some embodiments, the volume of extractant may be about 3% to about 10% of the fermentor working volume.

[00214] In some embodiments of the processes and systems described herein, gas stripping may be used to remove product alcohol from the fermentation broth. Gas stripping may be performed by providing one or more gases such as air, nitrogen, or carbon dioxide to the fermentation broth, thereby forming a product alcohol-containing gas phase. For example, gas stripping may be performed by sparging one or more gases through the fermentation broth. In some embodiments, the gas may be provided by the fermentation reaction. As an example, carbon dioxide may be provided as a by-product of the metabolism of a fermentable carbon source by the microorganism. In some embodiments, gas stripping may be used concurrently with extractant to remove product alcohol from the fermentation broth. Product alcohol may be recovered from the product alcohol-containing gas phase using methods known in the art, such as using a chilled water trap to condense the product alcohol, or scrubbing the gas phase with a solvent. Recombinant microorganisms and biosynthetic pathways

[00215] While not wishing to be bound by theory, it is believed that the processes described herein are useful in conjunction with any microorganism capable of producing fermentation products including alcohol-producing microorganism, particularly recombinant microorganisms which produce alcohol at titers above their tolerance levels.

[00216] Alcohol-producing microorganisms are known in the art. For example, fermentative oxidation of methane by methane trophic bacteria (e.g., Melhyiosinus trichosporium) produces methanol, and the yeast strain CEN.PK113-7D (CBS 8340, the Centraal Buro voor Schimmelculture; van Dijken, et al., Enzyme Microb. Techno. 26:706- 714, 2000) produces ethanol. Recombinant microorganisms which produce alcohol are also known in the art (e.g., 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. Patent No. 5,514,583; U.S. Patent No. 5,712, 133; PCT Application Publication No. WO 1995/028476; Feldmann, et al., Appl. Microbiol. Biotechnol. 38: 354-361, 1992; Zhang, et al, Science 267:240-243, 1995; U.S. Patent Application Publication No. 2007/0031918 Al; U.S. Patent No. 7,223,575; U.S. Patent No. 7,741, 1 19; U.S. Patent No. 7,851, 188; U.S. Patent Application Publication No. 2009/0203099 Al ; U.S. Patent Application Publication No. 2009/0246846 Al; and PCT Application Publication No. WO 2010/075241, which are all herein incorporated by reference).

[00217] In addition, microorganisms may be modified using recombinant technologies to generate recombinant microorganisms capable of producing product alcohols such as ethanol and butanol. Microorganisms that may be recombinantly modified to produce a product alcohol via a biosynthetic pathway include members of the genera Clostridium, Zymomonas, Escherichia, Salmonella, Serratia, Erwinia, Klebsiella, Shigella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Schizosaccharomyces, Kluyveromyces, Yarrowia, Pichia, Candida, Hansenula, Issatchenkia, or Saccharomyces. In some embodiments, recombinant microorganisms may be selected from the group consisting of Escherichia coli, Lactobacillus plantarum, Kluyveromyces lactis, Kluyveromyces marxianus and Saccharomyces cerevisiae. In some embodiments, the recombinant microorganism is yeast. In some embodiments, the recombinant microorganism is crabtree-positive yeast selected from Saccharomyces, Zygosaccharomyces, Schizosaccharomyces, Dekkera, Torulopsis, Brettanomyces, and some species of Candida. Species of crabtree-positive yeast include, but are not limited to, Saccharomyces cerevisiae, Saccharomyces kiuyveri, Schizosaccharomyces pombe, Saccharomyces bayanus, Saccharomyces mikitae, Saccharomyces paradoxus, Zygosaccharomyces rouxii, and Candida glabrata.

[00218] Saccharomyces cerevisiae are known in the art and are available from a variety of sources including, but not limited to, American Type Culture Collection (Rockville, MD), Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre, LeSaffre, Gert Strand AB, Ferm Solutions, North American Bioproducts, Martrex, and Lallemand. Saccharomyces cerevisiae include, but are not limited to, BY4741, CEN.PK 113-7D, Ethanol Red® yeast, Ferm Pro™ yeast, Bio-Ferm® XR yeast, Gert Strand Prestige Batch Turbo alcohol yeast, Gert Strand Pot Distillers yeast, Gert Strand Distillers Turbo yeast, FerMax™ Green yeast, FerMax™ Gold yeast, Thermosacc® yeast, BG-1, PE-2, CAT-1, CBS7959, CBS7960, and CBS7961.

[00219] In some embodiments, the microorganism may be immobilized or encapsulated. For example, the microorganism may be immobilized or encapsulated using alginate, calcium alginate, or polyacrylamide gels, or through the induction of biofilm formation onto a variety of high surface area support matrices such as diatomite, celite, diatomaceous earth, silica gels, plastics, or resins. In some embodiments, ISPR may be used in combination with immobilized or encapsulated microorganisms. This combination may improve productivity such as specific volumetric productivity, metabolic rate, product alcohol yields, and tolerance to product alcohol. In addition, immobilization and encapsulation may minimize the effects of the process conditions such as shearing on the microorganisms.

[00220] The production of butanol utilizing fermentation, as well as microorganisms which produce butanol, is disclosed, for example, in U.S. Patent No. 7,851, 188, and U.S. Patent Application Publication Nos. 2007/0092957; 2007/0259410; 2007/0292927; 2008/0182308; 2008/0274525; 2009/0155870; 2009/0305363; and 2009/0305370, the entire contents of each are herein incorporated by reference. In some embodiments, the microorganism is engineered to contain a biosynthetic pathway. In some embodiments, the biosynthetic pathway is an engineered butanol biosynthetic pathway. In some embodiments, the biosynthetic pathway converts pyruvate to a fermentation product. In some embodiments, the biosynthetic pathway converts pyruvate as well as amino acids to a fermentation product. In some embodiments, at least one, at least two, at least three, or at least four polypeptides catalyzing substrate to product conversions of a pathway are encoded by heterologous polynucleotides in the microorganism. In some embodiments, all polypeptides catalyzing substrate to product conversions of a pathway are encoded by heterologous polynucleotides in the microorganism. In some embodiments, the polypeptide catalyzing the substrate to product conversions of acetolactate to 2,3-dihydroxyisovalerate and/or the polypeptide catalyzing the substrate to product conversion of isobutyraldehyde to isobutanol are capable of utilizing reduced nicotinamide adenine dinucleotide (NADH) as a cofactor.

Biosynthetic Pathways

[00221] Biosynthetic pathways for the production of isobutanol that may be used include those described in U.S. Patent No. 7,851,188, which is incorporated herein by reference. In one embodiment, the isobutanol biosynthetic pathway comprises the following substrate to product conversions:

a) pyruvate to acetolactate, which may be catalyzed, for example, by acetolactate synthase;

b) acetolactate to 2,3-dihydroxyisovalerate, which may be catalyzed, for example, by acetohydroxy acid reductoisomerase;

c) 2,3-dihydroxyisovalerate to a-ketoisovalerate, which may be catalyzed, for

example, by acetohydroxy acid dehydratase;

d) a-ketoisovalerate to isobutyraldehyde, which may be catalyzed, for example, by a branched-chain a-keto acid decarboxylase; and

e) isobutyraldehyde to isobutanol, which may be catalyzed, for example, by a

branched-chain alcohol dehydrogenase.

[00222] In another embodiment, the isobutanol biosynthetic pathway comprises the following substrate to product conversions:

a) pyruvate to acetolactate, which may be catalyzed, for example, by acetolactate synthase;

b) acetolactate to 2,3-dihydroxyisovalerate, which may be catalyzed, for example, by ketol-acid reductoisomerase; c) 2,3-dihydroxyisovalerate to a-ketoisovalerate, which may be catalyzed, for example, by dihydroxyacid dehydratase;

d) a-ketoisovalerate to valine, which may be catalyzed, for example, by transaminase or valine dehydrogenase;

e) valine to isobutylamine, which may be catalyzed, for example, by valine

decarboxylase;

f) isobutylamine to isobutyraldehyde, which may be catalyzed by, for example, omega transaminase; and

g) isobutyraldehyde to isobutanol, which may be catalyzed, for example, by a

branched-chain alcohol dehydrogenase.

[00223] In another embodiment, the isobutanol biosynthetic pathway comprises the following substrate to product conversions:

a) pyruvate to acetolactate, which may be catalyzed, for example, by acetolactate synthase;

b) acetolactate to 2,3-dihydroxyisovalerate, which may be catalyzed, for example, by acetohydroxy acid reductoisomerase;

c) 2,3-dihydroxyisovalerate to α-ketoisovalerate, which may be catalyzed, for example, by acetohydroxy acid dehydratase;

d) α-ketoisovalerate to isobutyryl-CoA, which may be catalyzed, for example, by branched-chain keto acid dehydrogenase;

e) isobutyryl-CoA to isobutyraldehyde, which may be catalyzed, for example, by acylating aldehyde dehydrogenase; and

f) isobutyraldehyde to isobutanol, which may be catalyzed, for example, by a branched-chain alcohol dehydrogenase.

[00224] Biosynthetic pathways for the production of 1-butanol that may be used include those described in U.S. Patent Application Publication No. 2008/0182308, which is incorporated herein by reference. In one embodiment, the 1-butanol biosynthetic pathway comprises the following substrate to product conversions:

a) acetyl-CoA to acetoacetyl-CoA, which may be catalyzed, for example, by acetyl- CoA acetyltransferase; b) acetoacetyl-CoA to 3-hydroxybutyryl-CoA, which may be catalyzed, for example, by 3-hydroxybutyryl-CoA dehydrogenase;

c) 3-hydroxybutyryl-CoA to crotonyl-CoA, which may be catalyzed, for example, by crotonase;

d) crotonyl-CoA to butyryl-CoA, which may be catalyzed, for example, by butyryl- CoA dehydrogenase;

e) butyryl-CoA to butyraldehyde, which may be catalyzed, for example, by

butyraldehyde dehydrogenase; and

f) butyraldehyde to 1-butanol, which may be catalyzed, for example, by butanol

dehydrogenase.

[00225] Biosynthetic pathways for the production of 2-butanol that may be used include those described in U.S. Patent Application Publication No. 2007/0259410 and U.S. Patent Application Publication No. 2009/0155870, which are incorporated herein by reference. In one embodiment, the 2-butanol biosynthetic pathway comprises the following substrate to product conversions:

a) pyruvate to alpha-acetolactate, which may be catalyzed, for example, by

acetolactate synthase;

b) alpha-acetolactate to acetoin, which may be catalyzed, for example, by acetolactate decarboxylase;

c) acetoin to 3-amino-2-butanol, which may be catalyzed, for example, acetonin

aminase;

d) 3-amino-2-butanol to 3-amino-2-butanol phosphate, which may be catalyzed, for example, by aminobutanol kinase;

e) 3-amino-2 -butanol phosphate to 2-butanone, which may be catalyzed, for example, by aminobutanol phosphate phosphorylase; and

f) 2-butanone to 2-butanol, which may be catalyzed, for example, by butanol

dehydrogenase.

[00226] In another embodiment, the 2-butanol biosynthetic pathway comprises the following substrate to product conversions: a) pyruvate to alpha-acetolactate, which may be catalyzed, for example, by acetolactate synthase;

b) alpha-acetolactate to acetoin, which may be catalyzed, for example, by acetolactate decarboxylase;

c) acetoin to 2,3-butanediol, which may be catalyzed, for example, by butanediol dehydrogenase;

d) 2,3-butanediol to 2-butanone, which may be catalyzed, for example, by dial

dehydratase; and

e) 2-butanone to 2-butanol, which may be catalyzed, for example, by butanol

dehydrogenase.

[00227] Biosynthetic pathways for the production of 2-butanone that may be used include those described in U.S. Patent Application Publication No. 2007/0259410 and U.S. Patent Application Publication No. 2009/0155870, which are incorporated herein by reference. In one embodiment, the 2-butanone biosynthetic pathway comprises the following substrate to product conversions:

a) pyruvate to alpha-acetolactate, which may be catalyzed, for example, by

acetolactate synthase;

b) alpha-acetolactate to acetoin, which may be catalyzed, for example, by acetolactate decarboxylase;

c) acetoin to 3-amino-2-butanol, which may be catalyzed, for example, acetonin

aminase;

d) 3-amino-2-butanol to 3-amino-2-butanol phosphate, which may be catalyzed, for example, by aminobutanol kinase; and

e) 3-amino-2 -butanol phosphate to 2-butanone, which may be catalyzed, for example, by aminobutanol phosphate phosphorylase.

[00228] In another embodiment, the 2-butanone biosynthetic pathway comprises the following substrate to product conversions:

a) pyruvate to alpha-acetolactate, which may be catalyzed, for example, by

acetolactate synthase; b) alpha-acetolactate to acetoin which may be catalyzed, for example, by acetolactate decarboxylase;

c) acetoin to 2,3-butanediol, which may be catalyzed, for example, by butanediol dehydrogenase; and

d) 2,3-butanediol to 2-butanone, which may be catalyzed, for example, by diol

dehydratase.

[00229] The terms "acetohydroxyacid synthase," "acetolactate synthase," and "acetolactate synthetase" (abbreviated "ALS") may be used interchangeably herein to refer to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of pyruvate to acetolactate and CO ₂ . Example acetolactate synthases are known by the EC number 2.2.1.6 (Enzyme Nomenclature 1992, Academic Press, San Diego). These unmodified enzymes are available from a number of sources, including, but not limited to, Bacillus subtilis (GenBank Nos: CAB15618 (SEQ ID NO: 1), Z99122 (SEQ ID NO: 2), NCBI (National Center for Biotechnology Information) amino acid sequence, NCBI nucleotide sequence, respectively), Klebsiella pneumoniae (GenBank Nos: AAA25079 (SEQ ID NO: 3), M73842 (SEQ ID NO: 4)), and Lactococcus lactis (GenBank Nos: AAA25161 (SEQ ID NO: 5), L16975 (SEQ ID NO: 6)).

[00230] The term "ketol-acid reductoisomerase" ("KARI"), "acetohydroxy acid isomeroreductase," and "acetohydroxy acid reductoisomerase" may be used interchangeably and refer to a polypeptide (or polypeptides) having enzyme activity that catalyzes the reaction of (S)-acetolactate to 2,3-dihydroxyisovalerate. Example KARI enzymes may be classified as EC number EC 1.1.1.86 (Enzyme Nomenclature 1992, Academic Press, San Diego), and are available from a vast array of microorganisms, including, but not limited to, Escherichia coli (GenBank Nos: NP_418222 (SEQ ID NO: 7), NC_000913 (SEQ ID NO: 8)), Saccharomyces cerevisiae (GenBank Nos: NP_013459 (SEQ ID NO: 9), NC_001 144 (SEQ ID NO: 10)), Methanococcus maripaludis (GenBank Nos: CAF30210 (SEQ ID NO: 1 1), BX957220 (SEQ ID NO: 12)), and Bacillus subtilis (GenBank Nos: CAB14789 (SEQ ID NO: 13), Z991 18 (SEQ ID NO: 14)). KARIs include Anaerostipes caccae KARI variants "K9G9" and "K9D3" (SEQ ID NOs: 15 and 16, respectively). Ketol-acid reductoisomerase (KARI) enzymes are described in U.S. Patent Application Publication Nos. 2008/0261230, 2009/0163376, and 2010/0197519, and PCT Application Publication No. WO/201 1/041415, which are incorporated herein by reference. Examples of KARIs disclosed therein are those from Lactococcus lactis, Vibrio cholera, Pseudomonas aeruginosa PAOl, and Pseudomonas fluorescens PF5 mutants In some embodiments, the KARI utilizes NADH. In some embodiments, the KARI utilizes reduced nicotinamide adenine dinucleotide phosphate (NADPH).

[00231] The term "acetohydroxy acid dehydratase" and "dihydroxyacid dehydratase" ("DHAD") refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion the conversion of 2,3-dihydroxyisovalerate to a-ketoisovalerate. Example acetohydroxy acid dehydratases are known by the EC number 4.2.1.9. Such enzymes are available from a vast array of microorganisms, including, but not limited to, Escherichia coli (GenBank Nos: YP_026248 (SEQ ID NO: 17), NC000913 (SEQ ID NO: 18)), Saccharomyces cerevisiae (GenBank Nos: NP_012550 (SEQ ID NO: 19), NC 001142 (SEQ ID NO: 20), M. maripaludis (GenBank Nos: CAF29874 (SEQ ID NO: 21), BX957219 (SEQ ID NO: 22)), B. subtilis (GenBank Nos: CAB14105 (SEQ ID NO: 23), Z99115 (SEQ ID NO: 24)), L. lactis, and N. crassa. U.S. Patent Application Publication No. 2010/0081154, and U.S. Patent No. 7,851, 188, which are incorporated herein by reference, describe dihydroxyacid dehydratases (DHADs), including a DHAD from Streptococcus mutans.

[00232] The term "branched-chain a-keto acid decarboxylase," "a-ketoacid decarboxylase," "a-ketoisovalerate decarboxylase," or "2-ketoisovalerate decarboxylase" ("KIVD") refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of a- ketoisovalerate to isobutyraldehyde and CO ₂ . Example branched-chain a-keto acid decarboxylases are known by the EC number 4.1.1.72 and are available from a number of sources, including, but not limited to, Lactococcus lactis (GenBank Nos: AAS49166 (SEQ ID NO: 25), AY548760 (SEQ ID NO: 26); CAG34226 (SEQ ID NO: 27), AJ746364 (SEQ ID NO: 28), Salmonella typhimurium (GenBank Nos: NP_461346 (SEQ ID NO: 29), NC_003197 (SEQ ID NO: 30)), Clostridium acetobutylicum (GenBank Nos: NP_149189 (SEQ ID NO: 31), NC_001988 (SEQ ID NO: 32)), M. caseolyticus (SEQ ID NO: 33), and L. grayi (SEQ ID NO: 34).

[00233] The term "branched-chain alcohol dehydrogenase" ("ADH") refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of isobutyraldehyde to isobutanol. Example branched-chain alcohol dehydrogenases are known by the EC number 1.1.1.265, but may also be classified under other alcohol dehydrogenases (specifically, EC 1.1.1.1 or 1.1.1.2). Alcohol dehydrogenases may be NADPH-dependent or NADH- dependent. Such enzymes are available from a number of sources, including, but not limited to, Saccharomyces cerevisiae (GenBank Nos: NP_010656 (SEQ ID NO: 35), NC_001136 (SEQ ID NO: 36), NP_014051 (SEQ ID NO: 37), NC_001145 (SEQ ID NO: 38)), Escherichia coli (GenBank Nos: NP_417484 (SEQ ID NO: 39), NC_000913 (SEQ ID NO: 40)), C. acetobutylicum (GenBank Nos: NP_349892 (SEQ ID NO: 41), NC_003030 (SEQ ID NO: 42); NPJ49891 (SEQ ID NO: 43), NC_003030 (SEQ ID NO: 44)). U.S. Patent Application Publication No. 2009/0269823 describes SadB, an alcohol dehydrogenase (ADH) from Achromobacter xylosoxidans. Alcohol dehydrogenases also include horse liver ADH and Beijerinkia indica ADH (as described by U.S. Patent Application Publication No. 201 1/0269199, which is incorporated herein by reference).

[00234] The term "butanol dehydrogenase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of isobutyraldehyde to isobutanol or the conversion of 2-butanone and 2-butanol. Butanol dehydrogenases are a subset of a broad family of alcohol dehydrogenases. Butanol dehydrogenase may be NAD- or NADP- dependent. The NAD-dependent enzymes are known as EC 1.1.1.1 and are available, for example, from Rhodococcus ruber (GenBank Nos: CAD36475, AJ491307). The NADP dependent enzymes are known as EC 1.1.1.2 and are available, for example, from Pyrococcus furiosus (GenBank Nos: AAC25556, AF013169). Additionally, a butanol dehydrogenase is available from Escherichia coli (GenBank Nos: NP 417484, NC_000913) and a cyclohexanol dehydrogenase is available from Acinetobacter sp. (GenBank Nos: AAG 10026, 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 cofactor. Butanol dehydrogenases are available from, for example, C. acetobutylicum (GenBank Nos: NP_149325, NC_001988; this enzyme possesses both aldehyde and alcohol dehydrogenase activity); NPJ49891, NC_003030; and NP_349892, NC_003030) and Escherichia coli (GenBank Nos: NP_417-484, NC_000913).

[00235] The term "branched-chain keto acid dehydrogenase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of a-ketoisovalerate to isobutyryl-CoA (isobutyryl-coenzyme A), typically using NAD ⁺ (nicotinamide adenine dinucleotide) as an electron acceptor. Example branched-chain keto acid dehydrogenases are known by the EC number 1.2.4.4. Such branched-chain keto acid dehydrogenases are comprised of four subunits and sequences from all subunits are available from a vast array of microorganisms, including, but not limited to, Bacillus subtilis (GenBank Nos: CAB14336 (SEQ ID NO: 45), Z991 16 (SEQ ID NO: 46); CAB14335 (SEQ ID NO: 47), Z99116 (SEQ ID NO: 48); CAB 14334 (SEQ ID NO: 49), Z991 16 (SEQ ID NO: 50); and CAB 14337 (SEQ ID NO: 51), Z991 16 (SEQ ID NO: 52)) and Pseudomonas putida (GenBank Nos: AAA65614 (SEQ ID NO: 53), M57613 (SEQ ID NO: 54); AAA65615 (SEQ ID NO: 55), M57613 (SEQ ID NO: 56); AAA65617 (SEQ ID NO: 57), M57613 (SEQ ID NO: 58); and AAA65618 (SEQ ID NO: 59), M57613 (SEQ ID NO: 60)).

[00236] The term "acylating aldehyde dehydrogenase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of isobutyryl-CoA to isobutyraldehyde, typically using either NADH or NADPH as an electron donor. Example acylating aldehyde dehydrogenases are known by the EC numbers 1.2.1.10 and 1.2.1.57. Such enzymes are available from multiple sources, including, but not limited to, Clostridium beijerinckii (GenBank Nos: AAD31841 (SEQ ID NO: 61), AF 157306 (SEQ ID NO: 62)), Clostridium acetobutylicum (GenBank Nos: NP_149325 (SEQ ID NO: 63), NC_001988 (SEQ ID NO: 64); NPJ49199 (SEQ ID NO: 65), NC_001988 (SEQ ID NO: 66)), Pseudomonas putida (GenBank Nos: AAA89106 (SEQ ID NO: 67), U13232 (SEQ ID NO: 68)), and Thermus thermophilus (GenBank Nos: YP_145486 (SEQ ID NO: 69), NC_006461 (SEQ ID NO: 70)).

[00237] The term "transaminase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of α-ketoisovalerate to L-valine, using either alanine or glutamate as an amine donor. Example transaminases are known by the EC numbers 2.6.1.42 and 2.6.1.66. Such enzymes are available from a number of sources. Examples of sources for alanine-dependent enzymes include, but are not limited to, Escherichia coli (GenBank Nos: YP_026231 (SEQ ID NO: 71), NC_000913 (SEQ ID NO: 72)) and Bacillus licheniformis (GenBank Nos: YP_093743 (SEQ ID NO: 73), NC_006322 (SEQ ID NO: 74)). Examples of sources for glutamate-dependent enzymes include, but are not limited to, Escherichia coli (GenBank Nos: YP_026247 (SEQ ID NO: 75), NC_000913 (SEQ ID NO: 76)), Saccharomyces cerevisiae (GenBank Nos: NP_012682 (SEQ ID NO: 77), NC_001 142 (SEQ ID NO: 78)) and Methanobacterium thermoautotrophicum (GenBank Nos: NP_276546 (SEQ ID NO: 79), NC_000916 (SEQ ID NO: 80)).

[00238] The term "valine dehydrogenase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of α-ketoisovalerate to L-valine, typically using NAD(P)H as an electron donor and ammonia as an amine donor. Example valine dehydrogenases are known by the EC numbers 1.4.1.8 and 1.4.1.9 and such enzymes are available from a number of sources, including, but not limited to, Streptomyces coelicolor (GenBank Nos: NP_628270 (SEQ ID NO: 81), NC_003888 (SEQ ID NO: 82)) and Bacillus subtilis (GenBank Nos: CAB 14339 (SEQ ID NO: 83), Z991 16 (SEQ ID NO: 84)).

[00239] The term "valine decarboxylase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of L-valine to isobutylamine and CO ₂ . Example valine decarboxylases are known by the EC number 4.1.1.14. Such enzymes are found in Streptomyces, such as for example, Streptomyces viridifaciens (GenBank Nos: AAN10242 (SEQ ID NO: 85), AY116644 (SEQ ID NO: 86)).

[00240] The term "omega transaminase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of isobutylamine to isobutyraldehyde using a suitable amino acid as an amine donor. Example omega transaminases are known by the EC number 2.6.1.18 and are available from a number of sources, including, but not limited to, Alcaligenes denitrificans (AAP92672 (SEQ ID NO: 87), AY330220 (SEQ ID NO: 88)), Ralstonia eutropha (GenBank Nos: YP_294474 (SEQ ID NO: 89), NC_007347 (SEQ ID NO: 90)), Shewanella oneidensis (GenBank Nos: NP_719046 (SEQ ID NO: 91), NC_004347 (SEQ ID NO: 92)), and Pseudomonas putida (GenBank Nos: AAN66223 (SEQ ID NO: 93), AE016776 (SEQ ID NO: 94)).

[00241] The term "acetyl-CoA acetyltransferase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of two molecules of acetyl-CoA to acetoacetyl-CoA and coenzyme A (CoA). Example acetyl-CoA acetyltransferases are acetyl- CoA acetyltransferases with substrate preferences (reaction in the forward direction) for a short chain acyl-CoA and acetyl-CoA and are classified as E.C. 2.3.1.9 [Enzyme Nomenclature 1992, Academic Press, San Diego]; although, enzymes with a broader substrate range (E.C. 2.3.1.16) will be functional as well. Acetyl-CoA acetyltransferases are available from a number of sources, for example, Escherichia coli (GenBank Nos: NP_416728, NC_000913; NCBI amino acid sequence, NCBI nucleotide sequence), Clostridium acetobutylicum (GenBank Nos: NPJ49476.1, NC_003030; NP_149242, NC_001988, Bacillus subtilis (GenBank Nos: NP_390297, NC_000964), and Saccharomyces cerevisiae (GenBank Nos: NP_015297, NC_001 148). [00242] The term "3-hydroxybutyryl-CoA dehydrogenase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of acetoacetyl-CoA to 3- hydroxybutyryl-CoA. Example 3-hydroxybutyryl-CoA dehydrogenases may be NADH- dependent, with a substrate preference for (S)-3-hydroxybutyryl-CoA or (R)-3- hydroxybutyryl-CoA. Examples may be classified as E.C. 1.1.1.35 and E.C. 1.1.1.30, respectively. Additionally, 3-hydroxybutyryl-CoA dehydrogenases may be NADPH- dependent, with a substrate preference for (S)-3-hydroxybutyryl-CoA or (R)-3- hydroxybutyryl-CoA and are classified as E.C. 1.1.1.157 and E.C. 1.1.1.36, respectively. 3- Hydroxybutyryl-CoA dehydrogenases are available from a number of sources, for example, Clostridium acetobutylicum (GenBank Nos: NP_349314, NC_003030), Bacillus subtilis (GenBank Nos: AAB09614, U29084), Ralstonia eutropha (GenBank Nos: YP_294481, NC_007347), and Alcaligenes eutrophus (GenBank Nos: AAA21973, J04987).

[00243] The term "crotonase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of 3-hydroxybutyryl-CoA to crotonyl-CoA and Ι¾0. Example crotonases may have a substrate preference for (S)-3-hydroxybutyryl-CoA or (R)-3- hydroxybutyryl-CoA and may be classified as E.C. 4.2.1.17 and E.C. 4.2.1.55, respectively. Crotonases are available from a number of sources, for example, Escherichia coli (GenBank Nos: NP_41591 1, NC_000913), Clostridium acetobutylicum (GenBank Nos: NPJ49318, NC_003030), Bacillus subtilis (GenBank Nos: CAB13705, Z991 13), and Aeromonas caviae (GenBank Nos: BAA21816, D88825).

[00244] The term "butyryl-CoA dehydrogenase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of crotonyl-CoA to butyryl-CoA. Example butyryl-CoA dehydrogenases may be NADH-dependent, NADPH-dependent, or flavin-dependent and may be classified as E.C. 1.3.1.44, E.C. 1.3.1.38, and E.C. 1.3.99.2, respectively. Butyryl-CoA dehydrogenases are available from a number of sources, for example, Clostridium acetobutylicum (GenBank Nos: NP_347102, NC_ 003030), Euglena gracilis (GenBank Nos: Q5EU90, AY741582), Streptomyces collinus (GenBank Nos: AAA92890, U37135), and Streptomyces coelicolor (GenBank Nos: CAA22721, AL939127).

[00245] The term "butyraldehyde dehydrogenase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of butyryl-CoA to butyraldehyde, using NADH or NADPH as cofactor. Butyraldehyde dehydrogenases with a preference for NADH are known as E.C. 1.2.1.57 and are available from, for example, Clostridium beijerinckii (GenBank Nos: AAD31841, AF 157306) and Clostridium acetobutylicum (GenBank Nos: NP.sub.-149325, NC.sub.-001988).

[00246] The term "isobutyryl-CoA mutase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of butyryl-CoA to isobutyryl-CoA. This enzyme uses coenzyme B12 as cofactor. Example isobutyryl-CoA mutases are known by the EC number 5.4.99.13. These enzymes are found in a number of Streptomyces , including, but not limited to, Streptomyces cinnamonensis (GenBank Nos: AAC08713 (SEQ ID NO: 95), U67612 (SEQ ID NO: 96); CAB59633 (SEQ ID NO: 97), AJ246005 (SEQ ID NO: 98)), Streptomyces coelicolor (GenBank Nos: CAB70645 (SEQ ID NO: 99), AL939123 (SEQ ID NO: 100); CAB92663 (SEQ ID NO: 101), AL939121 (SEQ ID NO: 102)), and Streptomyces avermitilis (GenBank Nos: NP_824008 (SEQ ID NO: 103), NC_003155 (SEQ ID NO: 104); NP_824637 (SEQ ID NO: 105), NC_003155 (SEQ ID NO: 106)).

[00247] The term "acetolactate decarboxylase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of alpha-acetolactate to acetoin. Example acetolactate decarboxylases are known as EC 4.1.1.5 and are available, for example, from Bacillus subtilis (GenBank Nos: AAA22223, L04470), Klebsiella terrigena (GenBank Nos: AAA25054, L04507) and Klebsiella pneumoniae (GenBank Nos: AAU43774, AY722056).

[00248] The term "acetoin aminase" or "acetoin transaminase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of acetoin to 3-amino-2- butanol. Acetoin aminase may utilize the cofactor pyridoxal 5 '-phosphate or NADH or NADPH. The resulting product may have (R) or (S) stereochemistry at the 3 -position. The pyridoxal phosphate-dependent enzyme may use an amino acid such as alanine or glutamate as the amino donor. The NADH- and NADPH-dependent enzymes may use ammonia as a second substrate. A suitable example of an NADH-dependent acetoin aminase, also known as amino alcohol dehydrogenase, is described by Ito, et al. (U.S. Patent 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).

[00249] The term "acetoin kinase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of acetoin to phosphoacetoin. Acetoin kinase may utilize ATP (adenosine triphosphate) or phosphoenolpyruvate as the phosphate donor in the reaction. Enzymes that catalyze the analogous reaction on the similar substrate dihydroxyacetone, for example, include enzymes known as EC 2.7.1.29 (Garcia-Alles, et al, Biochemistry 43 : 13037-13046, 2004).

[00250] The term "acetoin phosphate aminase" refers to a polypeptide (or polypeptides) having enzyme 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 an amino acid such as alanine or glutamate. The NADH-dependent and NADPH-dependent enzymes may use ammonia as a second substrate. Although there are no reports of enzymes catalyzing this reaction on phosphoacetoin, there is a pyridoxal phosphate-dependent enzyme that is proposed to carry out the analogous reaction on the similar substrate serinol phosphate (Yasuta, et al, Appl. Environ. Microbial. 67:4999-5009, 2001).

[00251] The term "aminobutanol phosphate phospholyase," also called "amino alcohol diphosphate lyase," refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of 3-amino-2-butanol O-phosphate to 2-butanone. Amino butanol phosphate phospho-lyase may utilize the cofactor pyridoxal 5 '-phosphate. There are reports of enzymes that catalyze the analogous reaction on the similar substrate l-amino-2-propanol phosphate (Jones, et al, Biochem J. 134: 167-182, 1973). U.S. Patent Application Publication No. 2007/0259410 describes an aminobutanol phosphate phospho-lyase from the organism Erwinia carotovora.

[00252] The term "aminobutanol kinase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of 3-amino-2-butanol to 3-amino-2-butanol diphosphate. Amino butanol kinase may utilize ATP as the phosphate donor. Although there are no reports of enzymes catalyzing this reaction on 3-amino-2-butanol, there are reports of enzymes that catalyze the analogous reaction on the similar substrates ethanolamine and 1- amino-2-propanol (Jones, et al., supra). U.S. Patent Application Publication No. 2009/0155870 describes, in Example 14, an amino alcohol kinase of Erwinia carotovora subsp. Atroseptica.

[00253] The term "butanediol dehydrogenase" also known as "acetoin reductase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of acetoin to 2,3 -butanediol. Butanedial dehydrogenases are a subset of the broad family of alcohol dehydrogenases. Butanediol dehydrogenase enzymes may have specificity for production of (R)- or (S)-stereochemistry in the alcohol product. (S)-specific butanediol dehydrogenases are known as EC 1.1.1.76 and are available, for example, from Klebsiella pneumoniae (GenBank Nos: BBA13085, D86412). (R)-specific butanediol dehydrogenases are known as EC 1.1.1.4 and are available, for example, from Bacillus cereus (GenBank Nos. NP 830481, NC_004722; AAP07682, AE017000), and Lactococcus lactis (GenBank Nos. AAK04995, AE006323).

[00254] The term "butanediol dehydratase," also known as "dial dehydratase" or "propanediol dehydratase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of 2, 3 -butanediol to 2-butanone. Butanediol dehydratase may utilize the cofactor adenosyl cobalamin (also known as coenzyme Bw or vitamin B 12; although vitamin B 12 may refer also to other forms of cobalamin that are not coenzyme B 12). Adenosyl cobalamin-dependent enzymes are known as EC 4.2.1.28 and are available, for example, from Klebsiella oxytoca (GenBank Nos: AA08099 (alpha subunit), D45071 ; BAA08100 (beta subunit), D45071; and BBA08101 (gamma subunit), D45071 (all three subunits are required for activity), and Klebsiella pneumonia (GenBank Nos: AAC98384 (alpha subunit), AF102064; GenBank Nos: AAC98385 (beta subunit), AF102064, GenBank Nos: AAC98386 (gamma subunit), AF102064). Other suitable dial dehydratases include, but are not limited to, B12-dependent dial dehydratases available from Salmonella typhimurium (GenBank Nos: AAB84102 (large subunit), AF026270; GenBank Nos: AAB84103 (medium subunit), AF026270; GenBank Nos: AAB84104 (small subunit), AF026270); and Lactobacillus collinoides (GenBank Nos: CAC82541 (large subunit), AJ297723; GenBank Nos: CAC82542 (medium subunit); AJ297723; GenBank Nos: CAD01091 (small subunit), AJ297723); and enzymes from Lactobacillus brevis (particularly strains CNRZ 734 and CNRZ 735, Speranza, et al, J. Agric. Food Chem. 45:3476-3480, 1997), and nucleotide sequences that encode the corresponding enzymes. Methods of dial dehydratase gene isolation are well known in the art (e.g., U.S. Patent No. 5,686,276).

[00255] The term "pyruvate decarboxylase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the decarboxylation of pyruvic acid to acetaldehyde and carbon dioxide. Pyruvate dehydrogenases are known by the EC number 4.1.1.1. These enzymes are found in a number of yeast, including Saccharomyces cerevisiae (GenBank Nos: CAA97575 (SEQ ID NO: 107), CAA97705 (SEQ ID NO: 109), CAA97091 (SEQ ID NO: 1 11)).

[00256] It will be appreciated that microorganisms comprising an isobutanol biosynthetic pathway as provided herein may further comprise one or more additional modifications. U.S. Patent Application Publication No. 2009/0305363 (incorporated by reference) discloses increased conversion of pyruvate to acetolactate by engineering yeast for expression of a cytosol-localized acetolactate synthase and substantial elimination of pyruvate decarboxylase activity. In some embodiments, the microorganisms may comprise modifications to reduce glycero 1-3 -phosphate dehydrogenase activity and/or disruption in at least one gene encoding a polypeptide having pyruvate decarboxylase activity or a disruption in at least one gene encoding a regulatory element controlling pyruvate decarboxylase gene expression as described in U.S. Patent Application Publication No. 2009/0305363 (incorporated herein by reference), and/or modifications that provide for increased carbon flux through an Entner- Doudoroff Pathway or reducing equivalents balance as described in U.S. Patent Application Publication No. 2010/0120105 (incorporated herein by reference). Other modifications include integration of at least one polynucleotide encoding a polypeptide that catalyzes a step in a pyruvate-utilizing biosynthetic pathway. Other modifications include at least one deletion, mutation, and/or substitution in an endogenous polynucleotide encoding a polypeptide having acetolactate reductase activity. In some embodiments, the polypeptide having acetolactate reductase activity is YMR226C (SEQ ID NOs: 127, 128) of Saccharomyces cerevisiae or a homolog thereof. Additional modifications include a deletion, mutation, and/or substitution in an endogenous polynucleotide encoding a polypeptide having aldehyde dehydrogenase and/or aldehyde oxidase activity. In some embodiments, the polypeptide having aldehyde dehydrogenase activity is ALD6 from Saccharomyces cerevisiae or a homolog thereof. A genetic modification which has the effect of reducing glucose repression wherein the yeast production host cell is pdc- is described in U.S. Patent Application Publication No. 2011/0124060, incorporated herein by reference. In some embodiments, the pyruvate decarboxylase that is deleted or down-regulated is selected from the group consisting of: PDC1, PDC5, PDC6, and combinations thereof. In some embodiments, the pyruvate decarboxylase is selected from those enzymes in Table 1. In some embodiments, microorganisms may contain a deletion or down-regulation of a polynucleotide encoding a polypeptide that catalyzes the conversion of glyceraldehyde-3- phosphate to glycerate 1,3, bisphosphate. In some embodiments, the enzyme that catalyzes this reaction is glyceraldehyde-3 -phosphate dehydrogenase.

Table 1. SEQ ID Numbers of PDC Target Gene coding regions and Proteins

0257] In some embodiments, any particular nucleic acid molecule or polypeptide may be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence or polypeptide sequence described herein. The term "percent identity" as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. Identity and similarity can be readily calculated by known methods, including but not limited to those disclosed in: Computational Molecular Biology (Lesk, A. M., Ed.) Oxford University: NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.) Academic: NY (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., Eds.) Stockton: NY (1991).

[00258] Methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the MegAlign™ program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, WI). Multiple alignment of the sequences may be performed using the Clustal method of alignment which encompasses several varieties of the algorithm including the Clustal V method of alignment corresponding to the alignment method labeled Clustal V (disclosed by Higgins and Sharp, CABIOS. 5: 151-153, 1989;

Higgins, et al, Comput. Appl. Biosci. 8: 189-191, 1992) and found in the MegAlign™ program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). For multiple alignments, the default values correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of the sequences using the Clustal V program, it is possible to obtain a percent identity by viewing the sequence distances table in the same program. Additionally the Clustal W method of alignment is available and corresponds to the alignment method labeled Clustal W (Higgins and Sharp, CABIOS. 5: 151-153, 1989;

Higgins, et al, Comput. Appl. Biosci. 8: 189-191, 1992) and found in the MegAlign™ v6.1 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). Default parameters for multiple alignment (GAP PENALTY=10, GAP LENGTH PENALTY=0.2, Delay Divergen Seqs(%)=30, DNA Transition Weight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB ). After alignment of the sequences using the Clustal W program, it is possible to obtain a percent identity by viewing the sequence distances table in the same program.

[00259] Standard recombinant DNA and molecular cloning techniques are well known in the art and are described by Sambrook, et al. (Sambrook, J., Fritsch, E. F. and Maniatis, T. (Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989, here in referred to as Maniatis) and by Ausubel, et al. (Ausubel, et al, Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and Wiley- Interscience, 1987). Examples of methods to construct microorganisms that comprise a butanol biosynthetic pathway are disclosed, for example, in U.S. Patent No. 7,851, 188, and U.S. Patent Application Publication Nos. 2007/0092957; 2007/0259410; 2007/0292927; 2008/0182308; 2008/0274525; 2009/0155870; 2009/0305363; and 2009/0305370, the entire contents of each are herein incorporated by reference.

[00260] Further, while various embodiments of the present 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 persons skilled in the relevant art that various changes in form and detail can be made therein without departing from the spirit and scope of the invention. Thus, 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 claims and their equivalents.

[00261] 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 are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

EXAMPLES

[00262] The following nonlimiting examples will further illustrate the invention. It should be understood that, while the following examples involve corn as feedstock, other biomass sources such as cane may be used for feedstock without departing from the present invention. Moreover, while the following examples involve ethanol and butanol, other alcohols or fermentation products may be produced without departing from the present invention.

[00263] The meaning of abbreviations is as follows: "atm" means atmosphere, "ccm" means cubic centimeter(s) per minute, "g/L" means gram(s) per liter, "g" means gram(s), "gpl" means gram(s) per liter, "gpm" means gallon(s) per minute, "h" or "hr" means hour(s), "HPLC" means high performance liquid chromatography, "kg" means kilogram(s), "L" means liter(s), "min" means minute(s), "mL" means milliliter(s), "ppm" means parts per million, "psig" means pound(s) per square inch, gauge, and "wt%" means weight percent.

Example 1

Process for Production and Recovery of Butanol Produced by Fermentation

[00264] The processes described herein may be demonstrated using computational modeling such as Aspen modeling (see, e.g., U.S. Patent No. 7,666,282). For example, the commercial modeling software Aspen Plus® (Aspen Technology, Inc., Burlington, MA) may be used in conjunction with physical property databases such as DIPPR, available from American Institute of Chemical Engineers, Inc. (New York, NY) to develop an Aspen model for an integrated butanol fermentation, purification, and water management process. This process modeling can perform many fundamental engineering calculations, for example, mass and energy balances, vapor/liquid equilibrium, and reaction rate computations. In order to generate an Aspen model, information input may include, for example, experimental data, water content and composition of feedstock, temperature for mash cooking and flashing, saccharification conditions (e.g., enzyme feed, starch conversion, temperature, pressure), fermentation conditions (e.g., microorganism feed, glucose conversion, temperature, pressure), degassing conditions, solvent columns, pre-flash columns, condensers, evaporators, centrifuges, etc.

[00265] An Aspen model was developed with rigorous material and energy balance in which 53400 kg/h of corn was mashed and fermented to produce isobutanol and in which most of the isobutanol was extracted during fermentation and distilled. This model included an approximation of sequenced batch fermentations as continuous processes. An example of this fermentation, extraction, and distillation process is illustrated in Figure 14.

[00266] Liquefied corn mash 601 that was clarified to comprise 1.5 wt% suspended solids was pumped at 170.7 tonnes/h and 85°C through a heat exchanger and a water cooler and fed to fermentation 600 at 32°C. Vapor stream 602 was vented at 17.2 tonnes/h at atmospheric pressure from fermentation 600 to a scrubber with an average continuous molar composition of 95.8% carbon dioxide, 3.4% water, and 0.8% isobutanol. An average beer stream 603 comprising 12.6 gpl isobutanol was discharged continuously from fermentation 600 and preheated through a heat exchanger by the mash 601 prior to being distilled for isobutanol recovery.

[00267] Stream 604 with 3875 tonnes/h combined average flow is removed from fermentation 600 at an average isobutanol concentration of 11.1 gpl and an average temperature of 32°C and circulated through an extractor 610 for partial removal of isobutanol. The exiting aqueous broth 605 containing 7.9 gpl isobutanol is cooled by heat exchange with cooler tower water (CTW) to 30°C prior to re-entering fermentation 600. A solvent comprising diisopropylbenzene enters the extractor 610 and exits as stream 606 comprising 30.1 gpl isobutanol. The extractor 610 provides effectively five theoretical liquid-liquid equilibrium stages for contacting fermentation broth with solvent. Stream 606 passes at 340 tonnes/h through a heat exchanger and enters the middle of twelve theoretical stages of distillation column 620. A reboiler is operating at 0.6 atm and 183°C using 150 psig steam to produce solvent stream 607 comprising diisopropylbenzene and essentially no isobutanol that exchanges heat with solvent stream 606 through a heat exchanger and is further cooled by cooling water CTW prior to re-entering extractor 610. The overhead vapor of distillation column 620 is cooled CTW and condensed 630 to form 23.1 tonnes/h of reflux, 0.2 tonnes/h of a residual vapor off-gas 608, and 13.2 tonnes/h of product distillate 609 that comprises 99.2% isobutanol, 0.6% water, and 0.2% diisopropylbenzene.

Example 2

Process for Recovery of Ethanol Using an Extractant Column

[00268] A 1 " diameter Karr® extraction column (Koch Modular Process Systems, Paramus, NJ) was used to process fermentation broth that was produced during ethanol fermentation. The column contains a series of plates that run down the length of the column and which are attached to a central shaft. The shaft is attached to a drive which can move the perforated plates (1/4" diameter perforations) up and down in a reciprocating motion. The frequency of the motion was a variable during testing, but both the stroke length of the oscillation (0.75") and the spacing of the trays (2") were fixed. The column used had a plate stack height of 3000 mm.

[00269] The top of the column was provided with an aqueous feed consisting of

fermentation broth, while the bottom of the column was provided with a feed of corn oil fatty acids (COFA) as the extractant. The two feeds flowed countercurrent to one another through the column, and were collected as product at opposite ends of the column.

[00270] The fermentation broth was obtained using a fermentation protocol for production of ethanol from liquefied and saccharified corn mash from which, in some cases, some of the solids had been removed via centrifugation. In some cases, the extraction testing was done over the course of several days, such that a portion of the testing was done while CO ₂ off- gassing was at or near its maximum, while another portion was done when off-gassing had effectively stopped. The COFA used in this work was distilled grade from Emery

Oleochemicals (Cincinnati, OH).

[00271] Some experiments were run with COFA as the continuous phase in the column, while others were run with continuous aqueous phase. Experiments were also conducted with or without internals in place. Two types of internals were tested: stainless steel and polytetrafluoroethylene (PTFE). A range of flow rates were examined, in order to determine the flow regimes under which the column could be operated without flooding.

Impact of dynamic feed from fermentation

[00272] During the course of the testing, it was determined that in some cases the column performance varied as the fermentation progressed. Early in the fermentation, the fermentation broth comprising the feed is high in sugar, at intermediate times a considerable amount of CO ₂ (which can impact fluid flows) evolves from the fermentation broth, while at later times the concentration of ethanol in the fermentation broth is high. This temporal variation in the feed was reflected in variations in the capacity of the extraction column.

[00273] With conditions using PTFE plates and continuous COFA phase (no agitation), a difference was noted in performance when using fermentation broth collected when the fermentation was near the period of peak gas evolution ("intermediate broth") and toward the end of fermentation ("end broth"). Using end broth, a liquid throughput rate of 14 gpm/ft ² (Sample 3E) was achieved without flooding the column. The maximum throughput for intermediate broth that could be achieved prior to flooding was less than to 9 gpm/ft ² (Sample 4D), with noticeable differences in the size and appearance of the aqueous droplets. The droplet size of the aqueous phase was larger (with the formation of globules) in end broth as compared to intermediate broth.

Continuous Phase

0274] The maximum column throughput was also impacted by the nature of the continuous phase. For end of fermentation conditions, running with continuous aqueous phase and stainless steel (S. Steel) internals, a total liquid capacity of almost 14 gpm/ft ² was achieved (Sample 2B). For continuous organic phase and PTFE internals, the rate was less than 9 gpm/ft ² (Sample 4D). 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 Phase was continuous, Sample refers to run conditions, Internals refer to the material of the internals, Nom. AQ refers to the nominal aqueous flow rate, Nom. ORG refers to nominal organic flow rate, Total Flow (ccm) refers to the total flow of the aqueous and organic feeds, and Total Flow (gpm/ft ² ) refers to total flow per unit cross-sectional area.

Table 2

ORG 3G PTFE 180 60 240 11.6

ORG 3G PTFE 180 60 240 11.6

ORG 4A PTFE 110 70 180 8.7

ORG 4B PTFE 100 30 130 6.3

ORG 4C PTFE 100 60 160 7.7

ORG 4D PTFE 100 80 180 8.7

ORG 4E PTFE 85 60 145 7.0

ORG 4F PTFE 90 40 130 6.3

ORG 4G PTFE 70 40 110 5.3

ORG 4M PTFE 60 60 120 5.8

ORG 4N PTFE 80 80 160 7.7

[00275] When the column was operated without internals using feed comprised of

fermentation broth near the end of fermentation, the choice of the continuous phase affected the column capacity. For continuous aqueous phase, it was possible to operate at approximately 25 gpm/ft ² (Samples 2G and 2H). With continuous COFA phase, however, problems with flooding occurred at 18 gpm/ft ² (Sample 21). Results are shown in Table 3.

Table 3

Example 3

Effect of Fermentation Conditions on Extraction Column Capacity

[00276] The nature of fermentation broth is not static, but changes as the fermentation process progresses. In fermentation, the concentration of carbohydrates decreases as the carbohydrates are metabolized by microorganisms. This compositional change in the fermentation broth will alter physical parameters such as viscosity and surface tension of the fermentation broth, which have an effect on the extraction process. In addition to the changes in concentration, at intermediate times a considerable amount of CO ₂ is evolved; and this CO ₂ will impact the flow of the aqueous and organic liquids through the column.

[00277] A 1 " diameter glass Karr® extraction column (Koch Modular Process Systems, Paramus, NJ), outfitted with PTFE internals, was used to process fermentation broth from an ethanol fermentation. The processing was done at several timepoints during the course of the fermentation. Organic extractant (COFA) was the continuous phase in the column, with the fermentation broth passing through the column as droplets. Prior to the introduction of the fermentation broth to the column, the fermentation broth was passed through a tee in the line where CO ₂ bubbles present in the feed were removed through a vent.

[00278] With static internals (no agitation), a difference was noted in performance when using fermentation broth taken during the period of peak gas evolution ("intermediate broth") compared to broth taken toward the end of fermentation ("end broth"). Using intermediate broth, a liquid throughput rate of 14 gpm/ft ² was achieved. The maximum throughput (before column flooding) for the end broth was less than to 9 gpm/ft ² . There were noticeable differences in the size and appearance of the aqueous droplets. The droplet size of the aqueous phase was visibly larger for end broth as compared to intermediate broth.

Example 4

Effect of Isobutanol Concentration on Extraction Column Efficiency

[00279] During a typical fermentation process, the levels of product change with time. This dynamic concentration change can affect the mass transfer in an extraction process.

[00280] To demonstrate the effect of isobutanol concentration, a 1 " diameter glass Karr® extraction column (Koch Modular Process Systems, Paramus, NJ), outfitted with stainless steel internals, was used to process fermentation broth from a fermentation that contained approximately 3 g/L of isobutanol. The fermentation broth formed the continuous phase in the extractor, while the organic extractant (COFA) passed through the column as droplets. Although CO ₂ production had essentially ceased, the fermentation broth was passed through a tee in the line where any CO2 bubbles present in the feed were removed prior to the feed entering the extraction column. [00281] Samples of the feed and exiting streams were analyzed for isobutanol by liquid chromatography (LC) or gas chromatography (GC). Results are shown in Table 4. Mass balances were done, and the height of an equilibrium transfer stage (HETS) calculated using Kremser equations. For the two data points on as-is fermentation broth, the HETS values were 10 and 13 feet.

[00282] Isobutanol was then added to the fermentation broth to bring the concentration to 20 g/L. An extraction test was conducted and from the data, the HETS was found to be 18 feet. This value was some 50% higher than the values obtained on plain broth, and is in line with data obtained using thin stillage spiked with approximately 20 g/L isobutanol (see Figure 15).

Table 4

Example 5

ISPR Using an External Extraction Column

[00283] Fermentation broth from an isobutanol fermentation (10-liter scale) was circulated to a 5/8" diameter bench top Karr® column. The extraction solvent (COFA) was recycled from an extractant reservoir to the Karr® column. A control fermentation was run in which a volume of COFA was added to the fermentor to continuously extract isobutanol from the fermentation broth.

[00284] The Karr® column was run twice during the fermentation. The first run was at timepoint 4 to 7 hours of the fermentation and the second run was at timepoint 22 to 33 hours of the fermentation. Parameters such as p02 and pH were monitored for both fermentations. The measured p02 was lower for the run in which the Karr® column was used, as compared to the control run that did not use the Karr® column. Absolute pH values were similar for the Karr® column and the control, but the pH profiles were different for the two runs. The pH in the Karr® column run peaked early, flattened, then peaked again, versus a single gradual peak for the control.

[00285] Two aliquots of extraction solvent (1.8 liters each) were analyzed from the Karr® column. Samples were taken from each aliquot and analyzed for isobutanol content. The amount of isobutanol produced in the fermentation with the Karr® column was comparable to that produced in the control fermentation. The fermentation using the Karr® column produced a total of 82.4 grams isobutanol: approximately 34 grams were in 3.6 liters of organic phase and 48 grams in the aqueous phase. The control (30% by volume organic phase added to the fermentor) produced 90 g/L, 60 grams in 3 liters of organic phase and 30 grams in the aqueous phase. 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 a non-zero start of extraction in the Karr® column run. For the Karr® column at 22 hours, isobutanol was extracted from the fermentor more quickly than it was being produced. Glucose profiles were generated for the control and Karr® column. The profiles were similar, indicating cell growth and metabolism were comparable. Results are shown in Figures 16A and 16B. Brackets indicate the time points (4 to 7 hours and 22 to 33 hours) when the Karr® column was in operation.

Example 6

ISPR Using Mixer-Settler

[00286] An external mixer settler system was used to continuously remove isobutanol from an active fermentation broth containing a microorganism that produced isobutanol (i.e., isobutanologen). The study used approximately 100 liters of fermentation broth inoculated with an isobutanol-producing microorganism (i.e., isobutanologen). The contents of the fermentor were re-circulated from the fermentor through the mixer-settler extraction system. The extractant, comprising distilled COFA which contained no isobutanol, was used on a once-through basis.

[00287] Two static mixers were tested. The majority of the test used a Kenics® stainless steel static mixer (½" in diameter with 36 mixing elements). Between hours 12 and 24 of the run, a plastic mixer was used (StaMixCo HT-1 1-12.6-24, StaMixCo LLC, Brooklyn, NY). Fermentation broth and COFA were fed to opposite sides of a tee, from which the mixture flowed through the static mixer. The material exiting the static mixer was fed to the settler. The settler was made from a five-liter glass tank. A dip tube passed through the top of the settler, near the perimeter, and extended approximately halfway down the settler. The organic phase was withdrawn through a port at the top of the settler, while fermentation broth was removed from the bottom of the settler. The settler was fitted with an agitator that provided gentle mixing to the aqueous-organic interface in order to aid disengagement of the two liquid phases and thereby minimize accumulation of solids at the interface. Data collected during the run is presented in Table 5, and Figure 17 shows the isobutanol removal rates that were achieved during the course of the fermentation. As can be seen from the data, isobutanol levels in the aqueous broth remained relatively constant, indicating that isobutanol was removed from the fermentation broth at about the same rate as it is being produced. Referring to Table 5, Elapsed Time is time from start of fermentation, AQ Flow is aqueous feed flow, ORG flow is organic feed flow, iB in AQ feed is isobutanol in the aqueous feed, and iB in ORG product is isobutanol in the rich organic product.

Table 5

*A: ½" stainless Kenics® mixer, 32 elements

B: StaMixCo HT-1 1-12.6-24, plastic mixer

Example 7

On-Line, At-Line, and Real-Time Measurements

0288] A mash stream prepared from corn feedstock was conducted to a three-phase centrifuge generating three streams: mash, corn oil, and wet cake. On-line or at-line process measurements are employed, for example, to improve the recovery of starch/sugars and the quality of corn oil, and to maximize the amount of starch/sugars extracted from wet cake. Real-time measurements are used, for example, to control the addition of backset, cookwater, or water to slurry tanks to maintain a starch/sugar concentration set-point. The amount of starch/sugar extracted from the wet cake is maximized using the minimum amount of added water, and reducing the hydraulic load on the three-phase centrifuge.

[00289] Corn mash samples were analyzed using Fourier transform infrared spectroscopy (FTIR) with a diamond attenuated total reflectance (ATR) probe that allows for measurements in the presence of solids. The FTIR was calibrated by collecting spectra of standard samples in which total starch/sugar determinations using HPLC had been completed. The HPLC data was used to create a multivariate partial least squares (PLS) model for the FTIR. FTIR spectra were collected and a total starch concentration generated. Figure 18 illustrates the range of starch concentrations used to calibrate the FTIR.

[00290] Corn mash with an average starting concentration of 250 g/L was fed to a three- phase centrifuge. The subsequent wet cake was re-slurried and the concentration of starch was measured on two samples: 80 g/L and 70 g/L. This slurry was then separated using a three-phase centrifuge and the wet cake re-slurried. The starch concentration of this slurry was determined to be 28.9 g/L. Results are shown in Figure 19. These measurements were used to determine the correct amount of water to re-slurry the wet cake at each stage. Optimizing the water addition maximized the starch concentration and minimized the hydraulic load on the separation step. Moisture content of the wet cake was measured using near-infrared spectroscopy (NIR).

[00291] Corn oil quality is monitored in real-time and the data is used to control the three- phase centrifuge variables (e.g., feed rate, g forces, inlet flow rate, scroll speed). The quality of corn oil generated by the three-phase centrifuge was measured by monitoring the concentration of water carried into the corn oil during the separation. FTIR with a diamond ATR probe was used to collect corn oil spectra as it exited the three-phase centrifuge. The detection limit for water using the diamond ATR probe approach was approximately 500 ppm. Lower detection limits are achieved with the use of a flow cell with a longer effective path length.

[00292] Figure 20 contains a series of infrared spectra of corn oils that contain a range of water concentrations in excess of percent level concentrations down to 100's of ppm. Water concentration was determined using the -OH stretching region between 3700 cm-1 and 3050 cm-1. The data indicated that a process FTIR may be used to generate real-time water concentration in oil data. Real-time water concentration data may be used to control the process variables of the three-phase centrifuge (e.g., feed rate, g forces, inlet flow rate, scroll speed). The operation of the three-phase centrifuge may be controlled to yield the highest quality corn oil or to maximize throughput while not exceeding a water set point.

[00293] Real-time extractant monitoring was used to detect and monitor thermal breakdown of the extractant. Detection of these thermal breakdown products in real-time is used to trigger remediation of the extractant or purging of the contaminated extractant from the process.

[00294] Figure 21 is an example of the real-time measurement of isobutanol-rich COFA. The data was collected using a Metter-Toledo ReactIR™ 247 using a diamond ATR sampling probe in a flow cell. The COFA stream was collected from the outlet of a 1-inch diameter Karr® column and pumped to the FTIR using a peristaltic pump. The FTIR was calibrated by creating COFA standards spiked with isobutanol and generating a multivariate PLS model.

Example 8

Droplet size Analysis

[00295] This example describes the analysis of liquid extractant droplets after conducting a process stream containing fermentation broth and extractant (COFA) to a static mixer. A PVM® probe (Mettler-Toledo, LLC, Columbus OH) was inserted into the process stream approximately 24 hr after the process stream exited the static mixer. The PVM® probe was used to collect images every two minutes during a fermentation run. The images showed the presence of both COFA droplets ranging in size from 50 to 80 μιη in diameter and CO ₂ bubbles ranging in size from 200 and 400 μιη in diameter. Monitoring droplet size in the process stream containing fermentation broth and COFA after the static mixer is used to ensure that the droplets remain below a particular average diameter to ensure good mass transfer of isobutanol into the COFA droplets

[00296] The PVM® probe was also used to image the COFA droplets in the lean broth stream prior to return of the stream to the fermentor. The detection of COFA droplets in this stream is an indication of the amount of COFA returning to the fermentor. The PVM® probe was used to collect an image of the stream every two minutes during a fermentation. Unlike the stream exiting the static mixer, the lean broth stream had fewer and smaller droplets (10- 40 μηι). These measurements demonstrate the feasibility of using process imaging to monitor the amount of COFA returning to the fermentor.

[00297] Real-time average droplet size data from both sample points are used to monitor the phase separation of fermentation broth and COFA. An increase in the concentration or number of small COFA droplets detected in the lean fermentation broth recycle stream (after isobutanol extraction) can be an indicator that the phase separation of fermentation broth and COFA has degraded and too much COFA is exiting the extractor. To improve the quality of the phase separation and reduce the number or concentration of the COFA droplets returning to the fermentor in the lean broth stream, the average COFA droplet size is increased post static mixer.

[00298] Additional process variables that can impact average COFA droplet size include the concentration of polysaccharides in the fermentation broth, the ratio of fermentation broth to COFA, and total flow rate through the static mixer. As the fermentation progresses, flow rate and/or fermentation broth to COFA ratios may be changed to maintain a constant average COFA droplet size.

Example 9

Extractor Design

[00299] This example describes a method to design a large-scale extractor unit. Data from a pilot-scale extraction is used to estimate the size of the large-scale extractor unit. The effects of flow rate, agitation rate, and the presence or absence of internals on phase separation of the streams of the extractor unit from a pilot-scale extraction are determined. The total flow and ratio of fermentation broth flow to extractant flow is varied at fixed temperature over the course of the fermentation, and the conditions under which phase separation discontinues are observed. The maximum achievable flow to the extractor unit per square foot of extractor unit flow surface area is recorded. The following equation is used to determine flow per unit area:

Λ (Equation 1)

U = flow per unit area (gallons/minute/square foot)

F = total flow of fermentation broth and extractant to the extractor unit (gallons/minute) A = cross-sectional area in direction of flow (square feet) nD ¹

for an extraction column this is given by

4

D = column diameter (feet).

[00300] The diameter of a large-scale extractor unit is estimated by the expected flow of fermentation broth and extractant to the extractor unit using the following equation:

4 /7

D = ^{l e→cale} (Equation 2)

V πϋ

Fiarge-scaie ⁼ Total flow of fermentation broth and extractant to the large-scale extractor

(gallons/minute).

[00301] The height of the pilot-scale extractor unit is measured under different flow regimes including different flow rates, with and without internals present, different agitation rates, and at different concentrations of the product alcohol. Using this data, the number of theoretical stages achieved by the height of the extractor unit is estimated using the Kremser Equation (Seader and Henley, Separation Process Principles, 2 ^nd edition, John Wiley & Sons, 2006, pp. 358-359):

(Equation 3)

E = extraction factor = extrac t

Fbroth = flow of broth to the extractor unit (gallons/minute)

Fextractant = flow of extractant to the extractor unit (gallons/minute)

m = partition coefficient for product alcohol in fermentation broth and extractant phases (g/L per g/L)

Xf = concentration of product alcohol in fermentation broth feed (g/L) Xn = concentration of product alcohol in fermentation broth leaving the extractor unit

(g L)

Ys = concentration of product alcohol in extractant entering the extractor unit (g/L) n = number of theoretical stages achieved by the height of the extractor unit

Equation 3 is only valid when E≠ 1.

[00302] The height of a theoretical stage for the extractor unit is given by the height of the extraction column used in the pilot-scale extraction divided by the number of theoretical stages realized in a given experiment. The number of theoretical stages required to achieve the separation at large-scale is estimated using the operating conditions expected at large- scale in Equation 4:

(Equation 4) where ' indicates the condition of the large-scale extractor unit.

[00303] The product of the number of theoretical stages and height of a theoretical stage measured for similar flow conditions provides an estimate of the total height of the large- scale extractor unit. The flows and concentrations expected at a large-scale extractor unit are estimated using a dynamic fermentation model (e.g., Daugulis, et al, Biotech. Bioeng. 27: 1345-1356, 1985).

[00304] While various embodiments of the present 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 persons skilled in the relevant art that various changes in form and detail can be made therein without departing from the spirit and scope of the invention. Thus, 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. 0305] 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 are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.