MX2012014551A

MX2012014551A - Supplementation of fatty acids for improving alcohol productivity.

Info

Publication number: MX2012014551A
Application number: MX2012014551A
Authority: MX
Inventors: Robert Dicosimo; Michael Charles Grady; Ranjan Patnaik; Keith H Burlew
Original assignee: Butamax Tm Advanced Biofuels
Priority date: 2010-06-18
Filing date: 2011-06-17
Publication date: 2013-02-07
Also published as: BR112012032352A2; MX2012014784A; ZA201208372B; WO2011159962A1; EP2582826A2; MX2012014687A; CN103476936A; AU2011268215B2; WO2011159998A3; CA2801498A1; BR112012032166A2; EP2582827A2; AU2011268215A1; AU2011268169A1; KR20130116858A; CA2800542A1; ZA201208676B; EP2582825A1; NZ603036A; CN102947457B

Abstract

Fatty acids derived from biomass at a step in a fermentation process can be added to a fermentation medium comprising a recombinant microorganism that produces a product alcohol. At least one of growth rate and fermentable carbon consumption of the microorganism is greater in the presence of the fatty acids than the growth rate and the fermentable carbon consumption of the microorganism in the absence of the fatty acids. The addition of the fatty acids can increase glucose consumption, and can improve microorganism biomass production (cell growth/density) and growth rate, thereby reducing production time and increasing productivity of the fermentation process.

Description

SUPPLEMENTATION OF FATTY ACIDS TO IMPROVE THE PRODUCTIVITY OF ALCOHOL FIELD OF THE INVENTION The present invention relates to the production of fermentative alcohols, such as butanol and, particularly, to the processes of alcohol fermentation to achieve an improved alcohol productivity, wherein the fermentative growth of recombinant microorganisms occurs in the presence of derived fatty acids. of biomass in a stage of the fermentation process.

BACKGROUND OF THE INVENTION Alcohols have various applications in industry and science, such as beverages (ie, ethanol), fuels, reagents, solvents and antiseptics. For example, butanol is an alcohol that is an important industrial chemical with a variety of applications, including use as a fuel additive, such as. chemical substance used as a raw material in the plastics industry and as a food grade extractant in the food and flavor industry. Consequently, there is a high demand for alcohols, such as butanol, as well as more efficient production methods that do not harm the environment.

REF. : 237084 The production of alcohol, such as butanol, through fermentation by microorganisms is a production method that does not harm the environment. Microorganisms, such as yeasts, have been used for the production of alcohol products, where naturally occurring pyruvate is used as the initial substrate in the biosynthetic pathways. Butanol can be produced biologically as a byproduct of yeast fermentation, but the yield is typically very low. In order to improve the production of the desired products, such as butanol, the yeasts have been modified to express enzymes that alter the endogenous biosynthetic pathways or introduce new routes, and / or interrupt the expression of endogenous enzymes to alter the flow of metabolites. Introduced routes using cellular pyruvate as a substrate include routes, for example, for the production of butanol isomers. The disruption of pyruvate decarboxylase has been used to increase the availability of pyruvate for routes that produce the desired products, such as butanol. Additionally, recombinant microbial production hosts expressing a 1-butanol biosynthetic pathway (US Patent Application Publication No. 2008 / 0182308A1), a 2-butanol biosynthetic pathway (publication of the application for U.S. Patent Nos. 2007 / 0259410A1 and 2007/0292927) and an isobutanol biosynthetic route (U.S. Patent Application Publication No. 2007/0092957).

For example, the yeast Saccharomyces cerevisiae can be metabolically modified with switching mutations in the two primary genes of pyruvate decarboxylase (PDC). These genes, commonly called PDC1 and PDC5, produce enzymes that are directly related to the production of ethanol and the interruption of these genes has a negative impact on growth. The elimination of pyruvate decarboxylase and alcohol dehydrogenase (ADH) alters the biosynthetic pathway, resulting in lower production of fatty acids. Fatty acids are necessary for the formation of the cell wall and, therefore, for cell growth. The importance of fatty acids for cell growth is demonstrated, for example, in Otoguro, et al., (J. Biochem. 89: 523-529, 1981), which describes the effect of the antibiotic cerulenin, a known inhibitor of the synthesis of fatty acids, in cell growth. Cerulenin was added to a culture of S. cerevisiae that caused the inhibition of growth, but this was restored by adding oleic acid with certain saturated fatty acids (specifically, myristic acid, palmitic acid or pentadecanoic acid).

The metabolism of glucose in yeast generally follows a route of conversion of glucose into pyruvate, into acetyl-CoA, into cell mass. Consequently, there may be the conversion of pyruvate to acetaldehyde, in ethanol, or a conversion of acetaldehyde to acetyl-CoA, in the synthesis of fatty acids. With respect to recombinant microorganisms, a single PDC suppression reduces maximum growth but to a much lesser extent. When only one PDC gene is interrupted, the other PDC gene is active enough to allow the flow of carbon to acetaldehyde and, subsequently, ethanol and acetate. However, when a butanol product is desired, the production of ethanol reduces the yield of the butanol product in the substrate. The PDC genes are responsible for converting pyruvate to acetaldehyde, and the double mutation of PDCl and PDC5 prevents the production of acetaldehyde, thus altering the path to fatty acid biosynthesis and, therefore, inhibiting cell growth.

Therefore, there is a continuing need for methods for the production of fermentative alcohols by the use of recombinant microorganisms, wherein the growth rate and / or the biomass production of the microorganisms can be improved despite the reduction or elimination of the biosynthesis of fatty acids by the microorganism. The present invention provides other related advantages, as will be apparent from the description of the modalities included below.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to a method comprising: (a) providing a fermentation broth comprising a recombinant microorganism that produces an alcohol product from a fermentable carbon source, wherein the recombinant microorganism comprises a reduction or elimination of the pyruvate decarboxylase activity; (b) contacting the fermentation broth with a fermentable carbon source, so that the recombinant microorganism consumes the fermentable carbon source and produces the alcohol product; and (c) contacting the fermentation broth with fatty acids derived from biomass in a step of the fermentation process, wherein at least one of (i) the growth index or (ii) the fermentable carbon consumption of the recombinant microorganism it is higher in the presence of fatty acids than the rate of growth and / or fermentable carbon consumption of the recombinant microorganism in the absence of fatty acids. In a further embodiment, steps (b) and (c) occur, practically, simultaneously. In one embodiment, the fatty acids are selected from oleic acid, palmitic acid, myristic acid and mixtures thereof and, in another embodiment, the biomass is derived from corn grains, corn cobs, crop residues, such as corn husks. , corn stubble, herbs, wheat, rye, wheat straw, barley, barley straw, hay, rice straw, needle grass, waste paper, sugar cane bagasse, hum, sugar cane, soybean, components obtained of the grinding of grains, cellulosic material, lignocellulosic material, trees, branches, roots, leaves, wood chips, sawdust, bushes and shrubs, vegetables, fruits, flowers, manure, and mixtures thereof. In another embodiment, the fermentable carbon source is derived from biomass. In one embodiment, the alcohol product is butanol and, in another embodiment, the fermentation broth further comprises ethanol. In another embodiment, the recombinant microorganism has one or more deletions of the pyruvate decarboxylase (PDC) gene.

The present invention also relates to a method for producing an alcohol product; the method comprises: (a) providing biomass comprising a fermentable carbon source and oil; (b) converting at least a portion of the oil into fatty acids to form a biomass comprising the fatty acids; (c) contacting the biomass with a fermentation broth comprising a recombinant microorganism capable of producing an alcohol product from a fermentable carbon source, and wherein the recombinant microorganism comprises a reduction or elimination of the pyruvate decarboxylase activity; (d) contacting the fatty acids with the fermentation broth, wherein at least one of (i) the growth index and (ii) the fermentable carbon consumption of the recombinant microorganism is higher in the presence of the fatty acids than the growth rate and / or the fermentable carbon consumption of the recombinant microorganism in the absence of the fatty acids. In another modality, the stage (b) converting at least a portion of the oil to fatty acids comprises contacting the oil with one or more substances capable of hydrolyzing the oil portion into fatty acids. In one embodiment, the substance (s) comprises one or more enzymes and, in another embodiment, the enzyme (s) comprise lipase enzymes. In a further embodiment, before step (c), the enzyme (s) can be inactivated after at least a portion of the oil is hydrolyzed. In one embodiment, one or more of the steps (b), (c) and (d) occur in the fermentation vessel and, in another embodiment, one or more of steps (b), (c) and (d) occur, virtually, simultaneously. In one embodiment, the method further comprises the step of separating the oil from the biomass before step (b) of converting at least a portion of the oil into fatty acids. In another embodiment, the method further comprises: liquefying the biomass to produce a liquefied biomass, wherein the liquefied biomass comprises oligosaccharides; and contacting the liquefied biomass with a saccharification enzyme capable of converting the oligosaccharides into fermentable sugar to form a saccharified biomass, and wherein step (c) comprises contacting the saccharified biomass with the fermentation broth comprising a microorganism recombinant. In one embodiment, the fatty acids are selected from oleic acid, palmitic acid, myristic acid and mixtures thereof and, in another embodiment, the biomass is derived from corn grains, corn cobs, crop residues, such as corn husks. , corn stubble, herbs, wheat, rye, wheat straw, barley, barley straw, hay, rice straw, needle grass, waste paper, sugar cane bagasse, sorghum, sugar cane, soybean, components obtained of the grinding of grains, cellulosic material, lignocellulosic material, trees, branches, roots, leaves, wood chips, sawdust, bushes and shrubs, vegetables, fruits, flowers, manure, and mixtures thereof. In one embodiment, the method further comprises the step of fermenting a fermentable carbon source to produce the alcohol product. In one embodiment, the alcohol product is butanol and, in another embodiment, the fermentation broth further comprises ethanol. In another embodiment, the recombinant microorganism has one or more deletions of the pyruvate decarboxylase (PDC) gene.

Another method of the present invention includes a method for producing an alcohol product; the method comprises: (a) providing a raw material; (b) liquefying the raw material to create a suspension of raw material; (c) separating the raw material suspension to produce a product comprising (i) an aqueous layer comprising a fermentable carbon source, (ii) an oil layer and (iii) a solids layer; (d) obtaining an oil from the oil layer and converting at least a portion of the oil to fatty acids; (e) introducing the aqueous layer of (c) into a fermentation vessel containing a fermentation broth comprising a recombinant microorganism capable of producing an alcohol product from a fermentable carbon source, wherein the recombinant microorganism comprises a reduction or elimination of pyruvate decarboxylase activity; (f) fermenting the fermentable carbon source of the aqueous layer to produce the alcohol product; and (g) contacting the fermentation broth with the fatty acids, wherein at least one of (i) the growth index and (ii) the fermentable carbon consumption of the recombinant microorganism is higher in the presence of fatty acids than the growth rate and / or the fermentable carbon consumption of the recombinant microorganism in the absence of fatty acids. In another embodiment, step (d) of converting at least a portion of the oil to fatty acids comprises contacting the oil with one or more substances capable of hydrolyzing the oil portion into fatty acids. In a modality, the one or more substances comprise one or more enzymes and, in another embodiment, the enzyme or enzymes comprise lipase enzymes. In one embodiment, the fatty acids are selected from oleic acid, palmitic acid, myristic acid and mixtures thereof and, in another embodiment, the biomass is derived from corn grains, corn cobs, crop residues, such as corn husks. , corn stubble, herbs, wheat, rye, wheat straw, barley, barley straw, hay, rice straw, needle grass, waste paper, sugar cane bagasse, sorghum, sugar cane, soybean, components obtained of grain grinding, cellulosic material, lignocellulosic material, trees, branches, roots, leaves, wood chips, sawdust, bushes and shrubs, vegetables, fruits, flowers, manure, and mixtures thereof. In one embodiment, the alcohol product is butanol. In another embodiment, the recombinant microorganism has one or more deletions of the pyruvate decarboxylase (PDC) gene.

The present invention also relates to a composition comprising a recombinant microorganism, comprising a reduction or elimination of pyruvate decarboxylase and fatty acid activity.

The fatty acids (eg, oleic acid, palmitic acid and mixtures thereof) derived from biomass in a stage of the fermentation process may be added in a fermentation medium comprising a recombinant microorganism which produces an alcohol product. The microorganism can be yeast or another microorganism producing alcohol. In addition, the microorganism may have one or more deletions of the PDC gene and / or reduced or eliminated pyruvate decarboxylase activity. The addition of fatty acids can increase glucose consumption and improve biomass production (cell growth) and the growth rate of the microorganism. The improvement in the growth rate can reduce the production time and, thus, increase the productivity of the alcohol fermentation process.

In some embodiments, a method for producing an alcohol product in a fermentation process includes (a) providing a fermentation broth that includes a recombinant microorganism that produces an alcohol product from a fermentable carbon source; (b) contacting the fermentation broth with a fermentable carbon source so that the microorganism consumes the fermentable carbon source and produces the alcohol product; and (c) contacting the fermentation broth with fatty acids derived from biomass in a stage of the fermentation process, wherein at least one of (i) the growth rate and (ii) the fermentable carbon consumption of the microorganism is higher in the presence of fatty acids than the rate of growth and / or the fermentable carbon consumption of the microorganism in the absence of fatty acids.

In some embodiments, fatty acids are free fatty acids (FFAs). In some embodiments, the fatty acids include oleic acid. In some embodiments, the fatty acids include saturated fatty acids. In some embodiments, the fatty acids include palmitic acid. In some embodiments, the fatty acids include myristic acid.

In some embodiments, the alcohol product is butanol.

In some embodiments, the fermentable carbon source is derived from biomass. In some embodiments, the biomass includes corn and the fatty acids are fatty acids from corn oil.

In some embodiments, the fermentation broth also includes ethanol. In some embodiments, the method further includes contacting the fermentation broth with ethanol.

In some embodiments, the step of contacting the fermentation broth with fatty acids includes contacting triglycerides derived from biomass with one or more enzymes capable of hydrolyzing triglycerides to free fatty acids, so that the triglycerides are hydrolyzed into free fatty acids; and contacting the fermentation broth with the free fatty acids, wherein at least one of (i) the growth rate and (ii) the fermentable carbon consumption of the microorganism is greater in the presence of free fatty acids than in absence of these. In some embodiments, the enzyme or enzymes include lipase enzymes.

In some embodiments, the recombinant microorganism exhibits a single suppression of the pyruvate decarboxylase (PDC) gene. In some embodiments, the recombinant microorganism presents a double deletion of the PDC gene. In some embodiments, the recombinant microorganism has reduced or eliminated pyruvate decarboxylase activity.

In some embodiments, the concentration of fatty acids in the fermentation broth is not greater than about 0.8 g / 1.

In some embodiments, a method for producing an alcohol product from the fermentation biomass includes: (a) providing an aqueous biomass feed stream that includes water, fermentable carbon source and an amount of oil, wherein the source fermentable carbon and oil are derived from biomass; (b) hydrolyzing at least a portion of the oil in free fatty acids to form a biomass feed stream that includes the free fatty acids; (c) contacting the fermentation medium with the biomass feed stream in a fermentation vessel; the fermentation medium includes a recombinant microorganism that produces an alcohol product; and (d) fermenting the fermentable carbon source in the fermentation vessel to produce the alcohol product, wherein at least one of (i) the growth rate and (ii) the fermentable carbon consumption of the microorganism is greater in the presence of of free fatty acids that the rate of growth and / or the fermentable carbon consumption of the microorganism in the absence of free fatty acids.

In some embodiments, step (b) of hydrolyzing at least a portion of the oil in free fatty acids includes contacting the oil with a composition that includes one or more enzymes capable of hydrolyzing the oil portion into free fatty acids. In some embodiments, the method further comprises, prior to step (c), inactivating the enzyme (s) after hydrolysing at least a portion of the oil.

In some embodiments, the feed stream of aqueous biomass is a liquefied paste formed from a non-fractionated ground grain. In some embodiments, the non-fractionated ground grain is corn and the oil is corn oil.

In some embodiments, a method for producing an alcohol product includes (a) providing biomass including glucose and oil, which includes an amount of triglycerides; (b) contacting the oil with a composition that includes one or more substances capable of converting the triglycerides into free fatty acids, so that at least a portion of the triglycerides in the oil are converted to free fatty acids; (c) contacting the biomass with a fermentation broth that includes a microorganism capable of converting the glucose into an alcohol product, to produce an alcohol product; and (d) contacting the free fatty acids with the fermentation broth, wherein at least one of (i) the growth rate and (ii) the glucose consumption of the microorganism is higher in the presence of the free fatty acids than the growth rate and / or glucose consumption of the microorganism in the absence of free fatty acids.

In some embodiments, the method further comprises separating the oil from (a) from the biomass before step (b) of contacting the oil with one or more substances.

In some embodiments, step (b) of contacting the oil with a composition that includes one or more substances includes contacting the oil with one or more catalysts capable of hydrolyzing triglycerides to free fatty acids.

In some embodiments, step (b) of contacting the oil with a composition that includes one or more substances includes contacting the oil with one or more reactants or solvents capable of reacting the triglycerides chemically to obtain a reaction product. which includes free fatty acids.

In some embodiments, a method for producing butanol includes (a) providing biomass, which includes starch and oil, wherein the oil includes an amount of glycerides; (b) liquefying the biomass to produce a liquefied biomass, wherein the liquefied biomass includes oligosaccharides hydrolyzed from starch; (c) contacting the biomass of step (a) or the liquefied biomass of step (b) with a composition that includes one or more enzymes capable of converting glycerides to free fatty acids, so that at least a portion of the glycerides in the oil are converted into free fatty acids; (d) contacting the liquefied biomass with a saccharification enzyme capable of converting the oligosaccharides to fermentable sugar, which includes monomeric glucose; (e) contacting the liquefied biomass with a recombinant microorganism capable of converting the fermentable sugar into butanol to produce butanol; and (f) contacting the free fatty acids with the recombinant microorganism, wherein at least one of (i) the growth rate and (ii) the glucose consumption of the recombinant microorganism is higher in the presence of the free fatty acids than the rate of growth and / or glucose consumption of the recombinant microorganism in the absence of free fatty acids.

In some embodiments, a fermentation process to produce an alcohol product from raw material includes: (a) liquefying the raw material to create a suspension of raw material; (b) centrifuging the raw material suspension to produce a centrifugation product that includes (i) an aqueous layer that includes glucose, (ii) an oil layer that includes glycerides, and (iii) a layer of solids.; (c) hydrolyzing at least a portion of the glycerides in free fatty acids; (d) introducing the aqueous layer of (b) into a fermentation vessel containing a fermentation broth that includes a recombinant microorganism capable of producing an alcohol product from glucose; (e) fermenting the glucose from the aqueous layer to produce the alcohol product; and (f) contacting the fermentation broth with the free fatty acids, wherein at least one of (i) the growth rate and (ii) the glucose consumption of the microorganism is higher in the presence of the free fatty acids than the growth rate and / or glucose consumption of the microorganism in the absence of free fatty acids.

In some embodiments, the process for producing an alcohol product from a raw material includes, prior to the step of hydrolyzing the glycerides, introducing the glycerides into the fermentation vessel.

In some embodiments, a fermentation process includes (a) providing a fermentation broth that includes a recombinant microorganism that produces an alcohol product from a fermentable carbon source, a fermentable carbon source, an alcohol product and oil derivative of biomass, where the oil includes glycerides; (b) contacting the fermentation broth with a first extractant to form a two-phase mixture, which includes an aqueous phase and an organic phase, wherein the alcohol product and the oil are divided into the organic phase to form an organic phase containing the alcohol product; (c) separating the organic phase containing the alcohol product from the aqueous phase; (d) separating the alcohol product from the organic phase to produce a poor organic phase; (e) contacting the lean organic phase with a composition comprising one or more catalysts capable of hydrolyzing the glycerides to free fatty acids to produce a second extractant including at least a portion of the first extractant and free fatty acids; and (f) repeating step (b) by contacting the fermentation broth with the second extractant of step (e), wherein at least one of (i) the growth index and (ii) the consumption of The fermentable carbon of the microorganism is higher in the presence of the free fatty acids than the growth rate and / or the fermentable carbon consumption of the microorganism in the absence of the free fatty acids.

BRIEF DESCRIPTION OF THE FIGURES The appended figures, which are incorporated in the present invention and form part of the description, illustrate the present invention and, together with the description, serve, further, to explain the principles of this and to enable a person with experience in the art relevant, make and use the invention.

Figure 1 schematically illustrates an illustrative method and system of the present invention, wherein a liquefied biomass comes into contact with one or more substances for the hydrolysis of lipids and is introduced into a fermentation vessel.

Figure 2 illustrates schematically an illustrative method and system of the present invention, wherein a liquefied and saccharified biomass comes into contact with one or more substances for the hydrolysis of lipids and is introduced into a fermentation vessel.

Figure 3 schematically illustrates an illustrative method and system of the present invention, wherein undissolved solids and lipids are extracted from a liquefied biomass prior to fermentation, and wherein the extracted lipids are hydrolyzed to free fatty acids by the use of one or more substances, and the free fatty acids are introduced into a fermentation vessel.

Figure 4 illustrates schematically an illustrative method and system of the present invention, wherein the lipids derived from the native oil are hydrolyzed into free fatty acids by the use of one or more substances, and the free fatty acids are introduced into a container of fermentation Figure 5 illustrates schematically an illustrative method and system of the present invention, wherein the biomass lipids present in an extractant leaving a fermentation container are converted to free fatty acids which are introduced into a fermentation vessel.

Figure 6 is a graph illustrating the effect that the presence of fatty acids in a fermentation vessel has on glucose consumption for the butanologen strain NGCI-047.

Figure 7 is a graph illustrating the effect that the presence of fatty acids in a fermentation vessel has on glucose consumption for the butanologen strain NGCI-049.

Figure 8 is a graph illustrating the effect that the presence of fatty acids in a fermentation vessel has on glucose consumption for NYLA84 butanologen strain.

DETAILED DESCRIPTION OF THE INVENTION Unless otherwise defined, all scientific and technical terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In case of dispute, the present application will prevail along with the relevant definitions. In addition, unless otherwise required, terms in the singular will include pluralities and terms in the plural will include the singular. All publications, patents and other references mentioned in the present description are fully incorporated as a reference for all purposes.

To further define this invention, the following terms and definitions are provided.

As used in the present description, the terms "comprising", "comprising", "including", "including", "having", "having", "containing" or "containing" or any variation thereof , involve the inclusion of a whole number or group of integers mentioned, but not the exclusion of any other whole number 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 only to those elements, but may include others that are not expressly listed or are inherent. to the composition, mixture, process, method, article or apparatus. In addition, unless expressly specified otherwise, the disjunction is related to an "or" inclusive and not with an "or" excluding. By. For example, a condition A or B is satisfied by any of the following criteria: A is true (or current) and B is false (or not current), A is false (or not current) and B is true (or current), and both A and B are true (or current).

In addition, the indefinite articles "a" and "ones" that precede an element or component of the invention are intended to be non-restrictive with respect to the number of instances, i.e. occurrences of the element or component . Therefore, "a" or "ones" must be construed to include one or at least one, and the singular form of the word of the element or component includes, in addition, the plural, unless the number, obviously, indicate that it is singular.

The term "invention" or "present invention", as used in the present description, is a non-limiting term and is not intended to refer to any particular embodiment of the particular invention, but encompasses all possible modalities as described in request.

As used in the present description, the term "about", which modifies the amount of an ingredient or reactant employed in the invention, refers to the variation that may occur in the numerical amount, for example, through handling procedures. of liquids and typical measurement used to prepare concentrates or solutions for use in the real world; through inadvertent errors in these procedures; through differences in the manufacture, origin or purity of the ingredients used to prepare the compositions or carry out the methods; and similar. The term "approximately" 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 "approximately", the claims include equivalents for the quantities. In one embodiment, the term "approximately" means an amount within 10% of the numerical value reported, alternatively, within 5% of the numerical value reported.

"Biomass", as used in the present description, refers to the natural product that contains hydrolysable polysaccharides that provide fermentable sugars, which include any sugar and starch derived from natural resources such as corn, sugarcane, wheat, cellulose or lignocellulosic material, and materials comprising cellulose, hemicellulose, lignin, starch, oligosaccharides, disaccharides and / or monosaccharides, and mixtures thereof. The biomass may also comprise other components such as proteins and / or lipids. The biomass can be derived from a single source or can comprise a mixture derived from more than one source. For example, the biomass may comprise a mixture of corn cobs and corn stubble, or a mixture of grasses and leaves. Biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, papermaking sediments, organic waste, forest and forestry waste. Examples of biomass include, but are not limited to, corn grains, corn cobs, crop residues, such as corn husks, corn stubbles, herbs, wheat, rye, wheat s, barley, barley s, hay, sugar cane, rice s, needle grass, waste paper, sugarcane bagasse, sorghum, soybeans, components obtained from the grinding of grains, trees, branches, roots, leaves, wood chips, sawdust, bushes and shrubs, vegetables, fruits, flowers, manure and mixtures of these. For example, pasta, juice, molasses or hydrolyzate can be formed from biomass by any process known in the art for processing the biomass for fermentation purposes, such as by grinding, treatment and / or liquefaction, and comprises fermentable sugar and can understand water For example, cellulosic and / or lignocellulosic biomass can be processed to obtain a hydrolyzate containing fermentable sugars by any method known to a person skilled in the art. A pretreatment with low level of ammonia is described in the publication of the United States patent application no. 2007 / 0031918A1, which is incorporated by reference in the present description. Enzymatic saccharification of cellulosic and / or lignocellulosic biomass typically uses a set of enzymes to break down cellulose and hemicellulose to produce a hydrolyzate containing sugars, including glucose, xylose, and arabinose. (Suitable saccharification enzymes for cellulosic and / or lignocellulosic biomass are discussed in Lynd, et al (Microbiol, Mol, Biol. Rev. 66: 506-577, 2002).

The dough, the juice, the molasses or the hydrolyzate may include the raw material 12 and the raw material suspension 16, as described in the present invention. The aqueous feed stream can be derived or formed from biomass by any process known in the art for processing the biomass for fermentation purposes, such as by grinding, treatment and / or liquefaction, and comprises fermentable carbon substrate (e.g. sugar) and can comprise water. An aqueous feed stream may include raw material 12 and suspension of raw material 16, as described in the present invention.

"Biomass production", as used in the present description, refers to the production of microorganism biomass (ie production of cellular biomass or cell growth), such as occurs during the cultivation of microorganisms prior to fermentation or during the fermentative growth of microorganisms.

"Raw material", as used in the present description, refers to a supply in a fermentation process; the supply contains a fermentable carbon source with or without undissolved solids and, where appropriate, the supply contains the fermentable carbon source before or after the fermentable carbon source has been released from the starch or obtained from the decomposition of complex sugars by additional processing, such as by liquefaction, saccharification or other process. The raw material includes or is derived from biomass. Matters < Premiums include, but are not limited to, rye, wheat, corn, cane, barley, cellulosic material, lignocellulosic material or mixtures thereof.

"Fermentation broth", as used in the present description, means the mixture of water, sugars, dissolved solids, optionally microorganisms that produce alcohol, alcohol product and all other constituents of the material included in the fermentation vessel where The alcohol product is elaborated by the reaction of sugars with alcohol, water and carbon dioxide (C02) by means of the microorganisms present. Occasionally, as used in the present description, the term "fermentation medium" and "fermented mixture" can be used as synonyms for "fermentation broth".

"Fermentable carbon source" or "fermentable carbon substrate", as used in the present disclosure, refers to a carbon source capable of being metabolized (or "consumed") by the microorganisms described in the present description for production of fermentative alcohol. 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; C5 sugars, such as xylose and arabinose; substrates of a single carbon, which includes methane; and mixtures of these. The term "consumed", as used in the present description, includes processes by which compounds, for example, organic compounds, such as glucose, are decomposed by the action of the enzymes of a cell, resulting in the production of energy that the cell can use.

"Fermentable sugar", as used in the present description, refers to one or more sugars capable of being metabolized (or "consumed") by the microorganisms described in the present description for the production of fermentative alcohol.

"Fermentation vessel", as used in the present description, means the vessel in which the fermentation reaction is performed, by which the alcohol product, such as butanol, is made from sugars.

"Liquification vessel", as used in the present description, refers to the vessel in which the liquefaction is performed. Liquification is the process in which the oligosaccharides are released from the raw material. In some embodiments in which the raw material is corn, the oligosaccharides are released from the corn starch content during liquefaction.

"Saccharification vessel", as used in the present description, refers to the vessel in which the saccharification is performed (i.e., the decomposition of oligosaccharides into monosaccharides). In cases where fermentation and saccharification occur simultaneously, the saccharification vessel and the fermentation vessel may be the same.

"Sugar", as used in the present description, refers to oligosaccharides, disaccharides, monosaccharides and / or mixtures thereof. The term "saccharide" also includes carbohydrates, which include starches, dextrans, glycogens, cellulose, pentosans, as well as sugars.

As used herein, "saccharification enzyme" means one or more enzymes capable of hydrolyzing polysaccharides and / or oligosaccharides, for example, alpha-1, 4-glucoside glycogen or starch bonds. In addition, saccharification enzymes may include enzymes capable of hydrolyzing cellulosic or lignocellulosic materials.

"Non-dissolved solids", as used in the present description, means non-fermentable portions of raw material, eg, germ, fiber and gluten.

"Alcohol product", as used in the present description, refers to any alcohol that can be produced by a microorganism in a fermentation process that uses biomass as a source of fermentable carbon substrate. The alcohol products include, but are not limited to, Ci to C8 alkyl alcohols. In some embodiments, the alcohol products are C2 to C8 alkyl alcohols. In other embodiments, the alcohol products are C2 to C5 alkyl alcohols. It is preferred that the alkyl alcohols of Ci to Ce include, but are not limited to, methanol, ethanol, propanol, butanol and pentanol. In addition, the C2 to C8 alkyl alcohols include, but are not limited to, ethanol, propanol, butanol and pentanol. In addition, "alcohol" is used in the present description with reference to the alcohol product.

"Butanol", as used in the present description, specifically refers to the isomers of butanol 1-butanol (1-BuOH), 2-butanol (2-BuOH), tert-butanol and / or isobutanol (iBuOH O i- BuOH or I-BUOH, also known as 2-methyl-1-propanol), individually or as mixtures thereof. Occasionally, with reference to the butanol esters, the terms "butyl esters" and "butanol esters" may be used interchangeably.

"Propanol", as used in the present description, refers to the isomers of propanol isopropanol or 1-propanol.

"Pentanol", as used in the present description, refers to the isomers of pentanol 1-pentanol, 3-methyl-1-butanol, 2-methyl-1-butanol, 2, 2-dimethyl-1-propanol, 3 pentanol, 2-pentanol, 3-methyl-2-butanol or 2-methyl-2-butanol.

The term "alcohol equivalent", as used in the present description, refers to the weight of the alcohol that would be obtained by perfect hydrolysis of an alcohol ester and the subsequent recovery of the alcohol from an amount of alcohol ester.

The term "aqueous phase title", as used in the present disclosure, refers to the concentration of a particular alcohol (eg, butanol) in the fermentation broth.

The term "effective title", as used in the present description, refers to the total amount of a particular alcohol (e.g., butanol) produced by fermentation or alcohol equivalent of the alcohol ester produced by the esterification of alcohol per liter of fermentation medium. For example, the effective title of butanol in a volume unit of a fermentation includes: (i) the amount of butanol in the fermentation medium; (ii) the amount of butanol recovered from the organic extractant; (iii) the amount of butanol recovered from the gas phase, if degassing is used; and (iv) the alcohol equivalent of the butyl ester in the organic phase or in the aqueous phase.

"Extraction of product in situ (ISPR)", as used in the present description, means the selective extraction of a fermentation product specific to a biological process, such as fermentation, to control the concentration of the product. product in the biological process as the product is made.

"Extractant agent" or "ISPR extractant", as used in the present disclosure, refers to an organic solvent used to extract any alcohol product, such as butanol, or used to extract any ester of alcohol product produced by a catalyst from an alcohol product and a carboxylic acid or lipid. Occasionally, as used in the present description, the term "solvent" can be used as a synonym for "extractant agent". For the processes described in the present invention, the extractant agents are immiscible in water.

The terms "water-immiscible" or "insoluble" refer to a chemical component, such as an extractant or solvent, which is incapable of mixing with an aqueous solution, such as a fermentation broth, to form a single liquid phase.

The term "aqueous phase", as used in the present description, refers to the aqueous phase of a biphasic mixture obtained by contacting the fermentation broth with a water-immiscible organic extractant. In one embodiment of a process described in the present invention that includes extraction by fermentation, the term "fermentation broth" refers specifically to the aqueous phase in the extraction by biphasic fermentation.

The term "organic phase", as used in the present description, refers to the non-aqueous phase of a biphasic mixture obtained by contacting a fermentation broth with a water-immiscible organic extractant.

The term "fatty acid", as used in the present description, refers to a carboxylic acid (eg, aliphatic monocarboxylic acid) with C4 to C2e carbon atoms (more commonly, C12 to C24 carbon atoms), saturated or unsaturated. In addition, the fatty acids can be branched or unbranched. The fatty acids can be derived or included in esterified form in fat, oil or animal or vegetable wax. Fatty acids can occur naturally in the form of glycerides in fats and fatty oils, or can be obtained by hydrolysis of fats or synthesis. The term "fatty acid" can describe a single chemical species or a mixture of fatty acids. In addition, the term "fatty acid" encompasses free fatty acids.

The term "fatty alcohol", as used in the present description, refers to an alcohol with an aliphatic chain of C4 to C22 carbon atoms, saturated or unsaturated.

The term "fatty aldehyde", as used in the present description, refers to an aldehyde with an aliphatic chain of C4 to C22 carbon atoms, saturated or unsaturated.

The term "carboxylic acid", as used in the present description, refers to any organic compound with the general chemical formula -COOH, wherein a carbon atom is attached to an oxygen atom by a double bond to form a group carbonyl (-C = 0) and a hydroxyl group (-0H) by a single bond. The carboxylic acid may be in the form of a protonated carboxylic acid, in the form of a salt of a carboxylic acid (eg, ammonium, sodium or potassium salt), or as a mixture of protonated carboxylic acid and a salt of a carboxylic acid. The term "carboxylic acid" can describe a single chemical species (eg, oleic acid) or a mixture of carboxylic acids that can be produced, for example, by the hydrolysis of fatty acid esters derived from biomass or triglycerides, diglycerides, monoglycerides and phospholipids.

"Native oil", as used in the present description, refers to lipids obtained from plants (e.g., biomass) or animals. "Plant-origin oil", as used in the present description, refers to lipids obtained particularly from plants. Occasionally, "lipids" can be used as a synonym for "oil" and "acylglycerides". Native oils include, but are not limited to, tallow, corn, cañola, capric / caprylic triglycerides, castor, coconut, cottonseed, fish, jojoba, lardo, flax, oxtail, oiticica, palma, peanut, rapeseed, rice, safflower, soy, sunflower, tung, jatropha and mixtures of vegetable oils.

The term "separation", as used in the present description, is synonymous with "recovery" and refers to the extraction of a chemical compound from an initial mixture to obtain the compound in a higher purity or concentration than the purity or concentration of the product. composed in the initial mix.

As used in the present description, "recombinant microorganism" refers to microorganisms such as bacteria or yeast, which are modified by recombinant DNA techniques, for example, by modifying a host cell to comprise a biosynthetic path, such as a pathway. biosynthetic to produce an alcohol, such as butanol.

The present invention provides methods for producing an alcohol product (eg, fermentative alcohol) wherein the alcohol producing microorganisms in a fermentation vessel are contacted with fatty acids derived from native oil, such as biomass lipids, in a stage of the fermentation process. This supplementation of fatty acids during the fermentative growth of the microorganism can increase the fermentable carbon consumption of the microorganisms, the rate of growth and the production of biomass, particularly in relation to the recombinant microorganisms in which the pyruvate activity has been reduced or eliminated. decarboxylase.

In some embodiments, the glycerides in the oil can be chemically converted to fatty acids which are contacted with a fermentation broth, which includes a recombinant microorganism that produces an alcohol product from a fermentable carbon source. In other embodiments, the glycerides in the oil can be hydrolyzed catalytically (eg, enzymatically) in fatty acids that are contacted with a fermentation broth that includes a recombinant microorganism; In some embodiments, fatty acids can be obtained from the hydrolysis of lipids present in the biomass, which provides the fermentable carbon source for fermentation. The fatty acids can also be used as an ISPR extractant to extract the alcohol product from the fermentation broth.

Fatty acids can be saturated, monounsaturated, polyunsaturated, and mixtures of these. For example, oil with a naturally occurring fatty acid composition that includes a mixture of palmitic acid and oleic acid (eg, corn oil) can be hydrolyzed to produce a mixture of free oleic acid and free palmitic acid, which can be in contact with a fermentation broth in a fermentation vessel. In some embodiments, the concentration of carboxylic acid (such as fatty acid) in the fermentation vessel exceeds the solubility limit in the aqueous phase and results in the production of a two-phase fermentation mixture comprising an organic phase and a phase. watery In some embodiments, the concentration of carboxylic acids in the fermentation broth is typically less than about 0.8 g / 1 and is limited by the solubility of the carboxylic acid in the broth.

The growth rate and / or the fermentable carbon consumption of the microorganism is higher in the presence of fatty acids than the growth rate and the fermentable carbon consumption of the microorganism in the absence of fatty acids. Consequently, the supplementation of acid grades according to the methods of the present invention can achieve a higher cell concentration and a higher alcohol production than would be achieved without the supplementation of fatty acids. In some embodiments, the microorganism may be a microorganism producing butanol or other microorganism that typically requires supplementation of a 2-carbon substrate, e.g., ethanol, to survive and grow. In embodiments, fatty acid supplementation in accordance with the methods of the present invention may allow the 2-carbon dependent microorganisms to survive and grow without the supplementation of ethanol. In some modalities, microorganisms may be deficient in the production of acetyl-CoA from pyruvate. In some embodiments, the microorganism is metabolically modified with disruptive mutations in one or more pyruvate decarboxylase (PDC) genes, so that the route to fatty acid biosynthesis is modified. In some embodiments, the microorganism is metabolically modified with disruptive mutations in two PDC genes, such as PDC1 and PDC5, which results in the alteration of the pathway to fatty acid biosynthesis. Therefore, the methods of the present invention can achieve higher alcohol productivity by providing an optimal environment for the fermentative growth of recombinant microorganisms.

The present invention will be described with reference to the figures. Figure 1 shows an illustrative process flow diagram for the production of fermentative alcohol in accordance with one embodiment of the present invention. As shown, a raw material 12 can be introduced into an inlet of a liquefying vessel 10 and liquefied to produce a suspension of raw material 16. The raw material 12 contains hydrolysable starch which provides a fermentable carbon source (e.g., sugar fermentable such as glucose), and may be a biomass such as, but not limited to, rye, wheat, corn, cane, barley, cellulosic material, lignocellulosic material, or mixtures thereof, or it may be derived from a biomass. In some embodiments, the raw material 12 may be one or more components of a fractionated biomass and, in other embodiments, the raw material 12 may be a non-fractionated ground biomass. In some embodiments, the raw material 12 may be maize, such as ground unground corn kernels, and undissolved solids may include germ, fiber and gluten. The undissolved solids are non-fermentable portions of the raw material 12. For the purposes of the analysis of the present invention in relation to the embodiments shown in the figures, the raw material 12 will often be described as unfractionated ground corn, where the undissolved solids have not been separated from it. However, it should be understood that the illustrative methods and systems described in the present description can be modified for the different raw materials, whether fractioned or not, as is evident to a person skilled in the art. In some embodiments, the raw material 12 can be high oleic corn, so that the corn oil derived therefrom is a high oleic corn oil with an oleic acid content of at least about 55% by weight of oleic acid. In some embodiments, the content of oleic acid in high oleic corn oil can be up to 65% by weight relative to the content of oleic acid in conventional corn oil, which is about 24% by weight. High oleic oil may provide some advantages for use in the methods of the present invention, since hydrolysis of the oil provides free fatty acids with a high content of oleic acid to contact a fermentation broth. In some embodiments, the fatty acids or mixtures thereof comprise unsaturated fatty acids. The presence of unsaturated fatty acids reduces the melting temperature, which provides advantages for handling. Of the unsaturated fatty acids, those that are monounsaturated, that is, that have a carbon-carbon double bond, can provide advantages over the melting temperature without affecting the thermal and oxidative stability adequate according to the process considerations.

The process of liquefying the raw material 12 involves the hydrolysis of the polysaccharides in the raw material 12 into sugars, which include, for example, dextrins and oligosaccharides, and is a conventional process. Any known liquefaction process can be used, as well as the corresponding liquefaction vessel normally used in the industry including, but not limited to, acid process, acid-enzymatic process or enzymatic process. These processes can be used individually or in combination. In some embodiments, the enzymatic process may be used and a suitable enzyme 14, for example, alpha-amylase, is introduced into an inlet of the liquefaction vessel 10. In addition, water may be introduced into the liquefaction vessel 10. In some embodiments, it may be In addition, a saccharification enzyme, for example, glucoamylase, can be introduced into the liquefying vessel 10. In additional embodiments, a lipase can also be introduced into the liquification vessel 10 to catalyze the conversion of one or more components of the oil into free fatty acids.

The suspension of raw material 16 produced from the liquification of the raw material 12 includes sugar, oil 26 and undissolved solids derived from the biomass from which the raw material 12 was formed. In some embodiments, the oil is found in an amount of about 0% by weight to at least about 2% by weight of the composition of the fermentation broth. In some embodiments, the oil is in an amount of at least about 0.5% by weight of the raw material. The raw material suspension 16 can be discharged from an outlet of the liquefying vessel 10. In some embodiments, the raw material 12 is corn or corn kernels and, therefore, the raw material suspension 16 is a suspension of corn pulp. .

One or more substances 42 can be added to the suspension of raw material 16. The substances 42 are capable of hydrolyzing glycerides in the oil 26 in free fatty acids (FFA) 28. For example, when the raw is corn, oil 26 is corn oil constituent of the raw material and free fatty acids 28 are corn oil fatty acids (COFA). Therefore, after introducing the substances 42 into the raw material suspension 16, at least a portion of the glycerides in the oil 26 are hydrolyzed in FFA 28, which produces a suspension of raw material 18 with FFA 28.

In some embodiments, one or more substances 42 are one or more catalysts 42 capable of catalytically hydrolyzing the glycerides in the oil 26 in free fatty acids (FFA) 28. Therefore, after introducing the catalyst 42 in the suspension of raw material 16, at least a portion of the glycerides in the oil 26 are hydrolyzed in FFA 28, which produces a suspension of raw material 18 with FFA 28 and catalyst 42.

The acid / oil composition resulting from the hydrolysis of the oil 26 is typically at least about 17% by weight of FFA. In some embodiments, the acid / oil composition resulting from the hydrolysis of the oil 26 is at least about 20% by weight of FFA, at least about 25% by weight of FFA, at least about 30% by weight of FFA, at less about 35% by weight of FFA, at least about 40% by weight of FFA, at least about 45% by weight of FFA, at least about 50% by weight FFA, at least about 55% by weight of FFA, at least about 60% by weight of FFA, at least about 65% by weight of FFA, at least about 70% by weight of FFA, at least about 75% by weight of FFA, at least about 80% by weight of FFA, at least about 85% by weight of FFA, at least about 90% by weight of FFA, at least about 95% by weight of FFA, or at least about 99% by weight of FFA.

Alternatively, in some embodiments, the substance or substances 42 may constitute one or more reactants or solvents capable of chemically reacting the oil 26 in FFA 28 to bring it into contact with the recombinant microorganism 32. For example, the fatty acid grades of corn oil they can be synthesized from corn oil as oil 26 by base hydrolysis with NaOH and water as substances 42, as further described in the co-pending jointly-owned United States provisional application, no. of series 61 / 368,436, filed on July 28, 2010, and fully incorporated by reference in the present description. Further, for example, corn oil triglycerides as oil 26 can be reacted with aqueous ammonium hydroxide as reactant 42 to obtain fatty acids (and fatty amide) as further described in Roe, et al., (Am. Oil Chem. Soc. 29: 18-22, 1952), fully incorporated by reference in the present description. For the purposes of the analysis of the present invention in relation to the modalities shown in the figures, the substance (s) 42 will often be described as one or more catalysts as substances 42 for the hydrolysis of biomass lipids in FFA 28 supplemented during the fermentative growth of the recombinant microorganism 32. However, it should be understood that the illustrative methods and systems described in the present description can be modified so that the substances 42 are reactants and / or solvents capable of chemically converting the lipids of biomass into FFA 28.

In some embodiments, the catalyst 42 can be one or more enzymes, for example, hydrolase enzymes, such as lipase enzymes. The lipase enzymes used can 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 a Yarrowia strain. In a preferred aspect, the lipase source is selected from the group consisting of Absidia blakesleena, Absidia corymbifera, Achromobacter iophagus, Alcaligenes sp., Alternarla brassiciola, Aspergillus flavus, Aspergillus niger, Aureobasidium pullulans, Bacillus pumilus, Bacillus strearothermophilus, Bacillus subtilis, Brochothrix thermosohata, Candida cylindracea (Candida rugosa), Candida paralipolytica, Candida Antarctic lipase A, Candida antarctic lipase B, Candida ernobii, Candida deformans, Chromobacter viscosum, Coprinus cinerius, Fusarium oxysporum, Fusarium solani, Fusarium solani pisi, Fusariu roseum culmorum , Geotricum penicillatum, Anomalous Hansenula, Humicola brevispora, Humicola brevis var. thermoidea,. Humicola insolens, Lactobacillus curvatus, Rhizopus oryzae, Penicillium cyclopium, Penicillium crustosum, Penicillium expansum, 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 plantar! , Pseudomonas pseudoalcaligenes, Pseudomonas putida, Pseudomonas stutzeri and Pseudomonas wisconsinensis, Rhizoctonia solani, Rhizomucor miehei, Rhizopus japonicus, Rhizopus microsporus, Rhizopus nodosus, Rhodosporidium toruloides, Rhodotorula glutinis, Saccharomyces cerevisiae, Sporobolomyces shibatanus, Sus scrofa, Thermomyces lanuginosus. { previously Humicola lanuginose), Thiarosporella phaseolina, Trichoderma harzianum, Trichoderma reesei and Yarrowia lipolytica. In another preferred aspect, the lipase is selected from the group consisting of Thermomcyces lanuginosus, lipase from Aspergillus sp., Lipase from Aspergillus niger, lipase B from Candida antartica, lipase from Pseudomonas sp., Lipase from Penicillium roqueforti, lipase from Penicillium camembertii, lipase from Mucor javanicus, lipase from Burkholderia cepacia, lipase from Alcaligenes sp., lipase from Candida rugosa, lipase from Candida parapsilosis, lipase from Candida deformans, lipases A and B from Geotrichum candidum, lipase from Neurospora crassa, lipase from Nectria haematococca, lipase of Fusarium heterosporum, Rhizopus delemar lipase, Rhizomucor miehei lipase, Rhizopus arrhizus lipase and Rhizopus oryzae lipase. Commercially available lipase preparations suitable as enzyme catalyst 42 include, but are not limited to, Lipolase® 100 L, Lipex® 100L, Lipoclean® 2000T, Lipozyme® CALB L, Novozym® CALA L and Palatase 20000L, available from Novozymes, or of Pseudomonas fluorescens, Pseudomonas cepacia, Mucor miehei, porcine pancreas, Candida cylindracea, Rhizopus niveus, Candida antarctica, Rhizopus arrhizus or Aspergillus available from SigmaAldrich.

After hydrolysing at least a portion of the glycerides, in some embodiments, the catalyst 42 can be inactivated. Any method known in the art can be used to render the catalyst 42 inactive. For example, in some embodiments, the catalyst 42 can be inactivated by the application of heat and / or by adjusting the pH of the reaction mass to a pH at which the catalyst 42 is irreversibly inactivated, and / or by adding a chemical or biochemical species capable of selectively inactivating the activity of the catalyst. As shown, for example, in the embodiment of Figure 1, heat q is applied to the raw material suspension 18 so that the catalyst 42 becomes inactive. The application of heat q can be carried out in the suspension of raw material 18 before it is introduced into the fermentation vessel 30. The suspension of raw material 18 treated with heat (with the catalyst 42 inactive) is then introduced into a container of fermentation 30 together with the microorganism 32 to be included in a fermentation broth which is in the fermentation vessel 30. Alternatively, the raw material suspension 18 can be introduced into the fermentation vessel 30 and be subjected to heat while in the fermentation vessel 30. fermentation vessel, prior to inoculation of the fermentation vessel of the microorganism 32. For example, in some embodiments, the inactivation treatment of the catalyst can be carried out by heating the suspension of raw material 18 with heat to a temperature of at least about 75 °. C for at least about 5 minutes, at least about 75 ° C for at least about 10 minutes, at least about 75 ° C for at least about 15 minutes, at least about 80 ° C for at least about 5 minutes, at least about 80 ° C for at least about 10 minutes, at least about 80 ° C during at least about 15 minutes, at least about 85 ° C for at least about 5 minutes, at least about 85 ° C for at least about 10 minutes, or at least about 85 ° C for at least about 15 minutes. In some embodiments, after being subjected to the heat q, the raw material suspension 18 is cooled to a temperature suitable for fermentation before being introduced into the fermentation vessel 30 (or prior to inoculation of the fermentation vessel in case the application of heat q is carried out in the fermentation vessel). For example, in some embodiments, the temperature of the raw material suspension 18 is about 30 ° C before coming into contact with the fermentation broth.

The inactivation of the catalyst 42 is preferred when it is sought to prevent the catalyst 42 from esterifying the alcohol with fatty acids 28 in the fermentation vessel. In some embodiments, the production of alcohol ester is preferred by esterification of the alcohol product in a fermentation medium with an organic acid (eg, fatty acid) and a catalyst (eg, lipase), as further described in the joint and jointly owned United States Provisional Application, no. of series 61 / 368,429, filed on July 28, 2010; United States provisional application no. of series 61 / 379,546, filed on September 2, 2010; and United States provisional application no. of series 61 / 440,034, filed on February 7, 2011; fully incorporated as reference in the present description. For example, for the production of butanol, active catalyst 42 in the fermentation vessel (introduced by suspension 18) can catalyze the esterification of butanol with fatty acids 28 (introduced through suspension 18) to form butyl esters of fatty acids (FABE, for its acronym in English) in situ. In the embodiments where the fatty acid alcohol esters are preferred, the methods described in the present description can be modified to omit the inactivation of the catalyst 42 before contacting a fermentation broth including the alcohol product. Therefore, with reference to the illustrative flow diagrams of Figures 1-5, these alternative embodiments can be made by omitting the application of heat q in the process stream containing the catalyst 42, so that the catalyst 42 is esterified the alcohol product with the fatty acids 28 in the fermentation vessel 30. Furthermore, in some embodiments, the illustrative process flow diagrams of Figures 1-5 can be modified to omit the heat q if not necessary for the chemical conversion of oil 26 in FFA 28 by the use of one or more reactants or solvents such as substances 42, instead of catalysts 42.

The fermentation vessel 30 is configured to ferment the suspension 18 in order to produce an alcohol product, such as butanol. Particularly, the microorganism 32 metabolizes the fermentable sugar in suspension 18 and excretes an alcohol product. The microorganism 32 is selected from the group of bacteria, cyanobacteria, filamentous fungi and yeast. In some embodiments, the microorganism 32 can be a bacterium, such as E. coli. In some embodiments, the microorganism 32 can be a recombinant fermentative microorganism. The suspension may include sugar, for example, in the form of oligosaccharides and water, and may comprise less than about 20 g / 1 of monomeric glucose, more preferably less than about 10 g / 1 or less than about 5 g / 1 of monomeric glucose. The proper methodology for determining the amount of monomeric glucose is well known in the art. Suitable methods known in the art include high performance liquid chromatography (HPLC).

In some embodiments, suspension 18 is subjected to a saccharification process in order to decompose complex sugars (eg, oligosaccharides) in suspension 18 into monosaccharides that can be readily metabolized by microorganism 32. Any saccharification process can be used. It is routinely used in the industry, which includes, but is not limited to, acid process, acid-enzymatic process or the enzymatic process. In some embodiments, simultaneous saccharification and fermentation (SSF) can occur within the fermentation vessel 30, as shown in Figure 1. In some embodiments, an enzyme 38, such as glucoamylase, can be introduced into an entrance of the fermentation vessel 30 to decompose the starch or oligosaccharides into glucose capable of being metabolized by the microorganism 32.

Optionally, ethanol 33 may be provided in the fermentation vessel 30 to be included in the fermentation broth. In some embodiments, when a recombinant microorganism with a butanol biosynthetic pathway is used as the microorganism 32 for the production of butanol, the microorganism 32 may require the supplementation of a two carbon substrate (eg, ethanol) to survive and grow. Therefore, in some embodiments, ethanol 33 can be supplied in the fermentation vessel 30.

However, it has been determined that, surprisingly, the methods of the present invention, wherein there are free fatty acids (eg, FFA 28) in the fermentation vessel, can allow the reduction of the amount of ethanol 33 typically delivered by a recombinant microorganism determined without negatively affecting the vitality of the recombinant microorganism. Additionally, in some embodiments, the methods of the present invention demonstrate that the rate of production of alcohol (eg, butanol) without supplementation of ethanol is comparable to the production rate that can be achieved when it is supplemented with ethanol 33. As demonstrated additionally in the comparative examples presented in Examples 1-14 below, the rate of butanol production, when there is fatty acid but not ethanol in the fermentation vessel, may be higher than the rate of butanol production when there is no fatty acid nor ethanol in the fermentation vessel. Therefore, in some embodiments, the amount of ethanol supplementation 33 is reduced compared to conventional processes. For example, a typical amount of ethanol added in a fermentation vessel for microorganisms that require supplementation of a two-carbon substrate is about 5 g / 1 anhydrous ethanol (i.e., 5 g anhydrous ethanol per liter of fermentation medium). ). In some embodiments, the fermentation is not supplemented with ethanol 33. In the latter case, the ethanol stream 33 is omitted completely from the fermentation vessel. Therefore, in some embodiments of the present invention, it is possible to reduce or eliminate the cost associated with supplemental ethanol 33, as well as the discomfort associated with storing the ethanol vats 33 and supplying it in the fermentation vessel during the fermentation of the butanol. In addition, independently of the ethanol supplementation, in some embodiments, the methods of the present invention can provide a higher rate of glucose consumption by the microorganism 32 by virtue of the presence of fatty acids during fermentation. In accordance with the methods described in the present disclosure, fatty acids can be introduced into the fermentation vessel 30 as FFA 28, hydrolyzed from raw material oil 26 of the suspension 16, or hydrolyzed from native oil, such as biomass lipids, in a stage of the fermentation process. In addition, the fatty acids can be introduced into the fermentation vessel as an ISPR 29 extractant.

In the fermentation vessel 30, the microorganism 32 produces alcohol. In-place product extraction (ISPR) can be used to extract the alcohol product from the fermentation broth. In some modalities, ISPR includes liquid-liquid extraction. The liquid-liquid extraction can be carried out in accordance with the processes described in the publication of United States patent application no. 2009/0305370, the description of which is fully incorporated in the present description. The publication of United States patent application no. 2009/0305370 discloses methods for producing and recovering butanol from a fermentation broth by the use of liquid-liquid extraction; the methods comprise the step of contacting the fermentation broth with a water-immiscible extractant to form a two-phase mixture comprising an aqueous phase and an organic phase. Typically, the extractant may be an organic extractant selected from the group consisting of saturated, monounsaturated or polyunsaturated fatty alcohols (and mixtures thereof) of Ci2 to C2z, C12 to C22 fatty acids, C2 to C22 fatty acid esters, aldehydes Ci2 to C22 fatty acids, and mixtures thereof, which come in contact with a fermentation broth and form a two-phase mixture, comprising an aqueous phase and an organic phase. In addition, the extractant may be an organic extractant selected from the group consisting of saturated, monounsaturated and polyunsaturated fatty alcohols (and mixtures thereof) of C4 to C22I fatty acids of C4 to C2s, fatty acid esters of C4 to C2e, fatty aldehydes from C to C22, and mixtures thereof, which come into contact with a fermentation broth and form a two-phase mixture comprising an aqueous phase and an organic phase. The free fatty acids 28 of the suspension 18 can also act as an extractant ISPR 28. For example, when the free fatty acids 28 are corn oil fatty acids (COFA), the extractant ISPR 28 is COFA. The ISPR extractant (FFA) 28 comes into contact with the fermentation broth and forms a two phase mixture, comprising an aqueous phase 34 and an organic phase. The alcohol product present in the fermentation broth is preferably divided into the organic phase to form an organic phase containing alcohol 36. In some embodiments, the fermentation vessel 30 has one or more inlets to receive one or more extractant agents. ISPR 29, which form a two-phase mixture comprising an aqueous phase and an organic phase, and the alcohol product is divided into the organic phase.

The biphasic mixture can be extracted from the fermentation vessel 30 as the stream 39 and introduced into the vessel 35, where the organic phase containing alcohol 36 is separated from the aqueous phase 34. The organic phase containing alcohol 36 is separated from the phase aqueous 34 of the biphasic stream 39 by methods known in the art including, but not limited to, siphoning, decanting, aspiration, centrifugation, by the use of a gravity settler, membrane assisted phase separation, and the like. All or part of the aqueous phase 34 can be recycled in the fermentation vessel 30 as the fermentation medium (as shown), or it can be discarded and replaced by a new medium or treated for the extraction of any remaining alcohol product and, then, fermentation vessel 30 is recycled therein. Next, the organic phase containing alcohol 36 is treated in a separator 50 to recover the alcohol product 54, and the poor alcohol extractant 27 can be recycled back into the fermentation vessel. 30, generally combined with fresh FFA 28 of suspension 18 and / or with new extractant 29, for further extraction of the alcohol product. Alternatively, the new FFA 28 (of the suspension 18) and / or the extractant 29 can be added continuously in the fermentation vessel to replace the ISPR extractants extracted from the. current of the biphasic mixture 39.

In some embodiments, the extra ISPR 29 extractant may be an exogenous organic extractant such as oleyl alcohol, behenyl alcohol, cetyl alcohol, lauryl alcohol, myristyl alcohol, stearyl alcohol, 1-undecanol, oleic acid, lauric acid, myristic acid, stearic acid, methyl myristate, methyl oleate, undecanal, lauric aldehyde, 20-methylundecanal, and mixtures thereof. In some embodiments, the extractant ISPR 29 may be a carboxylic acid or free fatty acid and, in some embodiments, the carboxylic acid or free fatty acid has a chain of 4 to 28 carbons, 4 to 22 carbons in other embodiments, 8 to 22 carbons in other modalities, from 10 to 28 carbons in other modalities, from 7 to 22 carbons in other modalities, from 12 to 22 carbons in other modalities, from 4 to 18 carbons in other modalities, from 12 to 22 carbons in other modalities, and from 12 to 18 carbons, even in other modalities. In some embodiments, the extractant ISPR 29 is one or more of the following fatty acids: azaléic, capric, caprylic, castor, coconut (i.e., a combination of natural fatty acids including lauric, myristic, palmitic, caprylic, capric acid , stearic, caproic, arachidic, oleic and linoleic), dimer, isostearic, lauric, flax, myristic, oleic, olive, palm oil, palmitic, palm kernel, peanut, pelargonic, ricinoleic, sebacic, soy, stearic acid, oil of resin, tallow, 12 hydroxy stearic or any seed oil. In some embodiments, the extractant ISPR 29 is one or more diacids, for example, acelaic acid and sebacic acid. Therefore, in some embodiments, the extractant ISPR 29 may be a mixture of two or more different fatty acids. In some embodiments, the extractant ISPR 29 may be a free fatty acid derived from the chemical or enzymatic hydrolysis of glycerides derived from native oil. For example, in some embodiments, the extractant ISPR 29 may be free fatty acids 28 'obtained by enzymatic hydrolysis of the native oil, such as biomass lipids, as described below with reference to the embodiment of Figure 4. In some embodiments embodiments, the ISPR 29 extractant may be an extractant of fatty acids selected from the group consisting of fatty acids, fatty alcohols, fatty amides, methyl esters of fatty acids, low alcohol esters of fatty acids, glycol esters of fatty acids, hydroxylated triglycerides and mixtures thereof, obtained from the chemical conversion of the native oil, such as biomass lipids, as described, for example, in the co-pending jointly-owned United States provisional application, no. . series 61 / 368,436, filed July 28, 2010. In some embodiments, the biomass lipids to produce the extractant 29 may be obtained from the same biomass source or a different one from that obtained from the raw material 12. example, in some embodiments, the biomass lipids for producing the extractant 29 can be derived from soy, while the biomass source of the raw material 12 is corn. Any possible combination of different biomass sources can be used for the extractant 29 with respect to the raw material 12, as will be apparent to a person skilled in the art. In some embodiments, extra ISPR 29 extractants include COFA. The in situ extractive fermentation can be carried out in batches or in continuous mode in the fermentation vessel 30.

For in situ extractive fermentation, the organic extractant may come into contact with the fermentation medium at the start of fermentation and form a biphasic fermentation medium. Alternatively, the organic extractant may come into contact with the fermentation medium after the microorganism has reached the desired amount of growth, which can be determined by measuring the optical density of the culture. Additionally, the organic extractant may come into contact with the fermentation medium at the time when the level of alcohol product in the fermentation medium reaches a previously selected level. In the case of butanol production, for example, the ISPR extractant may come into contact with the fermentation medium before the butanol concentration reaches a level that could be toxic to the microorganism. After contacting the fermentation medium with the extractant ISPR, the butanol product is divided into the extractant and the concentration in the aqueous phase containing the microorganism decreases, which limits the exposure of the production microorganism to the inhibitory product. of butanol.

The volume of ISPR extractant that will be used depends on several factors including the volume of the fermentation medium, the size of the fermentation vessel, the partition coefficient of the extractant agent for the butanol product and the selected fermentation mode, as describe later. The volume of the extractant agent can be from about 3% to about 60% of the operating volume of the fermentation vessel. Depending on the efficiency of the extraction, the title in aqueous phase of the butanol in the fermentation medium can be, for example, from about 1 g / 1 to about 85 g / 1, from about 10 g / 1 to about 40 g / 1 , from about 10 g / 1 to about 20 g / 1, from about 15 g / 1 to about 50 g / 1 or from about 20 g / 1 to about 60 g / 1. In some embodiments, the resulting fermentation broth after esterification of the alcohol may comprise free (ie, non-esterified) alcohol and, in some embodiments, the concentration of free alcohol in the fermentation broth after the esterification of the alcohol is not greater than 1, 3, 6, 10, 15, 20, 25, 30 25, 40, 45, 50, 55 or 60 g / 1 when the alcohol product is butanol, or when the alcohol product is ethanol, the concentration of free alcohol in the fermentation broth after the esterification of the alcohol is not greater than 15, 20, 25, 30 25, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 g / 1. Without theoretical support, it is considered that a higher butanol titre can be obtained with the extractive fermentation method, in part, from the extraction of the toxic product of butanol from the fermentation medium, so that the level remains below the toxic level for the microorganism.

In a batch mode of in situ extractive fermentation, a volume of organic extractant is added to the fermentation vessel and the extractant is not removed during the process. This mode requires a larger volume of organic extractant to minimize the concentration of the butanol inhibitory product in the fermentation medium. Accordingly, the volume of the fermentation medium is smaller and the amount of product produced is less than that obtained by using the continuous mode. For example, the volume of the extractant in batch mode can be from 20% to about 60% of the operating volume of the fermentation vessel in one embodiment, and from about 30% to about 60% in another embodiment.

The degassing (not shown) can be used in conjunction with the extractant ISPR to extract the alcohol product from the fermentation medium.

In the embodiment of Figure 1, the alcohol product is extracted from the fermentation broth in situ, and the separation of the biphasic mixture 39 occurs in a separate vessel 35. In some embodiments, the separation of the biphasic mixture 39 may occur in the fermentation vessel, as shown in the embodiments of Figures 2 and 3 described below, wherein the stream of the organic phase containing alcohol 36 exits directly from the fermentation vessel 30. Furthermore, the stream of the aqueous phase 34 can exit directly from the fermentation vessel 30, treat for the extraction of any remaining alcohol product and recycle, or dispose of and replace with a new fermentation medium. The extraction of the alcohol product by the organic extractant agents can be carried out with or without the extraction of the microorganism from the fermentation broth. The microorganism can be extracted from the fermentation broth by methods known in the art including, but not limited to, filtration or centrifugation. For example, the stream of aqueous phase 34 may include microorganism 32, such as yeast. The microorganism 32 can be easily separated from the stream of the aqueous phase, for example, in a centrifuge (not shown). Then, the microorganism 32 can be recycled into the fermentation vessel 30 which, over time, can increase the rate of alcohol production, resulting in an increase in the efficiency of the production of alcohol.

In a continuous mode of in situ extractive fermentation, the volume of extractant may be from about 3% to about 50% of the operating volume of the fermentation vessel in one embodiment, from about 3% to about 30% in another embodiment, 3% to approximately 20% in another mode; and from 3% to approximately 10% in another modality. Because the product is continuously removed from the reactor, a smaller volume of extractant agent is required, which enables a larger volume of the fermentation medium to be used.

As an alternative to in situ extractive fermentation, the alcohol product can be extracted from the fermentation broth downstream of the fermentation vessel 30. In that case, the fermentation broth can be extracted from the fermentation vessel 30 and introduced into the vessel 35 to enter in contact with the extractant ISPR in order to obtain the biphasic mixture 39 in the container 35, which is then separated in the organic 36 and aqueous 34 phases. Alternatively, the extractant ISPR can be added in the fermentation broth in a separate container (not shown) before introduction into container 35.

In an intuitive, non-limiting example, in relation to the embodiment of Figure 1, an aqueous suspension of ground whole corn (as raw material 12) which can nominally contain about 4% by weight of corn oil, can be treated with amylase (as the liquefying enzyme 14) at a temperature of about 85 ° C to 120 ° C for 30 minutes to 2 hours, and the resulting liquefied paste 16 can be cooled to a temperature between 65 ° C and 30 ° C and treated with 0.1 ppm at 10 ppm (in some embodiments, from 0.5 ppm to 1.0 ppm) of lipase (as catalyst 42) at a pH of 4.5 to 7.5 (in some embodiments, at a pH of between 5.5 and 6.5) for a sufficient time to produce the minus 30% up to at least 99% of the conversion of the fatty acid content available in the lipids to free fatty acids. Optionally, the liquefied paste treated with lipase 18 can be heated to inactivate the lipase 42 prior to fermentation. The pulp 18 can be cooled to about 30 ° C (for example, with a heat exchanger) and loaded into the fermentation vessel 30 to about 25% to 30% by weight dry corn solids. Saccharification of the liquefied pasta 18 during fermentation by the addition of glucoamylase (such as saccharification enzyme 38) can result in the production of glucose. The resulting fermentation broth may contain a significantly lower amount of corn oil (e.g., about 1.2% by weight of corn oil) which may be present in the fermentation broth with a liquefied paste that has not been treated with lipase 42. Particularly, treatment with lipase 42 may result in the conversion of the oil lipids of maize 26 (triglycerides (TG)) in COFA as FFA 28 (and some diglycerides (DG) or monoglycerides (MG)) that come into contact with the fermentation broth. The growth rate and / or glucose consumption of the microorganism in the fermentation broth may be higher in the presence of COFA than the growth rate and the fermentable carbon consumption of the microorganism in the absence of fatty acids. For example, as described later in the examples, Figures 6-8 illustrate increased glucose consumption by the butanol producing microorganisms in the presence of supplemented fatty acids.

In some embodiments, the system and processes of Figure 1 may be modified so that the suspension of raw material 16 (containing oil 26) and catalyst 42 are introduced and contacted in the fermentation vessel 30 to produce the suspension 18 (containing FFA 28). Then, the temperature of the fermentation vessel can be increased to heat inactivate the catalyst 42. Subsequently, the temperature of the fermentation vessel can be reduced and the fermentation vessel can be inoculated with the microorganism 32, so that the sugars in the suspension 18 are fermented and produce an alcohol product.

In some embodiments, the system and processes of Figure 1 can be modified so that the simultaneous saccharification and fermentation (SSF) in the fermentation vessel 30 is replaced by a separate saccharification vessel 60 (see Figure 2) before the vessel. fermentation 30, as will be apparent to a person skilled in the art. Therefore, suspension 18 can be saccharified before or during fermentation in an SSF process. It will further be apparent that the catalyst 42 for the hydrolysis of the raw material oil 26 can be introduced before, after or at the same time with the saccharification enzyme 38. Thus, in some embodiments, the addition of the enzyme 38 and the catalyst 42 it can be carried out in stages (for example, catalyst 42, then enzyme 38 or vice versa), or practically simultaneously (that is, at exactly the same time that it takes a person or machine to perform the one-time addition, or an enzyme / catalyst immediately after the other catalyst / enzyme according to the time it takes a person or machine to perform the two-step addition).

For example, as shown in the embodiment of Figure 2, the system and processes of Figure 1 can be modified so that the simultaneous saccharification and fermentation (SSF) in the fermentation vessel 30 is replaced by a separate saccharification vessel 60. before the fermentation vessel 30. Figure 2 is practically the same as Figure 1, except for the inclusion of a separate saccharification vessel 60 receiving the enzyme 38, where the catalyst 42 is introduced into a stream of saccharified raw material and liquefied 62. The suspension of raw material 16 is introduced into the saccharification vessel 60 together with the enzyme 38, such as glucoamylase, so that the sugars in the form of oligosaccharides in the suspension 16 can be broken down into monosaccharides. A stream of saccharified liquefied raw material 62 leaves the saccharification vessel 60 into which the catalyst 42 is introduced. The raw material stream 62 includes monosaccharides, oil 26 and undissolved solids derived from the raw material. The oil 26 is hydrolysed with the introduction of catalyst 42, resulting in a suspension of saccharified and liquefied raw material 64 with free fatty acids 28 and catalyst 42.

Alternatively, in some embodiments, the catalyst 42 can be added with the saccharification enzyme 38 to simultaneously produce glucose and hydrolyze the lipids of the oil 26 into free fatty acids 28. The addition of the enzyme 38 and the catalyst 42 can be stepwise (by example, catalyst 42 and then enzyme 38, or vice versa) or simultaneously. Alternatively, in some embodiments, the suspension 62 can be introduced into a fermentation vessel, into which the catalyst 42 is directly added in the fermentation vessel 30.

In the embodiment of Figure 2, heat q is applied to the raw material suspension 64, so that the catalyst 42 becomes inactive, and the heat-treated suspension 64 is then introduced into the fermentation vessel 30 together with the producing microorganism. of alcohol 32, which metabolizes the monosaccharides to produce an alcohol product (eg, butanol). Alternatively, the suspension 64 can be introduced into the fermentation vessel 30 and subjected to heat q while it is in the fermentation vessel, prior to the inoculation of the microorganism 32.

As described above with reference to Figure 1, the free fatty acids 28 can also act as ISPR extractant to, preferably, separate the alcohol product from the aqueous phase. In some embodiments, one or more additional ISPR 29 extractant agents may also be introduced into the fermentation vessel 30. Separation of the two-phase mixture occurs in the fermentation vessel 30, so that the stream of the organic phase containing alcohol 36 and the stream of the aqueous phase 34 leaves directly from the fermentation vessel 30. Alternatively, the separation of the two-phase mixture can be carried out in a separate vessel 35, as provided in the embodiments of Figure 1. The remaining process operations of the The embodiment of Figure 2 are identical to those of Figure 1 and, therefore, will not be described in detail again.

Even in other embodiments, the oil 26 derived from the raw material 12 can be catalytically hydrolyzed in FFA 28 before or during liquefaction, so that the suspension of raw material containing FFA 28 leaves directly from the liquefying vessel 10 and can be introduced into the liquid. the fermentation vessel 30. For example, the raw material 12 containing oil 26 can be introduced into the liquefying vessel 10 together with the catalyst 42 for the hydrolysis of at least a portion of the glycerides in the oil 26 in FFA 28. enzyme 14 (for example, alpha-amylase) that hydrolyses the starch in the raw material 12 can also be introduced into the container 10 to produce a liquefied raw material. The addition of the enzyme 14 and the catalyst 42 can be carried out in stages or simultaneously. For example, catalyst 42 can be introduced, and then enzyme 14 can be introduced after at least a portion of oil 26 has been hydrolyzed. Alternatively, the enzyme 14 can be introduced and then the catalyst 42 can be introduced. The liquefaction process can include the application of heat q. In some embodiments, the catalyst 42 can be introduced before or during liquefaction when the process temperature is lower than the temperature that inactivates the catalyst 42, so that the oil 26 can be hydrolyzed. Subsequently, the application of heat q can provide a dual purpose of liquefaction and inactivation of the catalyst '42, if inactivation is preferred.

In some embodiments including any of the embodiments described above in relation to Figures 1 and 2, the undissolved solids can be extracted from the raw material suspension before it is introduced into the fermentation vessel 30. For example, as shown in FIG. embodiment of Figure 3, the raw material suspension 16 is introduced into an inlet of a separator 20 that is configured to discharge the undissolved solids as a solid phase or wet paste 24. For example, in some embodiments, the separator 20 may include a filter press, vacuum filtration or a centrifuge to separate undissolved solids from the raw material suspension 16. Optionally, in some embodiments, the separator 20 may be further configured to remove part or substantially all of the oil 26 present in the raw material suspension 16. In the embodiments, the separator 20 can be any suitable separator known in the art. ca to extract the oil from an aqueous feed stream including, but not limited to, siphoning, suction, decanting, centrifugation, gravity sedimentation, membrane assisted phase separation, and the like. The remaining raw material, which includes sugar and water, is discharged as an aqueous stream 22 into the fermentation vessel 30.

In some embodiments, the separator 20 extracts the oil 26 but not the undissolved solids. Therefore, the aqueous stream 22 which is introduced into the fermentation vessel 30 includes undissolved solids. In some modalities, the separator 20 includes a tricanter centrifuge 20 that agitates or swirls the stock suspension 15 to produce a spin product comprising an aqueous layer containing sugar and water (i.e. stream 22), a solids layer containing undissolved solids (i.e., wet paste 24) and an oily layer (i.e., oil stream 26). The methods and systems for extracting the undissolved solids from the suspension of raw material 16 by centrifugation are described in detail in the co-pending jointly owned United States Provisional Application, no. series 61 / 356,290, filed on June 18, 2010, which is fully incorporated by reference in the present description.

In any case, the catalyst 42 can be contacted with the extracted oil 26 to produce a stream of FFA 28 that includes the catalyst 42, as shown in Figure 3. Then, heat q can be applied to the FFA stream 28, so that the catalyst 42 becomes inactive. The FFA stream 28 and the inactive catalyst 42 can then be introduced into the fermentation vessel 30 together with the stream 22 and the microorganism 32. Alternatively, FFA 28 and the active catalyst 42 can be introduced into the fermentation vessel 30 from the container 40, and the active catalyst 42 can then be subjected to heat and inactivated while it is in the fermentation vessel prior to the inoculation of the microorganism 32.

FFA 28 can act as an extractant agent ISPR 28 and form a biphasic mixture in the fermentation vessel 30. The alcohol product obtained by .SSF is divided into the organic phase 36 constituted by FFA 28. In some embodiments, they can also be introduced one or more additional ISPR 29 extractants in the fermentation vessel 30. Therefore, the oil 26 (for example, from the raw material) can be catalytically hydrolyzed in FFA 28, to reduce the rate of lipid accumulation in an extractant ISPR and, at the same time, produce an ISPR extractant agent. The organic phase 36 can be separated from the aqueous phase 34 of the biphasic mixture 39 in the container 35. In some embodiments, the separation of the biphasic mixture 39 can occur in the fermentation vessel, as shown in the embodiments described in the Figures. 2 and 3, wherein the stream of the organic phase containing alcohol 36 exits directly from the fermentation vessel 30. The organic phase 36 can be introduced into the separator 50 for the recovery of the alcohol product 54 and optionally recycle the recovered extractant 27 , as shown in Figure 1. The remaining process operations of the modality of Figure 3 are identical to those of Figure 1 and, therefore, will not be described in detail again.

When the wet pulp 24 is extracted by the centrifuge 20, in some embodiments, a portion of the raw material oil 12, such as corn oil when the raw material is corn, remains in the wet pulp 24. The wet pulp 24 it can be washed with additional water in the centrifuge once the aqueous solution 22 has been discharged from the centrifuge 20. The washing wet paste 24 will recover the sugar (for example, oligosaccharides) present in the wet paste, and the sugar and the water recovered can be recycled in the liquefying vessel 10. After washing, the wet pulp 20 can be dried to form dry distillers grains with solubles (DDGS) by any suitable known process. The formation of the DDGS from the wet paste 24 formed in the centrifuge 20 has several benefits. As the undissolved solids do not pass into the fermentation vessel, the DDGS have no trapped extractant and / or alcohol product, such as butanol, are not subject to the conditions of the fermentation vessel and do not come into contact with the microorganisms present in the fermentation vessel. the fermentation vessel.

All these benefits facilitate the process and sale of DDGS, for example, as animal feed. In some embodiments, the oil 26 is not separately discharged from the wet pulp 24, but the oil 26 is included as part of the wet pulp 24 and, finally, is present in the DDGS. In these cases, the oil can be separated from the DDGS and converted into an ISPR 29 extractant agent for later use in the same alcohol fermentation process or in a different one. The methods and systems for extracting dissolved solids from the raw material 16 by centrifugation are described in detail in the co-pending and jointly owned United States patent application no. 61 / 356,290, filed June 18, 2010, which is fully incorporated by reference in the present description.

Even in other embodiments (not shown), saccharification can occur in a separate saccharification vessel (see Figure 2) located between the separator 20 and the liquefying vessel 10, as will be apparent to a person skilled in the art. .

Even in other embodiments shown, for example, in the embodiment of Figure 4, a native oil 26 'is supplied to a container 40 to which a catalyst 42 is also supplied, so that at least a portion of the glycerides in the 26 'oil are hydrolyzed to form FFA 28'. Subsequently, the catalyst 42 can be inactivated, for example, by the application of heat q. Then, a product stream from the container 40 containing FFA 28 'and inactive catalyst 42 is introduced into the fermentation vessel 30, together with the stream of aqueous raw material 22 in which the raw material oil 26 and, in some embodiments , undissolved solids, have been previously extracted by means of a separator 20 (see, for example, the embodiment of Figure 3). In addition, the saccharification enzyme 38 and the microorganism 32 are introduced into the fermentation vessel 30, so that the alcohol product is produced by SSF.

Alternatively, the oil 26 'and the catalyst 42 can be introduced directly into the fermentation vessel 30, where the oil 26' is hydrolysed in FFA 28 ', instead of using the vessel 40. Subsequently, the active catalyst 42 can be subjected to heat quenching while in the fermentation vessel prior to inoculation of the microorganism 32. Alternatively, FFA 28 'and the active catalyst 42 can be introduced into the fermentation vessel 30 from the vessel 40, and the active catalyst 42 can be subjected to subsequently, heat is inactivated while it is in the fermentation vessel prior to the inoculation of the microorganism 32. In some embodiments, the suspension of raw material 16 including oil 26, instead of stream 22, wherein the oil 26 it was extracted, it can be introduced into the fermentation vessel 30 and contacted with the active catalyst 42. The active catalyst 42 can therefore be used to hydrolyse the oil 26 in FFA 28, to reduce the loss and / or degradation of the partition coefficient of the extractant agent over time, which is attributed to the presence of the oil in the fermentation vessel.

In some embodiments, the system and processes of Figure 4 can be modified so that simultaneous saccharification and fermentation in the fermentation vessel 30 is replaced by a separate fermentation vessel 60 prior to the fermentation vessel 30, as will be apparent to one person. with experience in the technique.

In some embodiments, the native oil 26 'may be tallow, corn, cañola, capric / caprylic triglycerides, castor, coconut, cottonseed, fish, jojoba, lardo, flax, oxen, oiticica, palma, peanut, rapeseed, rice, safflower, soybean, sunflower, tung, jatropha, mixtures of vegetable oils, and mixtures of these. In some embodiments, the native oil 26 'is a mixture of two or more native oils, for example, a mixture of palm and soybean oils. In some embodiments, the native oil 26 'is a vegetable oil. In some embodiments, the vegetable oil, although not necessarily, can be derived from biomass that can be used in a fermentation process. The biomass can be the same or a different source from which the raw material 12 is obtained (shown in Figure 4 as stream 22). Therefore, in some embodiments, for example, the oil 26 'can be derived from corn, while the raw material 12 can be cane. For example, in some embodiments, the oil 26 'can be derived from corn and the biomass source from the raw material 12 can also be corn. Any possible combination of different biomass sources for the oil 26 'can be used with respect to the raw material 12, as will be apparent to a person skilled in the art. The remaining process operations of the embodiment of Figure 4 are identical to those of Figure 1 and, therefore, will not be described in detail again.

In some embodiments of the present invention, the biomass oil present in the raw material 12 can be converted to FFA 28 in a step subsequent to the alcoholic fermentation. Then, FFA 28 can be introduced into the fermentation vessel and contacted with the fermentation broth to achieve improved growth rate and / or fermentable carbon consumption of the alcohol producing microorganism. FFA 28 can also act as extractant agent ISPR 28. For example, in the embodiment of Figure 5, the raw material 12 is liquefied to produce the suspension of raw material 16 which includes the oil 26 derived from the raw material. The raw material suspension 16 may also include undissolved solids from the raw material. Alternatively, undissolved solids can be separated from suspension 16 by a separator, such as a centrifuge (not shown). The suspension of raw material 16 containing the oil 26 is directly introduced into the fermentation vessel 30 which contains a fermentation broth which includes the saccharification enzyme 38 and the microorganism 32. An alcohol product is produced by SSF in the fermentation vessel. fermentation 30. Alternatively, in some embodiments, the process can be modified to include a separate saccharification container, as discussed in relation to Figure 2.

The extractant ISPR 29 is introduced into the fermentation vessel 30 to form a biphasic mixture, and the alcohol product is extracted by partition in the organic phase of the extractant ISPR 29. In addition, the oil 26 is separated into the organic phase. The separation of the biphasic mixture occurs in the fermentation vessel 30, so that the stream of the organic phase containing alcohol 36 and the stream of the aqueous phase 34 leave directly from the fermentation vessel 30. Alternatively, the separation of the two-phase mixture it can be carried out in a separate container 35, as provided in the embodiments of Figure 1. The stream of the organic phase 36 including oil 26 is introduced into the separator 50 to recover the alcohol product 54 from the extractant agent 29. The agent Poor alcohol extractant 27 includes the recovered extractant 29 and the oil 26. The extractant 27 is contacted with the catalyst 42, so that at least a portion of the glycerides in the oil 26 are hydrolyzed to form FFA 28. Then, heat q can be applied to the extractant agent 27 which includes FFA 28 to inactivate the catalyst 42 before being recycled back into the container. fermentation 30. The recycled extractant stream 27 may be a separate stream or a stream combined with a fresh stream of auxiliary extractant 29. Subsequent extraction of the alcohol-containing organic phase 36 from the fermentation vessel 30 may include FFA 28 and ISPR 29 extractant (as a new extractant 29 and recycled extractant 27), in addition, of the alcohol product and the additional oil 26 of the freshly introduced stock suspension 16. Then, the organic phase 36 can be treated to recover the alcohol product, and recycled back into the fermentation vessel 30 after coming into contact with the catalyst 42 for hydrolysis of the additional oil 26, in the same manner as described above. In some embodiments, the use of auxiliary ISPR extractant 29 can be phased out as the fermentation process takes place over time because the process itself can produce FFA 28 as an auxiliary ISPR extractant to remove the alcohol product. . Therefore, the ISPR extractant may be the stream of recycled extractant 27 with FFA 28.

Thus, Figures 1-5 provide the various non-limiting modes of methods and systems related to the fermentation and FFA 28 processes produced by the hydrolysis of oil-derived biomass., and FFA 28 'produced from the catalytic hydrolysis of native oil 26', such as vegetable oil, which can be used to come into contact with a microorganism during fermentative growth, so that growth rate and / or carbon consumption fermentable microorganism is greater in the presence of free fatty acids, resulting in improved alcohol productivity. From the foregoing description and examples, a person skilled in the art can determine the essential characteristics of this invention and, without departing from it, introduce various changes and modifications of the invention to adapt it to the various uses and conditions. For example, in some embodiments, the fatty acid supplementation according to the present invention can be performed prior to fermentation, ie, during the seed culture of the microorganisms 32 prior to fermentation in the fermentation vessel 30. Typically, microorganisms 32, such as yeast, can be cultured in a seed culture to the desired cell concentration before being harvested and inoculated into the fermentation vessel 30, as is known in the art. Therefore, according to some embodiments, the seed culture medium can be contacted with FFA 28 so that improved growth and biomass production rates of the microorganism are obtained, which can reduce the time prior to fermentation associated with the Phase of seed culture in an alcohol fermentation process. Thus, it will be apparent that fatty acid supplementation according to the present invention can be carried out in several stages in an alcohol fermentation process, for example, during cultivation prior to fermentation and fermentation, to improve overall efficiency of the process without departing from the present invention.

In some embodiments, including any of the modalities mentioned above described with reference to Figures 1-5, the fermentation broth in the fermentation vessel 30 includes at least one recombinant microorganism 32 that is genetically modified to produce butanol by a biosynthetic route of at least one source of fermentable carbon to butanol. Particularly, recombinant microorganisms can be cultured in a fermentation broth containing suitable carbon substrates. Additional carbon substrates may include, but are not limited to, monosaccharides, such as fructose; oligosaccharides, such as lactose, maltose and sucrose; polysaccharides, such as starch or cellulose; or mixtures of these and non-purified mixtures of renewable raw materials, such as cheese whey permeate, fermented corn liquor, sugar beet molasses and barley malt. Other carbon substrates may include ethanol, lactate, succinate or glycerol.

Additionally, carbon substrates can be single carbon substrates, such as carbon dioxide or methanol, for which metabolic conversion in key biochemical intermediates has been demonstrated. In addition to the one and two carbon substrates, it is known that methylotrophic organisms use various additional carbon-containing compounds such as methylamine, glucosamine and various amino acids for metabolic activity. For example, methylotrophic yeasts are known to use the carbon of methylamine to form trehalose or glycerol (Bellion, et al., Microb Growth Cl Compd., [Int. Symp.], 7th (1993), 415- 32, Editor (s): Murrell, J. Collin, Kelly, Don P. Publisher: Intercept, Andover, United Kingdom of Great Britain). In addition, various species of Candida will metabolize alanine or oleic acid (Sulter, et al., Arch. Microbiol., 153: 485-489, 1990). Therefore, it is contemplated that the carbon source employed in the present invention can encompass a wide variety of carbon-containing substrates and will only be limited by the choice of organism.

Although it is contemplated that all of the above-mentioned carbon substrates and mixtures thereof are suitable, in some embodiments, the carbon substrates are glucose, fructose and sucrose, or mixtures of these with C5 sugars, such as xylose and / or arabinose. for the modified yeast to use C5 sugars. Sucrose can be derived from renewable sugar sources, such as sugar cane, sugar beet, cassava, sweet sorghum, and mixtures of these. Glucose and dextrose can be derived from renewable grain sources through the saccharification of starch-based raw materials including grains, such as corn, wheat, rye, barley, oats, and mixtures thereof. In addition, fermentable sugars can be derived from renewable cellulosic or lignocellulosic biomass through pretreatment and saccharification processes, as described, for example, in U.S. Patent Application Publication no. 2007/0031918 Al, which is incorporated by reference in the present description. In addition to the suitable carbon source (from the aqueous stream 22), the fermentation broth should contain minerals, salts, cofactors, buffer solutions and other suitable components known to those skilled in the art, suitable for the growth of crops. and the promotion of an enzymatic pathway comprising dihydroxy acid dehydratase (DHAD).

Recombinant microorganisms that produce butanol by a biosynthetic route may include a member of the genus Clostridium, Zymomonas, Escherichia, Sal onella, Serratia, Erwinia, Klebsiella, Shigella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthro &; acter, Corynejbacterium, Brevibacterium, Schizosaccharomyces, Kluyveromyces, Yarrowia, Pichia, Candida, Hansenula or Saccharomyces. In one embodiment, the recombinant microorganisms can be selected from the group consisting of Escherichia coli, Lactobacillus plantarum and Saccharomyces cerevisiae. In one embodiment, the recombinant microorganism is a positive crabtree yeast selected from Saccharomyces, Zygosaccharomyces, Schizosaccharomyces, Dekkera, Torulopsis, Brettanomyces and some species of Candida. Positive crabtree yeast species include, but are not limited to, Saccharo yces cerevisiae, Saccharomyces kluyveri, Sc izosaccharomyces pombe, Saccharomyces bayanus, Saccharomyces mikitae, Saccharomyces paradoxus, Zygosaccharomyces rouxii and Candida glabrata. For example, the production of butanol by fermentation by a microorganism, as well as the microorganisms that produce butanol, are known and described, for example, in U.S. Patent Application Publication no. 2009/0305370, incorporated by reference in the present description. In some embodiments, the microorganisms comprise a butanol biosynthetic pathway. Suitable isobutanol biosynthetic routes are known in the art (see, for example, U.S. Patent Application Publication No. 2007/0092957, incorporated by reference in the present description). In some embodiments, at least one, at least two, at least three or at least four substrate conversion catalytic polypeptides in product of a route are encoded by heterologous polynucleotides in the microorganism. In some embodiments, all polypeptides that catalyze substrate conversions in product of a pathway are encoded by heterologous polynucleotides in the microorganism. In some embodiments, the microorganism comprises a reduction or elimination of pyruvate decarboxylase activity. The. microorganisms practically free of pyruvate decarboxylase activity are described in the publication of United States patent application no. 2009/0305363, incorporated by reference in the present description.

The construction of certain strains, including those used in the examples, is provided in the present invention.

Construction of Saccharomyces cerevisiae strain BP1083 ("NGCI-070") Strain BP1064 was derived from CEN.PK 113-7D (CBS 8340; Cent aalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Center, The Netherlands) and contains deletions of the following genes: URA3, HIS3, PDC1, PDC5, PDC6 and GPD2. BP1064 was transformed with plasmids pYZ090 (sec.with ident.ident .: 1, described in US Provisional Application Serial No. 61 / 246,844) and pLH468 (sec.with Ident.No .: 2) to create strain NGCI-070 (BP1083, PNY1504).

The deletions, which completely eliminated the coding sequence, were created by homologous recombination with PCR fragments containing regions of homology in the 5 'direction and in the 3'-direction of the target gene and even a resistance marker G418 or URA3 gene for selection. of transformants. The resistance marker G418, flanked by loxP sites, was eliminated by the use of Cre recombinase. The URA3 gene was removed by homologous recombination to create a deletion without scar or, if flanked by loxP sites, was eliminated by the use of Cre recombinase.

The scarless suppression procedure was adapted based on Akada, et al., (Yeast 23: 399-405, 2006). Generally, the PCR cassette for each deletion without a scar was developed by combining four fragments, A-B-U-C, by means of superimposed PCR. The PCR cassette contained a selectable / counter-selectable marker, URA3 (Fragment U), which consisted of the native gene CEN.PK 113-7D URA3, together with the promoter (250 bp downstream of the URA3 gene) and terminator (150 bp in the 3 'direction of the URA3 gene). Fragments A and C, each 500 bp in length, corresponded to the 500 bp immediately downstream of the target gene (Fragment A) and 500 bp of the target gene (Fragment C). Fragments A and C were used to integrate the cassette into the chromosome by homologous recombination. Fragment B (500 bp in length) corresponded to the 500 bp immediately downstream of the target gene and was used for cleavage of the URA3 marker and Fragment C of the chromosome by homologous recombination, as a direct repeat was created. the sequence corresponding to Fragment B with the integration of the cassette into the chromosome. By using the ABUC cassette of the PCR product, the URA3 marker was first integrated and then excised from the chromosome by homologous recombination. The initial integration eliminated the gene, except the 500 bp of 3 '. After cleavage, the 500 bp of the 3 'region of the gene was also removed. For the integration of genes with this method, the gene that was wanted to integrate was included in the PCR cassette between Fragments A and B.

Deletion of URA3 To eliminate the coding region of endogenous URA3, a ura3:: loxP-kanMX-loxP cassette was amplified by PCR from the DNA template pLA54 (sec. With ident. No .: 3). pLA54 contains the TEF1 promoter of K. lactis and the kanMX marker, and is flanked by loxP sites to allow recombination with Cre recombinase and marker elimination. PCR was carried out using Phusion® DNA polymerase (New England BioLabs Inc., Ipswich, MA) and primers BK505 and BK506 (sec.with ident numbers: 4 and 5). The URA3 portion of each primer was derived from the 5 'region in the 5' direction of the URA3 promoter and the 3 'region in the 3' direction of the coding region so that the integration of the loxP-kanMX-loxP marker produced the region replacement. encoding of URA3. The PCR product was transformed into CEN.PK 113-7D by the use of standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202) and transformants were selected in YPD containing G418 (100 μ? / p ??) at 30 ° C. The transformants were analyzed by PCR to verify the correct integration by using LA468 and LA492 primers (sec. With ident.mixes: 6 and 7) and designated CEN.PK 113-7D Aura3:: kanMX.

Suppression of HIS3 The four fragments for the PCR cassette for the scarless suppression of HIS3 were amplified by using the Phusion® high fidelity PCR master mix (New England BioLabs Inc., Ipswich, MA) and CEN.PK 113- genomic DNA. 7D as a template, prepared with a yeast / bact kit. of Gentra® Puregene® (Qiagen, Valencia, CA). Fragment A of HIS3 was amplified with the primer oBP452 (sec. With ident. No .: 14) and the primer oBP453 (sec. With ident. No .: 15) containing a 5 'tail with homology to end 5 'from Fragment B of HIS3. Fragment B of HIS3 was amplified with the primer oBP454 (sec.with ident .: 16) containing a 5 'tail with homology to the 3' end of Fragment A of HIS3, and the primer oBP455 (sec. Ident .: 17) that contained a 5 'tail with homology to the 5' end of Fragment U of HIS3. Fragment U of HIS3 was amplified with primer 456 (Seq. No. 18) containing a 5 'tail with homology to the 3' end of Fragment B of HIS3, and primer oBP457 (sec. with ID No. 19) containing a 5 'tail with homology to the 5' end of Fragment C of HIS3. Fragment C of HIS3 was amplified with the OBP458 primer (sec.with ident. 20) containing a 5 'tail homologous to the 3' end of Fragment U of HIS3, and the primer oBP459 (SEQ ID No. 21). The PCR products were purified with a PCR purification kit (Qiagen, Valencia, CA). Fragment AB of HIS3 was created by PCR superimposed by mixing Fragment A and Fragment B of HIS3 and amplifying with primers? 452 (sec. With ID No. 14) and oBP455 (sec. ident: 17). The UC Fragment of HIS3 was created by PCR superimposed by mixing Fragment U and Fragment C of HIS3 and amplifying with primers OBP456 (sec. With ident. No .: 18) and oBP459 (sec. With ident. No .: twenty-one) . The resulting PCR products were purified on agarose gel followed by the use of a gel extraction kit (Qiagen, Valencia, CA). The resulting ABUC cassette of HIS3 was created by PCR superimposed by mixing Fragment AB and UC fragment of HIS3 and amplifying with primers oBP452 (sec.with ident.ID: 14) and oBP459 (sec.with ident. : twenty-one) . The PCR product was purified with a PCR purification kit (Qiagen, Valencia, CA).

Competent cells of CEN.PK 113-7D Aura3 :: kanMX were made and transformed with the ABUC PCR cassette of HIS3 using the Frozen-EZ Yeast Transformation II ™ kit (Zymo Research Corporation, Irvine, CA). The transformation mixtures were plated in complete synthetic media without uracil supplemented with 2% glucose at 30 ° C. Transformants with his3 elimination were analyzed by PCR with primers oBP460 (sec. With ident. No .: 22) and oBP461 (sec. With ident. No .: 23) by the use of genomic DNA prepared with a kit of yeast / bact. of Gentra® Puregene® (Qiagen, Valencia, CA). A suitable transformant was selected as the strain CEN.PK 113-7D Aura3 :: kanMX Ahis3 :: URA3.

Extraction of the KanMX marker from the Aura3 site and extraction of the URA3 marker from the Ahis3 site The KanMX marker was extracted by transforming CEN.PK 113-7D Aura3 :: kan X Ahis3 :: URA3 with pRS423:: PGALl-cre (sec.with ID .: 66, described in the United States provisional application no. 61 / 290,639) by the use of a Frozen-EZ Yeast Transformation II ™ kit (Zymo Research Corporation, Irvine, CA) and plated on a complete synthetic medium without histidine and uracil supplemented with 2% glucose at 30 °. C. The transformants were cultured in YP supplemented with 1% galactose at 30 ° C for approximately 6 hours to induce cleavage of Cre recombinase and the KanMX marker, and plated on YPD (2% glucose) at 30 ° C to the recuperation. An isolated strain was grown overnight in YPD and placed in a complete synthetic medium containing 5-fluoro-orotic acid (5-FOA, 0.1%) at 30 ° C to select isolated strains that lost the URA3 marker. The isolates resistant to 5-FOA were cultivated and plated in YPD for the extraction of plasmid pRS423:: PGALl-cre. The isolated strains were analyzed for loss of the KanMX marker, the URA3 marker and the pRS423 :: PGALl-cre plasmid by examining the growth on YPD + G418 plates, the complete synthetic media plates without uracil, and the plates of complete synthetic medium without histidine. The correct isolated strain that was sensitive to G418 and auxotrophic for uracil and histidine was selected as strain CEN.PK 113-7D Aura3 :: loxP Ahis3 and designated BP857. Deletions and marker extraction were confirmed by PCR and sequencing with the primers oBP450 (sec. With ID No. 24) and oBP451 (sec.with ID No.: 25) for Aura3 and the primers oBP460 ( sec. with Ident ID: 22) and oBP461 (sec. with Ident ID: 23) for ??? e3 by the use of genomic DNA prepared with a yeast / bact kit. of Gentra® Puregene® (Qiagen, Valencia, CA).

Deletion of PDC6 The four fragments for the PCR cassette for PDC6-free scar suppression were amplified by using the Phusion® high fidelity PCR master mix (New England BioLabs Inc., Ipswich, MA) and CEN.PK 113- genomic DNA. 7D as a template, prepared with a yeast / bact kit. of Gentra® Puregene® (Qiagen, Valencia, CA).

Fragment A of PDC6 was amplified with the primer oBP440 (sec. With ident. No .: 26) and the primer 0BP441 (sec.with ident.No .: 27) which contained a 5 'tail with homology to end 5 'from Fragment B of PDC6. Fragment B of PDC6 was amplified with the primer oBP442 (sec.with ident .: 28), which contained a 5 'tail with homology to the 3' end of Fragment A of PDC6, and the primer OBP443 (sec. Ident. no .: 29) which contained a 5 'tail with homology to the 5' end of Fragment U of PDC6. Fragment U of PDC6 was amplified with the primer oBP444 (sec.with ident .: 30) which contained a 5 'tail with homology to the 3' end of Fragment B of PDC6, and the primer OBP445 (sec. Ident .: 31) that contained a 5 'tail with homology to the 5' end of Fragment C of PDC6. Fragment C of PDC6 was amplified with the primer OBP446 (sec.ident .: 32) which contained a 5 'tail with homology to the 3' end of the PDC6 U fragment, and the primer 447 (sec. with identification number: 33). The PCR products were purified with a PCR purification kit (Qiagen, Valencia, CA). Fragment AB of PDC6 was created by PCR superimposed by mixing Fragment A and Fragment B of PDC6 and amplifying it with primers oBP440 (sec with ID number: 26) and oBP443 (sec. With ID no .: 29). The UC Fragment of PDC6 was created by PCR superimposed by mixing Fragment U and Fragment C of PDC6 and amplifying it with primers OBP444 (sec. With ident. No .: 30) and oBP447 (sec. With ident. No .: 33). The resulting PCR products were purified on agarose gel followed by the use of a gel extraction kit (Qiagen, Valencia, CA). The ABUC cassette of PDC6 was created by PCR superimposed by mixing Fragment AB and Fragment UC of PDC6 and amplifying with primers oBP440 (sec. With ident. No .: 26) and? 447 (sec. With no. ident .: 33). The PCR product was purified with a PCR purification kit (Qiagen, Valencia, CA).

The competent cells of CEN.PK 113-7D Aura3 :: loxP Ahis3 were made and transformed with the ABUC PCR cassette from PDC6 using the Frozen-EZ Yeast Transformation II ™ kit (Zymo Research Corporation, Irvine, CA). The transformation mixtures were plated in complete synthetic media without uracil supplemented with 2% glucose at 30 ° C. The transformants with elimination of pdc6 were analyzed by PCR with primers oBP448 (sec. With ident number: 34) and oBP449 (sec. With ident. No .: 35) by the use of genomic DNA prepared with a kit of yeast / bact. of Gentra® Puregene® (Qiagen, Valencia, CA). A correct transformant was selected as the strain CEN.PK 113-7D Aura3 :: loxP Ahis3 Apdc6:: URA3.

CEN.PK 113-7D Aura3 :: loxP Ahis3 Apdc6 :: URA3 was grown overnight in YPD and plated in a complete synthetic medium containing 5-fluoro-orotic acid (0.1%) at 30 ° C to select isolated strains that lost the URA3 marker. The deletion and extraction of the marker was confirmed by PCR and sequencing with primers oBP448 (sec. With ident. No .: 34) and oBP449 (sec. With ident. No .: 35) by the use of genomic DNA prepared with a yeast / bact kit. of Gentra® Puregene® (Qiagen, Valencia, CA). The absence of the PDC6 gene of the isolated strain was demonstrated by the negative PCR result by the use of specific primers for the coding sequence of PDC6, oBP554 (sec. With ident. No .: 36) and OBP555 (sec. Ident. no .: 37). The correct isolated strain was selected as the strain CEN.PK 113-7D Aura3 :: loxP Ahis3 Apdc6 and designated BP891.

Suppression of PDCl and integration of ilvDSm The PDCl gene was removed and replaced by the ilvD coding region of Streptococcus utans (ATCC No. 700610). Fragment A followed by the ilvD coding region of Streptococcus mutans for the PCR cassette for the deletion of PDCl-integration of ilvDSm was amplified by using the master mix for high fidelity PCR Phusion® (New England BioLabs Inc., Ipswich , MA) and NYLA83 genomic DNA (described in the present invention and in U.S. Provisional Application No. 61 / 246,709) as a template, prepared with a yeast / bact kit. of Gentra® Puregene® (Qiagen, Valencia, CA). The PDCl A-ilvDSm Fragment (sec. With ident. No .: 141) was amplified with the primer OBP513 (sec.with ident.ident .: 38) and the primer OBP515 (sec.with ident. : 39) that contained a 5 'tail with homology to the 5' end of Fragment B of PDCl. Fragments B, U and C of the PCR cassette for the deletion of PDC1-integration of ilvDSm were amplified with the master mix for high fidelity PCR Phusion® (New England BioLabs Inc., Ipswich, MA) and the genomic DNA of CEN. PK 113-7D as a template, prepared with a yeast / bact kit. of Gentra® Puregene® (Qiagen, Valencia, CA). Fragment B of PDCl was amplified with primer OBP516 (sec. With ident.ident .: 40) containing a 5 'tail with homology to the 3' end of Fragment A-ilvDSm of PDCl, and primer oBP517 (sec. with ID No.: 41) containing a 5 'tail with homology to the 5' end of Fragment U of PDCl. The PDCl Fragment U was amplified with the OBP518 primer (SEQ ID No. 42) containing a 5 'tail homologous to the 3' end of PDCl Fragment B, and the primer oBP519 (sec. Ident .: 43) containing a 5 'tail with homology to the 5' end of Fragment C of PDCl. The Fragment C of PDCl was amplified with the primer OBP520 (sec. With ident. No .: 44), which contained a 5 'tail with homology to the 3' end of the Fragment U of PDCl, and the primer OBP521 (sec. Ident. no .: 45). The PCR products were purified with a PCR purification kit (Qiagen, Valencia, CA). The A-ilvDSm-B Fragment of PDCl was created by PCR superimposed by mixing Fragment A-ilvDSm and Fragment B of PDCl and amplifying with primers oBP513 (sec. With ident. No .: 38) and oBP517 (sec. with ID number: 41). The UC fragment of PDCl was created by PCR superimposed by mixing Fragment U and Fragment C of PDCl and amplifying with primers OBP518 (sec. With ident. No .: 42) and OBP521 (sec. With ident. : Four. Five) . The resulting PCR products were purified on agarose gel followed by the use of a gel extraction kit (Qiagen, Valencia, CA). The PDCl A-ilvDSm-BUC cassette (sec. With ID: 142) was created by PCR superimposed by mixing Fragment A-ilvDSm-B and Fragment UC of PDCl and amplifying with OBP513 primers (sec. ID number: 38) and OBP521 (sec. with ID number: 45). The PCR product was purified with a PCR purification kit (Qiagen, Valencia, CA).

The competent cells of CEN.PK 113-7D Aura3 :: loxP Ahis3 Apdc6 were made and transformed with the PCR cassette A-ilvDSm-BUC of PDCl by using a Frozen-EZ Yeast Transormation II ™ kit (Zymo Research Corporation, Irvine , CA). The transformation mixtures were plated in complete synthetic media without uracil supplemented with 2% glucose at 30 ° C. Transformants with pdcl elimination and integration of ilvDSm were analyzed by PCR with primers oBP511 (sec. With ident. No .: 46) and 0BP512 (sec with ident. No .: 47) by using genomic DNA prepared with the yeast / bact kit. of Gentra® Puregene® (Qiagen, Valencia, CA). The absence of the PDC1 gene of the isolated strain was demonstrated by a negative PCR result by the use of primers specific for the coding sequence of PDC1, OBP550 (sec.with ident.ID: 48) and 0BP551 (sec. Ident. no .: 49). A correct transformant was selected as the strain CEN.PK 113-7D Aura3 :: loxP Ahis3 Apdc6 Apdcl:: ilvDSm-URA3.

CEN.PK 113-7D Aura3 :: loxP Ahis3 Apdc6 Apdcl :: ilvDSm-URA3 was grown overnight in YPD and plated in a complete synthetic medium containing 5-fluoro-orotic acid (0.1%) at 30 ° C to select isolated strains that lost the URA3 marker. The deletion of PDC1, the integration of ilvDSm and the extraction of the marker were confirmed by PCR and sequencing with primers 0BP511 (sec. With ident. No .: 46) and OBP512 (sec. With ident. No .: 47) by the use of genomic DNA prepared with the yeast / bact kit. of Gentra® Puregene® (Qiagen, Valencia, CA). The correct isolated strain was selected as the strain CEN.PK 113-7D Aura3:: loxP Ahis3 Apdc6 Apdcl :: ilvDSm and designated BP907.

Suppression of PDC5 and integration of sadB The PDC5 gene was removed and replaced by the sadB coding region of Achromobacter xylosoxidans. A segment of the PCR cassette for the deletion of PDC5-integration of sadB was first cloned into plasmid pUC19-URA3MCS. pUC19-URA3MCS is based on pUC19 and contains the sequence of the URA3 gene from Saccaromyces cerevisiae located in a multiple cloning site (MCS, for its acronym in English). pUC19 contains the pMBl replicon and a gene coding for beta-lactamase for replication and selection in Escherichia coli. In addition to the coding sequence for URA3, sequences upstream and downstream of this gene were included for the expression of the URA3 gene in the yeast. The vector can be used for cloning purposes and as a yeast integration vector.

DNA comprising the coding region of URA3 together with 250 bp 5 'and 150 bp downstream of the URA3 coding region of the Saccaromyces cerevisiae genomic DNA CEN.PK 113-7D was amplified with oBP438 primers (sec. with identification number: 12) containing the restriction sites BamHI, AscI, Pmel and Fsel and oBP439 (sec. with ident. no .: 13) containing the restriction sites Xbal, Pací and Notl, through the use of the Phusion® high fidelity PCR master mix (New England BioLabs Inc., Ipswich, MA). Genomic DNA was prepared by using the yeast / bact kit. of Gentra® Puregene® (Qiagen, Valencia, CA). The PCR product and pUC19 (sec. With ident. No .: 144) were ligated with T4 DNA ligase after digestion with BamHI and Xbal to create the vector pUC19-URA3MCS. The vector was confirmed by PCR and sequencing with primers OBP264 (SEQ ID NO: 10) and OBP265 (SEQ ID NO: 11).

The coding sequence of sadB and Fragment B of PDC5 were cloned into pUCl9-URA3MCS to create the sadB-BU portion of the A-sadB-BUC PCR cassette of PDC5. The coding sequence of sadB was amplified by the use of pLH468-sadB (sec.with ident.ID .: 67) as a template with the primer oBP530 (sec.with ident.ID .: 50) containing a restriction site AscI, and the primer oBP531 (sec. With ident. No .: 51) containing a 5 'tail with homology to the 5' end of Fragment B of PDC5. Fragment B of PDC5 was amplified with primer oBP532 (sec.with ident.ID .: 52) containing a 5 'tail with homology to the 3' end of sadB,. and the initiator oBP533 (sec.with ident.ID .: 53) containing a Pmel restriction site. The PCR products were purified with a PCR purification kit (Qiagen, Valencia, CA). Fragment B of sadB-PDC5 was created by PCR superimposed by mixing the PCR products of sadB and Fragment B of PDC5 and amplifying with primers OBP530 (sec. With ident. No .: 50) and oBP533 (sec. Ident .: 53). The resulting PCR product was digested with AscI and Pmel and ligated with T4 DNA ligase at the corresponding sites of pUC19-URA3MCS after digestion with the appropriate enzymes. The resulting plasmid was used as a template for the amplification of sadB-Fragment B-Fragment U by using the primers OBP536 (sec.with ident.ID: 54) and OBP546 (sec.with ident.no .: 55 ) containing a 5 'tail with homology to the 5' end of Fragment C of PDC5. Fragment C of PDC5 was amplified with primer OBP547 (sec.ident .: 56) which contained a 5 'tail with homology to the 3' end of sadB-Fragment B-Fragment U of PDC5, and the primer oBP539 (sec. with ident. no .: 57). The PCR products were purified with a PCR purification kit (Qiagen, Valencia, CA). SadB-Fragment B-Fragment U-Fragment C of PDC5 was created by PCR superimposed by mixing sadB-Fragment B-Fragment U and Fragment C of PDC5 and amplifying with primers oBP536 (sec. With ID No. 54) and oBP539 (sec. with ident. no .: 57). The resulting PCR product was purified on agarose gel followed by the use of a gel extraction kit (Qiagen, Valencia, CA). The A-sadB-BUC cassette of PDC5 (sec. With ID No. 143) was created by amplifying sadB-Fragment B-Fragment U-Fragment C of PDC5 with OBP542 primers (sec. With ident. No .: 58) containing a 5 'tail with homology at 50 nucleotides immediately downstream of the coding sequence of native PDC5, and oBP539 (SEQ ID NO: 57). The PCR product was purified with a PCR purification kit (Qiagen, Valencia, CA).

The competent cells of CEN.PK 113-7D Aura3 :: loxP Ahis3 Apdc6 Apdcl:: ilvDSm were elaborated and transformed with the PDC5 A-sadB-BUC PCR cassette using a Frozen-EZ Yeast Transormation II ™ kit (Zymo Research Corporation, Irvine, CA). The transformation mixtures were plated on complete synthetic media without uracil supplemented with 1% ethanol (not glucose) at 30 ° C. Transformants with pdc5 elimination and integration of sadB were analyzed by PCR with primers oBP540 (sec. With ident. No .: 59) and OBP541 (sec. With ident. No .: 60) by using genomic DNA prepared with a yeast / bact kit. of Gentra® Puregene® (Qiagen, Valencia, CA). The absence of the PDC5 gene of the isolated strain was demonstrated by a negative PCR result by the use of primers specific for the coding sequence of PDC5, oBP552 (sec. With ident. No .: 61) and oBP553 (sec. Ident. no .: 62). A correct transformant was selected as the strain CEN.PK 113-7D Aura3 :: loxP Ahis3 Apdc6 Apdcl:: ilvDSm Apdc5:: sadB-URA3.

CEN.PK 113-7D Aura3 :: loxP Ahis3 Apdc6 Apdcl:: ilvDSm Apdc5:: sadB-URA3 was grown overnight in YPE (1% ethanol) and plated on a complete synthetic medium supplemented with ethanol (not glucose) and containing 5-fluoro-orotic acid (0.1%) at 30 ° C to select isolated strains that lost the URA3 marker. The deletion of PDC5, the integration of sadB and the extraction of the marker were confirmed by PCR with the primers OBP540 (sec. With ident. No .: 59) and OBP541 (sec. With ident. No .: 60) by the use of genomic DNA prepared with a yeast / bact kit. of Gentra® Puregene® (Qiagen, Valencia, CA). The correct isolated strain was selected as the CEN strain. PK 113-7D Aura3 :: loxP Ahis3 Apdc6 Apdcl:: ilvDSm Apdc5 :: sadB and designated BP913.

Suppression of GPD2 To eliminate the coding region of endogenous GPD2, a gpd2:: loxP-URA3-loxP cassette (sec. With ID: 145) was amplified by PCR using loxP-URA3-loxP (sec. Ident .: 68) as DNA template. loxP-URA3-loxP contains the URA3 marker of (ATCC No. 77107) flanked by loxP recombinase sites. PCR was carried out using Phusion® DNA polymerase (New England BioLabs Inc., Ipswich, MA) and LA512 and LA513 primers (sec. With ident. No .: 8 and 9). The GPD2 portion of each primer was derived from the 5 'region in the 5' direction of the GPD2 coding region and the 3 'region in the 3' direction of the coding region, so that the integration of the loxP-URA3-loxP marker produced the replacement of the coding region of GPD2. The PCR product was transformed into BP913 and the transformants were selected in complete synthetic media without uracil supplemented with 1% ethanol (non-glucose). The transformants were analyzed by PCR to verify their correct integration through the use of the primers OBP582 and AA270 (sec.with ident numbers: 63 and 64).

The URA3 marker was recycled by transformation with pRS423:: PGALl-cre (sec.with ident.ID .: 66) and plated on complete synthetic media without histidine supplemented with 1% ethanol at 30 ° C. The transformants were grown according to the stria method in a complete synthetic medium supplemented with 1% ethanol and containing 5-fluoro-orotic acid (0.1%), and incubated at 30 ° C to select isolated strains that lost the marker URA3. The isolates resistant to 5-FOA were cultured in YPE (1% ethanol) for the extraction of plasmid pRS423 :: PGALl-cre. The deletion and extraction of the marker were confirmed by PCR with the primers OBP582 (sec. With ident. No .: 63) and oBP591 (sec. With ident. No .: 65). The correct isolated strain was selected as the strain CEN.PK 113-7D Aura3 :: loxP Ahis3 Apdc6 Apdcl:: ilvDSm Apdc5 :: sadB Agpd2:: loxP and designated PNY1503 (BP1064).

BP1064 was transformed with plasmids pYZ090 (sec.with ident.ident .: 1) and pLH468 (sec.with ident.ident .: 2) to create the strain NGCI-070 (BP1083; PNY1504).

Construction of the strains NYLA74, NYLA83 and NYLA84 Insertion-inactivation of the endogenous genes PDC1 and PDC6 of S. cerevisiae. The genes PDC1, PDC5 and PDC6 encode the three major isoenzymes of pyruvate decarboxylase, as described below: Construction of pRS425:: GPM-sadB A DNA fragment encoding a butanol dehydrogenase (SEQ ID NO: 70) of Achromobacter xylosoxidans (described in U.S. Patent Application Publication No. 2009/0269823) was cloned. The coding region of this gene, called sadB, for secondary alcohol dehydrogenase (sec .: Ident .: 69) was amplified by using standard conditions of the genomic DNA of A. xylosoxidans, prepared with the Gentra® Puregene kit ® (Qiagen, Valencia, CA) according to the recommended protocol for gram-negative organisms with direct and inverse primers N473 and N469 (sec. With ident. No .: 74 and 75), respectively. The PCR product was cloned with TOPO®-Blunt in pCR®4 BLUNT (Invitrogen ™, Carlsbad, CA) to produce pCR4Blunt:: sadB, which was transformed into E. coli Mach-1 cells. Subsequently, the plasmid was isolated from four clones, and the sequence was verified.

The coding region sadB was amplified by PCR from pCR4Blunt:: sadB. The PCR primers contained additional 5 'sequences that would overlap with the yeast GP 1 promoter and the ADH1 terminator (N583 and N584, presented as sec. With ident. Nos .: 76 and 77). Then, the PCR product was cloned by using the "recombination repair" methodology in Saccharomyces cerevisiae (a, et al., Gene 58: 201-216, 1987) as follows. The shuttle vector of yeast-E. coli pRS425:: GPM:: kivD:: ADH containing the GPM1 promoter (sec. with ID: 72), the KivD coding region of Lactococcus lactis (sec. with ident. no .: 71) and the ADH1 terminator (SEQ ID No. 73) (described in U.S. Patent Application Publication No. 2007/0092957 A1, Example 17) was digested with the restriction enzymes BbvCI and PacI to release the coding region kivD. Approximately 1] iq of the remaining fragment of the vector was transformed into the S. cerevisiae strain BY4741 together with 1 μg of the sadB PCR product. Transformants were selected in complete synthetic medium without leucine. The appropriate recombination event, which generated pRS425 :: GPM-sadB, was confirmed by PCR using the primers N142 and N459 (sec.with ident.s.:108 and 109).

Construction of the integration cassette PGPMl-sadB and suppression of PDC6: A pdc6 integration cassette was prepared:: PGPMl-sadB-ADHlt-URA3r by joining the segment GPM-sadB-ADHt (sec. With ident. No .: 79) of pRS425:: GPM-sadB (sec. Ident .: 78) with the URA3r gene of pUC19-URA3r. pUC19-URA3r (SEQ ID No. 80) containing the URA3 marker of pRS426 (ATCC No. 77107) flanked by 75 bp homologous repeat sequences to allow in vivo homologous recombination and URA3 marker extraction . The two DNA segments were joined by SOE PCR (as described in Horton, et al., Gene 77: 61-68, 1989) by using the plasmid DNA pRS425 :: GPM-sadB and pUC19-URA3r as a template , with Phusion® DNA polymerase (New England BioLabs Inc., Ipswich, MA) and primers from 114117-11A to 114117-11D (sec.with ident numbers: 81, 82, 83 and 84), and 114117-13A and 114117-13B (sec. with ident.s.:85 and 86).

The external primers for the SOE PCR (114117-13A and 114117-13B) contained 5 'and 3' regions of -50 bp homologous to the 5 'and 3' regions of the PDC6 promoter and terminator, respectively. The PCR fragment from the finished cassette was transformed into BY4700 (ATCC No. 200866) and the transformants were maintained in complete synthetic media without uracil and supplemented with 2% glucose at 30 ° C by the use of standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pgs. 201-202). Transformants were analyzed by PCR using primers 112590-34G and 112590-34H (sec. With ident.s.:87 and 88) and 112590-34F and 112590-49E (sec. With ident. No .: 89 and 90) to verify the integration in the PDC6 locus with suppression of the coding region of PDC6. The URA3r marker was recycled by plating with supplemental complete synthetic media with 2% glucose and 5-FOA at 30 ° C in accordance with standard protocols. The elimination of the marker was confirmed by placing patches of colonies on the plates with 5-FOA medium on SD-URA media to verify the absence of growth. The resulting identified strain has the genotype: BY4700 pdc6:: PGPMl-sadB-ADHlt.

Construction of the pdcl :: integration cassette PPDCl-ilvD and suppression of PDC1: An integrating cassette PPDCl-ilvD-FBAlt-URA3r was prepared by joining the ilvD-FBAlt segment (sec.with ident.ID: 91) of pLH468 (sec.with ident.ident .: 2) with the gene URA3r from pUC19-URA3r by SOE PCR (as described in Horton, et al., Gene 77: 61-68, 1989) by using the plasmid DNAs of pLH468 and pUC19-URA3r as template, with Phusion® DNA polymerase (New England BioLabs Inc., Ipswich, A) and initiators 114117-27A to 114117-27D (sec. With ident. No .: 111, 112, 113 and 114).

The external primers for the SOE PCR (114117-27A and 114117-27D) contained the 5 'and 3' regions of ~ 50 bp homologous to the 3 'regions of the PDC1 promoter and 3' of the coding sequence of the PDC1 promoter. PDC1. The PCR fragment from the finished cassette was transformed into BY4700 pdc6 :: PGPMl-sadB-ADHlt and the transformants were maintained in complete synthetic media without uracil and supplemented with 2% glucose at 30 ° C by the use of standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Fiarbor Laboratgry Press, Cold Spring Harbor, NY, pp. 201-202). The transformants were analyzed by PCR using the 114117-36D and 135 primers (sec. With ID Nos .: 92 and 93) and primers 112590-49E and 112590-30F (sec. With ident. and 94) to verify integration into the PDC1 locus with deletion of the coding sequence of PDC1. The URA3r marker was recycled by plating with complete synthetic media supplemented with 2% glucose and 5-FOA at 30 ° C in accordance with standard protocols. The elimination of the marker was confirmed by placing patches of colonies on the plates with 5-FOA medium on SD-URA media to verify the absence of growth. The resulting strain identified "NYLA67" has the genotype: BY4700 pdc6 :: PGPMl-sadB-ADHlt pdcl :: PPDCl-ilvD-FBAlt.

Suppression of HIS3 To suppress the coding region of the endogenous gene HIS3, a his3 :: URA3r2 cassette was amplified by PCR from the DNA template URA3r2 (sec. With ident. No .: 95). URA3r2 contains the URA3 marker of pRS426 (ATCC No. 77107) flanked by 500 bp homologous repeat sequences to allow homologous recombination in vivo and elimination of the ÜRA3 marker. PCR was performed by using Phusion® DNA polymerase (New England BioLabs Inc., Ipswich, MA) and primers 114117-45A and 114117-45B (sec.with ident Nos .: 96 and 97) which generated a product of PCR ~ 2.3 kb. The HIS3 portion of each primer was derived from the 5 'region 5' of the HIS3 promoter and 3 'region 3' of the coding region so that the integration of the URA3r2 marker produces the replacement of the coding region of HIS3. The PCR product was transformed into NYLA67 by the use of standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202) and the transformants were selected on complete synthetic media without uracil and supplemented with 2% glucose at 30 ° C. The transformants were analyzed to verify the correct integration by replication of plate culture of transformants in complete synthetic media without histidine and supplemented with 2% glucose at 30 ° C. The URA3r marker was recycled by plating in complete synthetic media supplemented with 2% glucose and 5-FOA at 30 ° C in accordance with standard protocols. The elimination of the marker was confirmed by placing patches of colonies on the plates with 5-FOA medium on SD-URA media to verify the absence of growth. The resulting identified strain, named NYLA73, has the genotype: BY4700 pdc6 :: PGPMl-sadB-ADHlt pdcl :: PPDCl-ilvD-FBAlt Ahis3.

Construction of the integration cassette pdc5 :: kanMX and suppression of PDC5: A pdc5:: kan X4 cassette of chromosomal DNA from strain YLR134W (ATCC No. 4034091) was amplified by PCR with Phusion® DNA polymerase (New England BioLabs Inc., Ipswich, MA) and the PDC5 primers:: KanMXF and PDC5 :: KanMXR (sec. With ident.s.:98 and 99) that generated a PCR product ~ 2.2 kb. The PDC5 portion of each primer was derived from the 5 'region upstream of the PDC5 promoter and 3' region downstream of the coding region so that the integration of the kanMX4 marker produces the replacement of the coding region of PDC5. The PCR product was transformed into NYLA73 through the use of standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202) and transformants were selected on YP media supplemented with 1% ethanol and geneticin (200 g / ml) at 30 ° C. The transformants are >; analyzed by PCR to verify the correct integration in the PDC locus with replacement of the coding region of PDC5 through the use of PDC5kofor and N175 primers (sec.with ident numbers: 100 and 101). The correct transformants identified have the genotype: BY4700 pdc6 :: PGPMl-sadB-ADHlt pdcl :: PPDCl-ilvD-FBAlt Ahis3 pdc5:: kanMX. The strain was named NYLA74.

The plasmid vectors pRS423:: CÜPl-alsS + FBA-budA and pRS426:: FBA-budC + GPM-sadB were transformed into NYLA74 to create a butanediol producing strain (NGCI-047).

The plasmid vectors pLH475-Z4B8 (sec.with ident.ID: 140) and pLH468 were transformed into NYLA74 to create an isobutanol producing strain (NGCI-049).

Suppression of HXK2 (hexokinase II): A hxk2 :: URA3r cassette was amplified from the URA3r2 template (described above) with Phusion® DNA polymerase (New England BioLabs Inc., Ipswich, A) and primers 384 and 385 (sec. : 102 and 103) that generated a PCR product -2.3 kb. The HXK2 portion of each primer was derived from the 5 'region upstream of the HXK2 promoter and 3' region downstream of the coding region so that the integration of the URA3r2 marker produces the replacement of the HXK2 coding region. . The PCR product was transformed into NYLA73 through the use of standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202) and the transformants were selected on synthetic media complete without uracil and supplemented with 2% glucose at 30 ° C. Transformants were analyzed by PCR to verify correct integration at the HXK2 locus with replacement of the HXK2 coding region by using primers N869 and N871 (sec.with ident nos .: 104 and 105). The URA3r2 marker was recycled by plating with complete synthetic media supplemented with 2% glucose and 5-FOA at 30 ° C in accordance with standard protocols. The elimination of the marker was confirmed by placing patches of colonies of the 5-FOA plates on the SD-URA media to verify the absence of growth, and by PCR to verify the elimination of the correct marker by means of the use of primers N946 and N947 (sec. with ID numbers: 106 and 107). The resulting identified strain, named NYLA83, has the genotype: BY4700 pdc6 :: PGPMl-sadB-ADHlt pdcl :: PPDCl-ilvD-FBAlt Ahis3 Ahxk2.

Construction of the integration cassette pdc5 :: kanMX and suppression of PDC5: A cassette A pdc5 :: kanMX4 was amplified by PCR as described above. The PCR fragment was transformed into NYLA83 and the transformants were selected and analyzed as described above. The identified correct transformants named NYLA84 have the genotype: BY4700 pdc6 :: PGPMl-sadB-ADHlt pdcl :: PPDCl-ilvD-FBAlt Ahis3 Ahxk2 pdc5:: kanMX.

The plasmid vectors pLH468 and pLH532 were simultaneously transformed into the strain NYLA84 (BY4700 pdc6:: PGPMl-sadB-ADHlt pdcl :: PPDCl-ilvD-FBAlt Ahis3 Ahxk2 pdc5:: kanMX4) by the use of standard genetic techniques (Methods in Yeast Genetics , 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) and the resulting "NYLA84 butanologen" was maintained in complete synthetic media without histidine and uracil, supplemented with 1% ethanol at 30 ° C.

Expression vector pLH468 The plasmid pLH468 (sec.with ident .: 2) was constructed for the expression of DHAD, KivD and HADH in yeast, as described in the publication of the United States patent application no. 2009/0305363, incorporated by reference in the present description. pLH486 was constructed to contain: a chimeric gene with the coding region of the ilvD gene of Streptococcus mutans (position nt 3313-4849) expressed from the FBA1 promoter of S. cerevisiae (nt 2109-3105) followed by the terminator FBA1 (nt 4858-5857 ) for the expression of DHAD; a chimeric gene with codon optimized horse liver dehydrogenase coding region (nt 6286-7413) expressed from the S. cerevisiae GP 1 promoter (nt 7425-8181) followed by the ADH1 terminator (nt 5962-6277) for the expression of ADH; and a chimeric gene with the coding region of the kivD gene optimized by codons of Lactococcus lactis (nt 9249-10895) expressed from the TDH3 promoter (nt 10896-11918) followed by the TDH3 terminator (nt 8237-9235) for the expression of KivD.

The coding regions for ketoisovalerate decarboxylase (KivD) of Lactococcus lactis and horse liver alcohol dehydrogenase (HADH) were synthesized by DNA2.0, Inc. (Menlo Park, CA) based on the codons that were optimized for expression in Saccharomyces cerevisiae (sec. with ident. no .: 71 and 118, respectively) and provided in the plasmids pKivDy-DNA2.0 and pHadhy-DNA2.0. The encoded proteins are sec. with numbers of ident .: 117 and 119, respectively. The individual expression vectors for KivD and HADH were constructed. To join pLH467 (pRS426:: PTDH3-kivDy-TDH3t), the vector pNY8 (sec.with ident.num .: 121; also referred to as pRS426.GPD-ald-GPDt, described in the patent application publication) from United States No. 2008/0182308, Example 17, which is incorporated by reference in the present description) was processed by digestion with the enzymes AscI and Sfil, thereby eliminating the GPD promoter and the ald coding region. A fragment of the TDH3 promoter (seq. No. 122) of pNY8 was amplified by PCR to add an AscI site to the 5 'end and a Spel site to the 3' end, by using the 5 'initiator OT1068 and the 3 'initiator OT1067 (sec. with ident. no .: 123 and 124). The fragment of the vector pNY8 digested with Ascl / Sfil was bound with the PCR product of the TDH3 promoter digested with AscI and Spel and the Spel-Sfil fragment containing the coding region optimized for codons isolated from the vector pKivD-DNA2.0. The triple union generated the vector pLH467 (pRS426:: PTDH3-kivDy-TDH3t). PLH467 was verified to determine restriction mapping and sequencing. pLH435 (pRS425:: PGPMl-Hadhy-ADHlt) was derived from vector pRS425:: GPM-sadB (sec.with ident.num .: 78) which is described in U.S. Provisional Application no. series 61 / 058,970, Example 3, which is incorporated by reference in the present description. pRS425 :: GPM-sadB is the vector pRS425 (ATCC No. 77106) with a chimeric gene containing the GPM1 promoter (sec. with ident.No .: 72), the coding region of a butanol dehydrogenase from Achromobacter xylosoxidans (sadB DNA with SEQ ID No. 69, Protein with SEQ ID No. 70: Described in U.S. Patent Application Publication No. 2009/0269823), and the ADH1 terminator (sec. with ident. no .: 73). pRS425:: GPMp-sadB contains the Bbvl and Pací sites at the 5 'and 3' ends of the sadB coding region, respectively. A Nhel site was added at the 5 'end of the sadB coding region by site-directed mutagenesis using primers OT1074 and OT1075 (sec.with ident Nos .: 126 and 127) to generate the vector pRS425-GPMp-sadB -NheI, which was verified by sequencing. pRS425:: PGPMl-sadB-Nhel was processed by digestion with Nhel and Pací to leave the coding region sadB, and joined with the Nhel-PacI fragment containing the coding region HADH optimized by codons of the vector pHadhy-DNA2.0 to create pLH435.

To combine the KivD and HADH expression cassettes in a single vector, the yeast vector pRS411 (ATCC No. 87474) was digested with SacI and NotI, and ligated with the Sacl-Sall fragment of pLH467 containing the cassette PTDH3-kivDy -TDH3t together with the Sall-Notl fragment of pLH435 containing the PGPMl-Hadhy-ADHlt cassette in a triple binding reaction. This produced the vector pRS411:: PTDH3-kivDy-PGPMl-Hadhy (pLH441) which was verified with restriction mapping.

To generate a co-expression vector for the three genes in the lower isobutanol route: ilvD, kivDy and Hadhy, pRS423 FBA ilvD (Strep) (sec. With ident. No .: 128) described in the publication of U.S. Patent Application No. 2010/0081154 as the source of the IlvD gene. This shuttle vector contains an origin of replication Fl (nt 1423 to 1879) for maintenance in E. coli and an origin of 2 microns (nt 8082 to 9426) for replication in yeast. The vector has an FBA1 promoter (nt 2111 to 3108; sec. with no. ID: 120) and an FBA terminator (nt 4861 to 5860; sec. with ident. no .: 129). In addition, it includes the His marker (nt 504 to 1163) for selection in yeast and the marker of ampicillin resistance (nt 7092 to 7949) for selection in E. coli. The ilvD coding region (nt 3116 to 4828; sec.with ident.ID: 115; Protein with sec.with ident.ind 116) of Streptococcus mutans UA159 (ATCC No. 700610) is between the FBA promoter and the FBA terminator and forms a chimeric gene for expression. In addition, a Lumio tag is fused to the ilvD coding region (nt 4829-4849).

The first step was to linearize pRS423 FBA ilvD (Strep) (also called pRS423-FBA (Spel) -IlvD { Streptococcus mutans) -Lumium) with SacI and SacII (with the SacII site generated with blunt ends by the use of T4 DNA polymerase), to give a vector with a total length of 9,482 bp. The second step was to isolate the kivDy-hADHy cassette from pLH441 with SacI and Kpnl (with the Kpnl site generated with blunt ends by the use of T4 DNA polymerase), which gives a fragment of 6.063 bp. This fragment was ligated with the 9,482 bp vector fragment of pRS423-FBA (Spel) -IlvD (Streptococcus mutans) -Lumium. This generated vector pLH468 (pRS423:: PFBA1-ilvD (Strep) Lumio-FBAlt-PTDH3-kivDy-TDH3t-PGPMl-hadhy-ADHlt) was confirmed with restriction mapping and sequencing.

Construction of pLH532 The pLH532 plasmid (sec.with ident.ID: 130) was constructed for expression of ALS and KARI in yeast. pLH532 is a pHR81 vector (ATCC No. 87541) containing the following chimeric genes: 1) the CUP1 promoter (sec with ident number: 139), coding region of the acetolactate synthase of Bacillus subtilis (AlsS; Ident No .: 137; Protein with sec. with no. of ident. 138) and terminator CYC1 2 (sec. With ident. No .: 133); 2) an ILV5 promoter region (sec. With ID: 134), coding region Pf5.IlvC (sec.with ID .: 132) and ILV5 terminator (sec. With ID: 135) ); and 3) the promoter region FBA1 (sec. with ident. no .: 136), KARI coding region of S. cerevisiae (ILV5, sec. with ident. no .: 131); and the CYC1 terminator.

The Pf5.IlvC coding region is a sequence encoding KARI derived from Pseudomonas fluorescens, as described in U.S. Patent Application Publication no. 2009/0163376, which is incorporated by reference in the present description.

The Pf5.IlvC coding region was synthesized by DNA2.0, Inc. (Menlo Park, CA, sec.with ident .: 132) based on the codons that were optimized for expression in Saccharomyces cerevisiae.

Construction of pYZ090 pYZ090 (sec. with ID No. 1) is based on the main strand of pHR81 (ATCC No. 87541) and was constructed to contain a chimeric gene with the coding region of the alsS gene of Bacillus subtilis (nt position 457- 2172) of the yeast CUPl promoter (nt 2-449) and followed by the CYC1 terminator (nt 2181-2430) for the expression of ALS, and a chimeric gene with the coding region of the ilvC gene of Lactococcus lactis (nt 3634- 4656) expressed from the yeast ILV5 promoter (2433-3626) and followed by the ILV5 terminator (nt 4682-5304) for the expression of KARI.

Construction of pYZ067 pYZ067 was constructed to contain the following chimeric genes: 1) the coding region of the ilvD gene of S. mutans UA159 (nt position 2260-3971) expressed from the yeast FBA1 promoter (nt 1161-2250) followed by the FBA terminator (nt 4005 -4317) for the expression of dihydroxy acid dehydratase (DHAD), 2) the coding region for horse liver ADH (nt 4680-5807) expressed from the yeast GPM promoter (nt 5819-6575) followed by the ADH1 terminator ( nt 4356-4671) for the expression of alcohol dehydrogenase, and 3) the coding region of the KivD gene of Lacrococcus lactis (nt 7175-8821) expressed from the yeast TDH3 promoter (nt 8830-9493) followed by the TDH3 terminator (nt 5682 -7161) for the expression of ketoisovalerate decarboxylase.

Construction of pRS423:: CUPl-alsS + FBA-budA and pRS426 :: FBA-budC + GPM-sadB and pLH475-Z4B8 The construction of pRS423 :: CUP1-alsS + FBA-budA and pRS426:: FBA-budC + GPM-sadB and pLH475-Z4B8 is described in the publication of the United States patent application no. 2009/0305363, incorporated by reference in the present description.

EXAMPLES The following non-limiting examples will illustrate the invention in more detail. It should be understood that, while the following examples involve the use of corn as a raw material and COFA as an ISPR extractant obtained from the hydrolysis of corn lipids, other sources of biomass can be used as a raw material and for the enzymatic hydrolysis of oil. of biomass, without departing from the present invention.

As used in the present description, the meaning of the abbreviations used is as follows: "g" means gram (s), "kg" means kilogram (s), "1" means liter (s), "mi" means milliliter (s), "μ?" means microliter (s), "ml / 1" means milliliter (s) per liter, "ml / min" means millilit or (s) per minute, "DI" means deionized, "uM" means micrometer (s), "nm "means nanometer (s)," p / v "means weight / volume," DO "means optical density," ?? e ?? " means an optical density at a wavelength of 600 nM, "psc" means cellular dry weight, "rpm" means revolutions per minute, "° C" means degrees Celsius, "° C / min" means degrees Celsius per minute, " "lepm" means standard liters per minute, "ppm" means part per million, "pdc" means pyruvate decarboxylase enzyme followed by the number of enzyme.

General methods Growing in sowing flask A strain of Saccharomyces cerevisiae that was modified to produce isobutanol from a carbohydrate source, with suppression of pdcl, pdc5 and pdc6, was cultured at 0.55-1.1 g / 1 psc (DO600 1.3-2.6 - Thermo Helios OÍ Thermo Fisher Scientific Inc., Waltham, Massachusetts) in seed flasks from a frozen culture. Culturing was carried out at 26 ° C in an incubator rotating at 300 rpm. The frozen culture was previously stored at -80 ° C. The composition of the medium of the first sowing flask was: 3. 0 g / 1 dextrose 3. 0 g / 1 of ethanol, anhydrous 3. 7 g / 1 of half full amino acid ForMedium ™ (Kaiser): without HIS, without URA (reference number DSCK162CK) 6.7 g / 1 of nitrogenous base without Difco amino acids (No. 291920) 12 milliliters of the culture from the first seed flask was transferred to a 2 liter flask and cultured at 30 ° C in an incubator rotating at 300 rpm. The second sowing flask has 220 ml of the following medium: 30. 0 g / 1 dextrose 5. 0 g / 1 of ethanol, anhydrous 3. 7 g / 1 of half full amino acid ForMedium ™ (Kaiser): without HIS, without URA (reference number DSCK162CK) 6.7 g / 1 of nitrogen base of yeast without Difco amino acids (No. 291920) 0. 2 M of MES buffer solution titrated at a pH of 5.5-6.0 The culture was performed at 0.55-1.1 g / 1 psc (?? ± 1.3-2.6). An addition of 30 ml of a solution containing 200 g / 1 of peptone and 100 g / 1 of yeast extract was made at this cell concentration. Then, an addition of 300 ml of 0.2 uM of filter sterilized Cognis was made, and 90-95% oleyl alcohol was added to the flask. The culture was allowed to grow > 4 g / 1 psc (D060o> 10) before being harvested and added to the fermentation.

Preparation of fermentation Initial preparation of the fermentation vessel A 2 liter glass jacketed fermentation vessel (Sartorius AG, Goettingen, Germany) was charged with internal water at 66% liquifying weight. A pH probe (Hamilton Easyferm Plus K8, part no .: 238627, Hamilton Bonaduz AG, Bonaduz, Switzerland) was calibrated using the control tower calibration menu Sartorius DCU-3. The zero was calibrated at pH = 7. The amplitude was calibrated at a pH = 4. Then, the probe was placed in a fermentation vessel through the stainless steel head plate. A dissolved oxygen probe (probe p02) was also placed in the fermentation vessel through the head plate. Used tubes were fixed to supply nutrients, the seed culture, the extraction solvent and the base in the head plate and the ends were laminated. The entire fermentation vessel was placed in a Steris autoclave (Steris Corporation, Mentor, Ohio) and sterilized in a liquid cycle for 30 minutes.

The fermentation vessel was removed from the autoclave and placed in a load cell. The cooling water supply and the return line were connected to the internal water and drained, respectively. The condenser inlet and outlet cooling water lines were connected to a 6-L recirculation temperature bath at 7 ° C. The ventilation line that transfers the gas from the fermentation vessel was connected to a line of passage that was connected to a Thermo mass spectrometer (Prima dB, Thermo Fisher Scientific Inc., Waltham, Massachusetts). The spray tube line was connected to the gas supply line. Tubes to add nutrients, extraction solvent, seed culture and base were connected through pumps or clamped.

The temperature of the fermentation vessel was controlled at 55 ° C with a thermocouple and a circulation circuit. internal water. The wet corn kernels (yellow notch No. 2) were milled by using a grinder mill with a 1.0 mm screen, and the resulting ground whole corn kernels were subsequently added in the fermentation vessel at a load of 29 to 30% (dry solids weight of the corn) of the liquefaction reaction mass.

Treatment with lipase prior to liquefaction A lipase enzyme stock in the fermentation vessel was added to a final lipase concentration of 10 ppm. The fermentation vessel was maintained at 55 ° C, 300 rpm and 0.3 lepm of N2 superimposed during > 6 hours. Once the lipase treatment was finished, the liquefaction was performed as described below (Liquefaction).

Liquefaction An alpha-amylase was added to the fermentation vessel according to its specification sheet while the fermentation vessel was mixing at 300-1200 rpm, with the addition of sterile internal N2 at 0.3 rpm through the spray tube. The determined temperature value from 55 ° C to 85 ° C was changed. When the temperature was > 80 ° C, the liquefaction cooking time was started and the liquefaction cycle was maintained at > 80 ° C for 90-120 minutes. The determined temperature value of the fermentation vessel was established at the fermentation temperature of 30 ° C after the liquefaction cycle was completed. N2 was redirected from the spray tube to the head space to prevent foaming without the addition of a defoaming guiding agent.

Treatment with lipase after liquefaction The temperature of the fermentation vessel was set at 55 ° C instead of 30 ° C after the liquefaction cycle was complete (Liquefaction). The pH was manually controlled at pH = 5.8 by adding boluses of acid or base as needed. A lipase enzyme stock in the fermentation vessel was added to a final lipase concentration of 10 ppm. The fermentation vessel was maintained at 55 ° C, 300 rpm and 0.3 lepm of N2 superimposed during > 6 hours. Once the lipase treatment was finished, the temperature of the fermentation vessel was set at 30 ° C.

Heat inactivation treatment of lipase (heat death treatment method) The temperature of the fermentation vessel was maintained a > 80 ° C during > 15 minutes to inactivate the lipase. After the heat inactivation treatment, the temperature of the fermentation vessel was set at 30 ° C.

Addition of nutrients prior to inoculation Ethanol (6.36 ml / 1, volume after inoculation, 200 graduation, anhydrous) was added to the fermentation vessel just before inoculation. Thiamin was added to the final concentration of 20 mg / 1 and, in addition, 100 mg / 1 of nicotinic acid was added just before inoculation.

Addition of oleyl alcohol or corn oil fatty acids prior to inoculation 1 1/1 (post-inoculation volume) of oleyl alcohol or corn oil fatty acids was added immediately after inoculation.

Inoculation of the fermentation vessel The probe p02 of the fermentation vessels was calibrated to zero while adding N2 in the fermentation vessel. The probe p02 of the fermentation vessels is. calibrated to its amplitude with sterile water treatment at 300 rpm. The fermentation vessel was inoculated after the second seed flask with > 4 g / l of psc. The shake flask was removed from the incubator / stirrer for 5 minutes to allow phase separation of the oleyl alcohol phase and the aqueous phase. The aqueous phase (110 ml) was transferred to a sterile inoculation bottle. The inoculum was pumped into the fermentation vessel through a peristaltic pump.

Operational conditions of the fermentation vessel The fermentation vessel was operated at 30 ° C during the complete growth and production stages. The pH was dropped from a pH between 5.7-5.9 to a determined control value of 5.2 without adding acid. The pH was monitored during the rest of the growth and production stages at a pH = 5.2 with ammonium hydroxide. Sterile air was added into the fermentation vessel through the spray tube, at 0.3 lepm during the rest of the growth and production stages. P02 was set to be controlled at 3.0% by the PID control circuit of the Sartorius DCU-3 control box, by using agitation control only, and a minimum of 300 rpm and a maximum of 2000 rpm was set for the agitator . Glucose was delivered through the simultaneous saccharification and fermentation of the liquefied corn paste by adding α-amylase (glucoamylase). An excess of glucose (1-50 g / 1) was maintained during the time that the starch was available for saccharification.

Analytical method Gas analysis The process air was analyzed in a Thermo Prima mass spectrometer (Thermo Fisher Scientific Inc., altham, assachusetts). It was the same process air that was sterilized and then added to each fermentation vessel. The gaseous effluent from each fermentation vessel was analyzed in the same mass spectrometer. A verification of the Thermo Prima dB calibration was carried out every Monday at 6:00 a.m. Calibration verification was scheduled through the Gas Works vl.O software (Thermo Fisher Scientific Inc., Waltham, Mass.) Associated with the mass spectrometer. The calibrated gas was: GAS% concentration in moles Frequency of calibration calibration Nitrogen 78% weekly Oxygen 21% weekly Isobutanol 0.2% annually Argon 1% weekly Dioxide 0.03% weekly carbon Carbon dioxide was checked at 5% and 15% during the calibration cycle with other known packaged gases. Oxygen was verified at 15% with other known packaged gases. Based on the analysis of the gaseous effluent from each fermentation vessel, the amount of stripped isobutanol, oxygen consumed and carbon dioxide breathed in the gaseous effluents was measured by the use of a molar fraction analysis of the mass spectrometer and flow rates. gaseous (mass flow controller) in the fermentation vessel. The gasification index was calculated per hour and then that index was integrated with the duration of the fermentation.

Biomass measurement A solution of 0.08% trypan blue was prepared from a 1: 5 dilution of 0.4% trypan blue in NaCl (VWR BDH8721-0) with IX PBS. A 1.0 ml sample was extracted from a fermentation vessel and placed in a 1.5 ml Eppendorf centrifuge tube and centrifuged in an Eppendorf, 5415C at 14,000 rpm for 5 minutes. After centrifugation, the upper solvent layer was extracted with a variable channel pipette of m200 BioHit with pipette tip of 20-200 μ? BioHit. Special attention was paid not to extract the layer between the solvent and the aqueous layers. After extracting the solvent layer, the sample was resuspended by using a Vortex-Genie® set at 2700 rpm.

A series of dilutions were required to prepare the ideal concentration for the hemocytometer counts. If the OD was 10, a 1:20 dilution was made to achieve 0.5 OD, which would provide the ideal number of cells for the count per square, 20-30. To reduce inaccuracies in dilution due to corn solids, several dilutions were required with pipette tips of 100-1000 μ? BioHit cut out. The tips were cut approximately 1 cm to increase the opening and prevent the tip from becoming clogged. For a final dilution of 1:20, a 1: 1 dilution of the fermentation sample and 0.9% NaCl solution was prepared. Next, a 1: 1 dilution of the above solution (i.e., the initial 1: 1 dilution) and the 0.9% NaCl solution (the second dilution) was generated followed by a 1: 5 dilution of the second dilution and the trypan blue solution. The samples were shaken between each dilution and the cut ends were rinsed in solutions of 0.9% NaCl and trypan blue.

The coverslip was carefully placed over the hemocyte (Hausser Scientific Bright-Line 1492). An aliquot (10 μ?) Of the final dilution of trypan blue was extracted with a variable channel pipette m20 BioHit with pipette tips of 2-20 μ? BioHit and injected into the hemocytometer. The hemocytometer was placed in the Zeis Axioskop 40 microscope at a magnification of 40x. The central quadrant was divided into 25 squares and the counting and registration of the four squares of the angles and the center in both chambers was performed. After counting the chambers, the average was obtained and multiplied by the dilution factor (20), then by 25 for the number of squares in the quadrant in the hemocyte, and was subsequently divided by 0.0001 mi, which is the quadrant volume that was posted. The sum of this calculation is the number of cells per me.

Liquid chromatography (LC) analysis of fermentation products in the aqueous phase The samples were refrigerated until they were ready for processing. The samples were extracted from the refrigeration and allowed to reach room temperature (approximately one hour). Approximately 300 μ? of the sample was transferred with a mlOOO BioHit variable channel pipette with pipette tips of 100-1000 μ? BioHit to a 0.2 um centrifugal filter (modified Nanosep® MF nylon centrifuge filter) and then centrifuged with Eppendorf, 5415C for five minutes at 14,000 rpm. Approximately 200 μ? of the filtered sample was transferred to a 1.8 autosampler vial with a 250 μm glass vial. insert with polymer support. A threaded cap with PT FE partition was used to cover the vial before shaking the sample with a Vort ex-Genie® assembly at 2700 rpm.

The sample was then subjected to an Agilent 1200-series LC equipped with isocratic binary pumps, vacuum desiccator, heated column compartment, sampling cooling system, UV DAD detector and RI detector. The column used was Aminex HPX-87H, 300 X 7.8 with a filling of cation H Bio-Rad, precolumn 30X4.6. The temperature of the column was 40 ° C, with a mobile phase of 0.01 N of sulfuric acid at a flow rate of 0.6 ml / min for 40 minutes. The results are shown in Table 1.

Table 1. Retention times of fermentation products in aqueous phase Gas chromatography (GC) analysis of the fermentation products in the solvent phase The samples were refrigerated until they were ready for processing. The samples were extracted from the refrigeration and allowed to reach room temperature (approximately one hour). Approximately 150 μ? of the sample was transferred by using a mlOOO BioHit variable channel pipette with pipette tips of 100-1000 μ? BioHit to a 1.8 autosampler vial with a 250 μm glass vial? insert with polymer support. A threaded cap with a PTFE partition was used to cover the vial.

The sample was subjected to the Agilent 7890A gas chromatograph with a 7683B injector and a G2614A autosampler. The column was an HP-InnoWax column (30m x 0.32mm ID, 0.25μm film). The carrier gas was helium at a flow rate of 1.5 ml / min measured at 45 ° C with a constant head pressure; the division of the injector was from 1:50 to 225 ° C; oven temperature was 45 ° C for 1.5 minutes, 45 ° C to 160 ° C at 10 ° C / min for 0 minutes, after 230 ° C at 35"C / min for 14 minutes for a run time 29 minutes The flame ionization detection was used at 260 ° C with auxiliary helium gas of 40 ml / min.The results are shown in Table 2.

Table 2. Retention times of fermentation products in the solvent phase Samples analyzed for fatty acid butyl esters were subjected to an Agilent 6890 gas chromatograph with a 7683B injector and a G2614A autosampler. The column was an HP-DB-FFAP column (15 meters x 0.53 mm ID (Megabore), 1 micron film thickness column (30 mx 0.32 mm ID, 0.25 μp film) .The carrier gas was helium at a flow rate of 3.7 mi / min measured at 45 ° C with a constant head pressure, the injector division was from 1:50 to 225 ° C, the oven temperature was 100 ° C for 2.0 minutes, 100 ° C at 250 ° C at 10 ° C / min, after 250 ° C for 9 minutes for a run time of 26 minutes Flame ionization detection at 300 ° C with helium 40 ml helium gas was used The following GC standards (Nu-Chek Prep, Elysian, MN) were used to confirm the identity of the isobutyl ester products of fatty acids: isobutyl palmitate, isobutyl stearate, isobutyl oleate, isobutyl linoleate, isobutyl linolenate, Isobutyl arachididate Examples 1 to 14 describe various fermentation conditions that can be used for the claimed methods. As an example, some fermentations were subjected to lipase treatment prior to liquefaction and others to lipase treatment after liquefaction. In other examples, the fermentation was subjected to the heat inactivation treatment. After the fermentation, the effective titration of isobutanol (tit. Ef of iso), ie the total grams of isobutanol produced per liter in aqueous volume, was measured. The results are shown in Table 3.

Example 1 (control) Experimental identifier 2010Y014 included: Growth method in seed flask, method of initial preparation of the fermentation vessel, liquefaction method, method of addition of nutrients prior to inoculation, method of inoculation of the fermentation vessel, method of operating conditions of the fermentation vessel, and all analytical methods. Oleyl alcohol was added in a single group between 0.1-1.0 hours after inoculation. The butanologen was NGCI-070.

Example 2 The experimental identifier 2010Y015 included: Growth method in seed flask, method of initial preparation of the fermentation vessel, liquefaction method, lipase treatment method after liquefaction, method of nutrient addition prior to inoculation, method of inoculation of the fermentation vessel, method of operating conditions of the fermentation vessel, and all analytical methods. Oleyl alcohol was added in a single group between 0.1-1.0 hours after inoculation. The butanologen was NGCI-070.

Example 3 The experimental identifier 2010Y016 included: Growth method in seed flask, initial preparation method of the fermentation vessel, liquefaction method, lipase treatment method after liquefaction, pre-inoculation method of nutrient addition except for the exclusion of ethanol, inoculation method of the fermentation vessel, method of operating conditions of the fermentation vessel, and all analytical methods. Oleyl alcohol was added in a single group between 0.1- 1.0 hour after inoculation. The butanologen was NGCI-070.

Example 4 Experimental identifier 2010Y017 included: Seed flask growth method, initial fermentation vessel preparation method, liquefaction method, post-liquefaction heat death treatment method, nutrient addition method prior to inoculation except for the exclusion of ethanol, method of inoculation of the fermentation vessel, method of operating conditions of the fermentation vessel, and all analytical methods. Oleyl alcohol was added in a single group between 0.1- 1.0 hour after inoculation. The butanologen was NGCI-070.

Example 5 Experimental identifier 2010Y018 included: Growth method in seed flask, initial preparation method of the fermentation vessel, liquefaction method, lipase treatment method after liquefaction except for the addition of 7.2 ppm of lipase after liquefaction, method of treatment of death by heat after liquefaction, method of addition of nutrients prior to inoculation, method of inoculation of the fermentation vessel, method of operating conditions of the fermentation vessel, and all analytical methods. Oleyl alcohol was added in a single group between 0.1-1.0 hours after inoculation. The butanologen was NGCI-070.

Example 6 (control) Experimental identifier 2010Y019 included: Seed flask growth method, initial fermentation vessel preparation method, post-liquefying heat death treatment method, pre-inoculation nutrient addition method, vessel inoculation method of fermentation, method of operating conditions of the fermentation vessel, and all analytical methods. Oleyl alcohol was added in a single group between 0.1-1.0 hours after inoculation. The butanologen was NGCI-070.

Example 7 (control) Experimental identifier 2010Y021 included: Growth method in seed flask, initial preparation method of the fermentation vessel, lipase treatment method prior to liquefaction, heat death treatment method during liquefaction, subsequent nutrient addition method to inoculation, method of inoculation of the fermentation vessel, method of operating conditions of the fermentation vessel, and all analytical methods. Oleyl alcohol was added in a single group between 0.1- 1.0 hour after inoculation. The butanologen was NGCI-070.

Example 8 Experimental identifier 2010Y022 included: Growth method in seed flask, method of initial preparation of fermentation vessel, liquefaction method, method of addition of nutrients prior to inoculation, method of inoculation of the fermentation vessel, method of operating conditions of the fermentation vessel, and all analytical methods. Oleyl alcohol was added in a single group between 0.1- 1.0 hour after inoculation. The butanologen was NGCI-070.

Example 9 Experimental identifier 2010Y023 included: Growth method in seed flask, method of initial preparation of the fermentation vessel, liquefaction method, l-ipase treatment method after liquefaction, without heat death treatment, method of addition of post-inoculation nutrients, inoculation method of the fermentation vessel, method of operating conditions of the fermentation vessel, and all analytical methods. The corn oil fatty acids made from crude corn oil were added in a single group between 0.1 and 1.0 hour after inoculation. The butanologen was NGCI-070.

Example 10 Experimental identifier 2010Y024 included: Growth method in seed flask, method of initial preparation of the fermentation vessel, lipase treatment method prior to liquefaction, liquefaction method, treatment of heat death during liquefaction, method of addition of nutrients prior to inoculation except that there is no addition of ethanol, method of inoculation of the fermentation vessel, method of operating conditions of the fermentation vessel, and all analytical methods. Oleyl alcohol was added in a single group between 0.1-1.0 hours after inoculation. The butanologen was NGCI-070.

Example 11 Experimental identifier 2010Y029 included: Growth method in seed flask, method of initial preparation of the fermentation vessel, lipase treatment method prior to liquefaction, liquefaction method, treatment of heat death during liquefaction, method of addition of nutrients prior to inoculation, method of inoculation of the fermentation vessel, method of operating conditions of the fermentation vessel, and all analytical methods. The corn oil fatty acids made from crude corn oil were added in a single group between 0.1 and 1.0 hour after inoculation. The butanologen was NGCI-070.

Example 12 Experimental identifier 2010Y030 included: Growth method in seed flask, method of initial preparation of the fermentation vessel, lipase treatment method prior to liquefaction, liquefaction method, treatment of heat death during liquefaction, method of addition of nutrients prior to inoculation except that there is no addition of ethanol, method of inoculation of the fermentation vessel, method of operating conditions of the fermentation vessel, and all analytical methods. The corn oil fatty acids made from crude corn oil were added in a single group between 0.1 and 1.0 hour after inoculation. The butanologen was NGCI-070.

Example 13 - (control) Experimental identifier 2010Y031 included: Growth method in seed flask, method of initial preparation of the fermentation vessel, liquefaction method, lipase treatment method after liquefaction, no heat death treatment, previous method of nutrient addition to inoculation except that there is no addition of ethanol, method of inoculation of the fermentation vessel, method of operating conditions of the fermentation vessel, and all analytical methods. The corn oil fatty acids made from crude corn oil were added in a single group between 0.1 and 1.0 hour after inoculation. The butanologen was NGCI-070.

Example 14 Experimental identifier 2010Y032 included: Growth method in seed flask, initial preparation method of the fermentation vessel, liquefaction method, lipase treatment method after liquefaction, no heat death treatment, subsequent nutrient addition method to inoculation, method of inoculation of the fermentation vessel, method of operating conditions of the fermentation vessel, and all analytical methods. The corn oil fatty acids made from crude corn oil were added in a single group between 0.1 and 1.0 hour after inoculation. The butanologen was NGCI-070.

Table 3. Fermentation conditions for examples 1-14 5 5 15 * The "tit. Ef of iso in g / 1" = total grams of isobutanol produced per liter in aqueous volume Example 15 The experimental identifier was GLNOR432A. NGCI-047 (butanediol producer) was grown in a 25 ml medium in a 250 ml flask of a frozen vial at ~ 1 OD. The previous seed culture was transferred to a 2 liter flask and cultured at 1.7-1.8 OD. The medium for both flasks was: 3. 0 g / 1 dextrose 3. 0 g / 1 of ethanol, anhydrous 6. 7 g / 1 nitrogenous base without Difco amino acids (No. 291920) 1. 4 g / 1 of yeast-deficient mixture (Sigma Y2001) 10 ml / 1 of 1% w / v of L-Leucine raw material 2 ml / 1 of 1% w / v raw material of L-tryptophan A 1 liter fermentation vessel Applikon was inoculated with 60 ml of the seed flask. The fermentation vessel contained 700 ml of the following sterile medium: 20. 0 g / 1 dextrose 8. 0 ml / 1 ethanol, anhydrous 6. 7 g / 1 nitrogenous base without Difco amino acids (No. 291920) 2. 8 g / 1 of yeast-deficient mixture (Sigma Y2001) 20 ml / 1 of 1% w / v of L-leucine raw material 4 ml / 1 of 1% w / v of raw material of L-tryptophan 0. 5 ml of antifoam Sigma 204 0. 8 ml / 1 of 1% w / v Ergesterol solution in l: l :: Tween 80: Ethanol The residual glucose was kept in excess with a glucose solution of 50% by weight. The dissolved oxygen concentration of the fermentation vessel was controlled at 30% under stirring control. The pH was controlled at pH = 5.5. The fermentation vessel was cleaned with 0.3 lpm of sterile internal air. The temperature was controlled at 30 ° C.

Example 16 The experimental identifier was GLNOR434A. This example is the same as Example 15 except for the addition of 3 g of oleic acid and the addition of 3 g of palmitic acid before inoculation. NGCI-047 (butanediol producer) was the biological catalyst.

Figure 6 shows that there were more grams per liter of glucose consumed in the fermentation vessel that received the fatty acids. The squares represent the fermentation vessel that received oleic acid and palmitic acid. The circles represent the fermentation vessel that did not receive additional fatty acids.

Example 17 The experimental identifier was GLNOR435A. This example is the same as Example 15 except that it was inoculated with NGCI-049 (producer of isobutanol).

Example 18 The experimental identifier was GLNOR437A. This example is the same as Example 16 except that it was inoculated with NGCI-049 (producer of isobutanol).

Figure 7 shows that it speaks more grams per liter of glucose consumed in the fermentation vessel that received the fatty acids. The squares represent the fermentation vessel that received oleic acid and palmitic acid. The circles represent the fermentation vessel that did not receive additional fatty acids.

Example 19 The experimental identifier was 090420_3212. This example was carried out in a manner similar to Example 15 except that it was inoculated with butanologen NYLA84 (isobutanol producer). This fermentation was carried out in a 1 liter Sartorius fermentation vessel.

Example 20 The experimental identifier was 2009Y047. This example was carried out in a manner similar to Example 16 except that it was inoculated with butanologen NYLA84 (isobutanol producer). This fermentation was carried out with a 1 liter Sartorius fermentation vessel.

Figure 8 shows that there were more grams per liter of glucose consumed in the fermentation vessel that received the fatty acids. The squares represent the fermentation vessel that received oleic acid and palmitic acid. The circles represent the fermentation vessel that did not receive additional fatty acids. The results of Examples 15 to 20 are shown in Table 4, which illustrates the addition of ± fatty acids, the maximum optical density and the glucose consumed in g / 1.

Table 4 Example 21 Lipase treatment of liquefied corn paste for simultaneous saccharification and fermentation with extraction of the product in situ by the use of oleyl alcohol Samples of broth and oleyl alcohol were taken from the fermentations performed as described in Examples 1, 2 and 3 and analyzed to determine the percentage by weight of lipids (derivatives such as methyl esters of fatty acids, FAME) and the percentage by weight of free fatty acids (FFA, methyl esters of fatty acids, FAME) according to the method described in EG Bligh and WJ Dyer (Canadian Journal of Biochemistry and Physiology, 37: 911-17, 1959, hereinafter, Reference 1). The liquefied corn paste prepared for each of the three fermentations was also analyzed to determine the percentage by weight of lipids and FFA after treatment with Lipolase® 100 L (Novozymes) (10 ppm of total soluble protein Lipolase® (analysis of BCA proteins, Sigma Aldrich)) per kg of liquefaction reaction mass containing 30% by weight ground corn kernels). No lipase was added to the liquefied corn paste in Example 1 (control), and the fermentations described in Examples 2 and 3 containing liquefied corn paste treated with lipase (without heat inactivation of lipase) were identical except that Ethanol was added in the fermentation described in Example 3.

The percentage of FFA in the liquefied corn pulp treated with lipase prepared for the fermentations that were carried out as described in Examples 2 and 3 was 88% and 89%, respectively, compared to 31% without the lipase treatment (Example 1) . At 70 hours (end of run (EOR)), the concentration of FFA in the OA phase of the fermentations performed as described in Examples 2 and 3 (containing active lipase) was 14% and 20%, respectively, and it was determined by GC / MS that the corresponding increase in lipids (measured as fatty acid methyl ester derivatives of corn oil) was due to the lysing catalyzed eduction of COFA by OA, where COFA was first produced by lipase-catalyzed hydrolysis of corn oil in liquefied corn pulp. The results are shown in Table 5.

Table 5. Content of lipids and free fatty acids of fermentations containing oleyl alcohol as ISPR solvent and active lipase Fermentation Lipase Time Lipids FFA Lipids FFA lipids% (h), (% in (% in (g) (g) + FFA shows weight) weight) FFA (g) Example 1 None Pasta 0.61 0.28 573 274 7.7 31 liquefied Example 1 None 0.8 h, 0.49 0.22 5.5 2.5 8.0 31 broth Fermentation Lipase Time Lipids FFA Lipids FFA Lipidos% (h), (% in (% in (g) (g) + FFA shows weight) weight) FFA (g) Example 1 None 31 h, 0.19 0.03 2.1 0.3 2.4 13 broth Example 1 None 31 h, 0.36 0.21 3.4 2.0 5.3 37 OA Example 1 None 70 h, 0.15 0.03 1.7 0.3 2.0 15 broth Example 1 None 70 h, 0.57 0.25 5.3 2.3 7.7 31 OA Example 2 10 ppm Paste 0.13 0.97 1.1 8.5 9.6 88 liquefied Example 2 10 ppm 0.8 h, 0.15 0.62 1.7 7.0 8.7 81 broth Example 2 10 ppm 31 h, 0.16 0.05 1.8 0.5 2.3 23 broth Example 2 10 ppm 31 h, 0.37 0.23 3.5 2.2 5.7 38 OA Example 2 10 ppm 70 h, 0.17 0.02 1.9 0.3 2.2 13 broth Example 2 10 ppm 70 h, 0.60 0.10 5.7 1.0 6.7 14 OA Example 3 10 ppm Paste 0.12 0.97 1.0 8.5 9.5 89 liquefied Fermentation Lipase Time Lipids FFA Lipids FFA Lipidos% (h), (% in (% in (g) (g) + FFA shows weight) weight) FFA (g) Example 3 10 ppm 0.8 h, 0.32 0.40 3.6 4.5 8.1 56 broth Example 3 10 ppm 31 h, 0.17 0.05 1.9 0.6 2.5 24 broth Example 3 10 ppm 31 h, 0.38 0.22 3.6 2.1 5.7 37 OA Example 3 10 ppm 70 h, 0.15 0.02 1.7 0.2 1.9 13 broth Example 3 10 ppm 70 h, 0.46 0.12 4.4 1.1 5.6 20 OA Example 22 Heat inactivation of lipase in liquefied corn paste treated with lipase to limit the production of oleyl alcohol esters of free fatty acids from corn oil Tap water (918.4 g) was added in a 2 liter resin jacketed kettle; subsequently, 474.6 g wet weight (417.6 g dry weight) of ground whole corn kernels (1.0 mm sieve in grinding mill) was added under agitation. The mixture was heated to 55 ° C under stirring at 300 rpm and the pH adjusted to 5.8 with 2 N of sulfuric acid. To the mixture was added 14.0 g of an aqueous solution containing 0.672 g of Spezyme®-FRED L (Genencor®, Palo Alto, CA), and the sample temperature was increased to 85 ° C under agitation at 600 rpm and a pH of 5.8. After 120 minutes at 85 ° C, the mixture was cooled to 50 ° C and aliquots of 45.0 ml of the resulting liquefied corn paste were transferred to 50 ml polypropylene centrifuge tubes, which were stored frozen at -80 ° C.

In a first reaction, 50 g of the liquefied corn paste prepared as described above was mixed with 10 ppm of Lipolase® 100 L (Novozymes) for 6 h at 55 ° C and without lipase inactivation at 85 ° C for 1 h; The mixture was cooled to 30 ° C. In a second reaction, 50 g of liquefied corn paste was mixed with 10 ppm of Lipolase® for 6 h at 55 ° C, then heated at 85 ° C for 1 h (inactivation of lipase) and subsequently cooled to 30 ° C. In a third reaction, 50 g of liquefied corn paste without added lipase was mixed for 6 h at 55 ° C, and unheated at 85 ° C for 1 h, the mixture was cooled to 30 ° C, 38 g of alcohol was added. of oleyl and the resulting mixture was stirred for 73 h at 30 ° C. In a fourth reaction, 50 g of liquefied corn paste without added lipase was mixed for 6 h at 55 ° C, then heated at 85 ° C for 1 h and subsequently cooled to 30 ° C. A sample was taken from each of the reaction mixtures at 6 hours, then, 38 g of oleyl alcohol was added and the resulting mixtures were stirred at 30 ° C and samples were taken at 25 and 73 hours. The samples (both liquefied paste and oleyl alcohol (OA)) were analyzed to determine the percentage by weight of lipids (derivatized as methyl esters of fatty acids, FAME) and the percentage by weight of acids Free fatty acids (FFA, derivatized as methyl esters of fatty acids, FAME) according to the method described in Reference 1.

The percentage of FFA in the OA phase of the second reaction that was performed with heat inactivation of lipase before the addition of OA was 99% at 25 hours and 95% at 73 hours, compared to only 40%. % FFA and 21% FFA at -25 hours and 73 hours, respectively, when the lipase in the liquefied corn paste treated with lipase is not inactivated by heat (first reaction). No significant changes were observed in the percentage of FFA in the two control reactions without added lipase. The results are shown in Table 6.

Table 6. Lipid and free fatty acid content of a mixture of liquefied corn paste and oleyl alcohol in the presence or absence of active or heat-inactivated lipase Lipid Time Conditions FFA Lipids FFA Lipids +% reaction (h), (% in (% in (mg) (mg) FFA (mg) FFA shows weight) weight) 10 ppm of 6 h, paste 0.08 0.71 41 345 386 89 active lipase, liquefied without treatment 25 h, 0.22 0.06 105 27 132 20 by heat to paste 85 ° C liquefied Lipid Time Conditions FFA Lipids FFA Lipids +% reaction (h), (% in (% in (mg) (mg) FFA (mg) FFA shows weight) weight) pasta treatment by heat to liquefied 85 ° C 25 h, OA 0.58 39 212 143 355 40 73 h, 0.25 05 121 22 143 18 pasta liquefied 73 h, OA 0.91 0.24 333 88 420 21 10 ppi of 6 h, paste 0.06 0.45 28 224 252 89 liquefied lipase inactive, Treatment 25 h, 0.10 0.11 49 54 103 53 by heat to paste 85 ° C liquefied 25 h, OA 0.02 0.96 8 366 374 99 73 h, 0.24 0.15 117 72 189 62 pasta liquefied 73 h, OA 0.06 1.11 23 424 447 95 Lipid Time Conditions FFA Lipids FFA Lipids +% reaction (h), (% in (% in (mg) (mg) FFA (mg) FFA shows weight) weight) without lipase, 6 h, paste 0.80 0.40 401 199 599 33 liquefied without 25 h, '0.30 0.05 147 25 173 15 pasta treatment by heat to liquefied 85 ° C 25 h, OA 0.55 0.36 212 139 351 40 73 h, 0.23 0.05 117 26 143 23 pasta liquefied 73 h, OA 0.79 0.42 305 162 467 34 without lipase, 6 h, paste 0.36 370 183 553 33 liquefied treatment 25 h, 0.31 0.05 156 27 183 15 by heat to paste 85 ° C liquefied 25 h, OA 0.60 0.35 233 136 369 37 73 h, 0.20 0.05 99 23 121 23 pasta liquefied 73 h, OA 0.84 0.41 326 159 486 33 Example 23 Heat inactivation of the lipase in liquefied corn paste treated with lipase for simultaneous saccharification and fermentation with in situ product extraction through the use of oleyl alcohol Three fermentations were performed as described in the Examples 4, 5 and 6. No lipase was added to the liquefied corn paste in Examples 4 and 6 before fermentation, and the lipase treatment of the liquefied corn paste in the fermentation described in Example 5 (with 7.2 ppm of total soluble protein Lipolase®) was immediately followed by the heat inactivation treatment (to completely inactivate the lipase) and, subsequently, by the addition of nutrients prior to inoculation and fermentation. The percentage of FFA in the liquefied corn paste prepared without the lipase treatment for the fermentations that were carried out as described in Examples 4 and 6 was 31% and 34%, respectively, compared to 89% with the lipase treatment (Example 5). In the course of the fermentations listed in Table 10, the concentration of FFA in the OA phase was not reduced in any of the three fermentations, including that which contained the heat-inactivated lipase. The percentage of FFA in the OA phase of the fermentation carried out according to Example 5 (with the heat inactivation of the lipase before fermentation) was 95% at 70 hours (end of execution (EOR)), in comparison with only 33% FFA for the other two fermentations (Examples 4 and 6), where the liquefied corn paste was not treated with lipase. The results are shown in Table 7.

Table 7. Content of lipids and free fatty acids of fermentations containing oleyl alcohol as ISPR solvent and heat-inactivated lipase (after Fermentation Lipase Time (h), Lipids FFA Lipids FFA Lipids +% sample (% by weight) (% by weight) (g) (g) FFA (g) FFA Example 4 None Liquefied paste 0.65 0.30 7.2 3.3 10.4 31 Example 4 None 0.2 h, broth 0.56 0.28 6.6 3.3 9.9 33 Example 4 None 4.3 h, broth 0.28 0.09 3.3 1.0 4.4 24 Example 4 None 4.3 h, OA 0.45 0.27 4.0 2.4 6.4 37 Example 4 None 30 h, broth 0.17 0.05 2.0 0.6 2.7 24 Example 4 None 30 h, OA 0.63 0.29 5.7 2.6 8.3 32 Example 4 None 53 h, broth 0.13 0.04 1.5 0.5 2.0 23 Example 4 None 53 h, OA 0.67 0.32 6.0 2.9 8.9 32 Example 4 None 70 h, broth 0.13 0.04 1.5 0.4 1.9 23 Example 4 None 70 h, OA 0.64 0.31 5.8 2.8 8.5 33 Example 5 7.2 ppm Liquified paste 0.11 0.89 1.3 9.9 11.2 89 Fermentation Lipase Time (), Lipids FFA Lipids FFA Lipids +% sample (% by weight) by weight) (g) (g) FFA (g) FFA Example 5 7.2 ppm 0.2 h, broth 0.25 0.83 2.9 9.8 12.8 77 Example 5 7.2 ppm 4.3 h, broth 0.14 0.17 1.6 2.1 3.7 56 5 Example 5 7.2 ppm 4.3 h, ?? 0.02 0.84 0.2 7.9 8.1 97 Example 5 7.2 ppm 30 h, broth 0.08 0.18 1.0 -2.1 3.1 68 Example 5 7.2 ppm 30 h, OA 0.04 0.92 0.3 8.6 8.9 96 Example 5 7.2 ppm 53 h, broth 0.07 0.11 0.9 1.3 2.2 61 Example 5 7.2 ppm 53 h, OA 0.08 0.95 0.7 8.9 9.6 93 10 Example 5 7.2 ppm 70 h, broth 0.08 0.10 0.9 1.2 2.1 55 Example 5 7.2 ppm 70 h, OA 0.05 0.94 0.4 8.8 9.2 95 Example 6 None Liquefied paste 0.66 0.34 7.3 3.8 11.1 34 Example 6 None 0.2 h, broth 0.63 0.34 7.6 4.0 11.6 34 15 Example 6 None 4.3 h, broth 0.33 0.10 3.9 1.2 5.1 23 Example 6 None 4.3 h, OA 0.45 0.27 4.0 2.4 6.4 38 Fermentation Lipase Time (h), Lipids FFA Lipids FFA Lipids +% sample (weight%) by weight) (g) (g) FFA (g) FFA Example 6 None 30, broth 0.17 0.06 2.1 0.8 2.8 26 Example 6 None 30, OA 0.69 0.33 6.2 3.0 9.1 32 5 Example 6 None 53, broth 0.14 0.05 1.6 0.5 2.2 25 Example 6 None 53 h, OA 0.72 0.35 6.4 3.1 9.5 33 Example 6 None 70 h, broth 0.15 0.05 1.8 0.6 2.4 25 Example 6 None 70 h, OA 0.70 0.34 6.2 3.0 9.2 33 10 Example 24 Lipase treatment of whole corn kernels ground before liquefaction Tap water (1377.6 g) was added to each of the kettles. 2 liter resin jacketed, then 711.9 g wet weight (625.8 g dry weight) of ground whole corn kernels (1.0 mm sieve in the grinder mill) was added to each kettle under stirring. Each mixture was heated to 55 ° C under agitation at 300 rpm and the pH was adjusted to 5.8 with 2 N of sulfuric acid. To each mixture was added 21.0 g of an aqueous solution containing 1,008 g of Spezyme®-FRED L (Genencor®, Palo Alto, CA). Then, to a mixture was added 10.5 ml of aqueous solution of Lipolase® 100L (21 mg of total soluble protein, 10 ppm of final concentration of lipase) and to the second mixture was added 1.05 ml of aqueous solution of Lipolase® 100L (2.1 mg of total soluble protein, 1.0 ppm final concentration of lipase). Samples of each reaction mixture were taken after 1 hour and at 2, 4 and 6 hours at 55 ° C subsequently, the temperature of the mixture was increased to 85 ° C under agitation at 600 rpm and a pH of 5.8, and took a sample when the mixture reached 85 ° C for the first time. After 120 minutes at 85 ° C a sample was taken, the mixtures were cooled to 50 ° C and the final samples of the resulting liquefied corn paste were transferred to centrifuge tubes of 50 ml polypropylene; all samples were stored frozen at -80 ° C.

In two separate reactions, a 50 g sample of the liquefied corn paste treated with 10 ppm lipase or a 55 g sample of the liquefied corn paste treated with 1.0 ppm lipase, prepared as described above, was mixed with alcohol. Oleyl (OA) (38 g) at 30 CC for 20 hours; subsequently, the liquefied pulp and the OA in each reaction mixture were separated by centrifugation and each phase was analyzed to determine the percentage by weight of lipids (derivatized as methyl esters of fatty acids, FAME) and the percentage by weight of fatty acids Free (FFA, methyl esters of fatty acids, FAME) in accordance with the method described in Reference 1. The percentage of FFA in the OA phase of the liquefied paste / OA mixture prepared by heat inactivation of 10 ppm of lipase during liquefaction was 98% at 20 hours, compared to only 62% of FFA in the OA phase of the liquefied pulp / OA mixture prepared by heat inactivation of 1.0 ppm lipase during liquefaction. The results are shown in Table 8.

Table 8. Lipid and free fatty acid content of a blend of liquefied corn paste and oleyl alcohol by using the lipase treatment of the ground corn suspension before liquefaction (heat inactivation of the lipase during liquefaction) Conditions Time (h), Lipids FFA Lipids FFA Lipids +% reaction sample (% in (% in (mg) (mg) FFA (mg) FFA weight) weight) 10 ppm of 1 h, before 0 .226 0. 627 112 311 424 74 lipase of the liquefaction at 55 ° C 2 h, before 0 .199 0. .650 99 323 422 77 before the liquefaction liquidation at 4 h, before 0 .151 0,, 673 75 334 410 82 from the liquefaction 85 ° C, 6 h, before 0 .101 0. .700 50 348 398 87 mixed from the with liquefaction OA for 0 h, 85 ° C, 0 .129 0. .764 64 380 444 86 20 h pasta liquefied Conditions Time (h), Lipids FFA Lipids FFA Lipids +% reaction sample (% in (% in (mg) (mg) FFA (mg) FFA weight) weight) 2 h, 85 ° C, 0.129 0.751 64 373 437 85 pasta liquefied 20, 30 ° C, 0.074 0.068 37 34 71 48 pasta liquefied 20 h, 30 ° C, 0.015 1.035 5.7 394 400 98 OA 1. 0 ppm of 1 h, before 0.408 0.480 226 266 492 54 lipase of the liquefaction at 55 ° C 2 h, before 0.401 0.424 222 235 457 51 before the liquefaction liquefaction at 4 h, before 0.299 0.433 165 240 405 58 of the liquefaction 85 ° C, 6 h, before 0.346 0.453 192 251 442 57 mixed from the with liquefaction Conditions Time (h), Lipids FFA Lipids FFA Lipids +% reaction sample (% in (% in (mg) (mg) FFA (mg) FFA weight) weight) OA for 0 h, 85 ° C, 0.421 0.407 233 225 458 49 20 h liquefied paste 2 h, 85 ° C, 0.424 0.429 235 237 472 50 liquefied pasta 20 h, 30 ° C, 0.219 0.054 121 30 151 20 liquefied paste 20 h, 30 ° C, 0.344 0.573 140 233 373 62 OA Example 25 Lipase analysis for the treatment of whole corn kernels milled before liquefaction Seven reaction mixtures containing tap water (67.9 g) and ground whole corn kernels (35.1 g wet weight, ground with a 1.0 mm sieve by the use of a grinder mill) at a pH of 5.8 were shaken at 55 ° C in stoppered flasks. A sample of 3 ml (t = 0 h) was taken from each flask and frozen immediately on dry ice, then 0.5 ml of 10 mm sodium phosphate buffer (pH 7.0) containing 1 mg of sodium phosphate was added. total soluble protein (10 ppm of the final concentration in the reaction mixture) of one of the following lipases (Novozymes) to each flask: Lipolase® 100 L, Lipex® 100L, Lipoclean® 2000T, Lipozyme® CALB L, Novozyme® CALA L and Palatase 20000L; no lipase was added in the seventh flask. The resulting samples were stirred at 55 ° C in stoppered flasks, and 3 ml samples were taken from each reaction mixture at the time and at 2, 4 and 6 hours, and immediately frozen on dry ice until the percentage was analyzed. by weight of lipids (derivatized as methyl esters of fatty acids, EAME) and the percentage by weight of free fatty acids (FFA, derivatized as methyl esters of fatty acids, EAME) in accordance with the method described in Reference 1 , and the percentage content of free fatty acids was calculated in relation to the total combined concentrations of lipids and free fatty acids for each sample. The results are shown in Table 9.

Table 9 Percent content of free fatty acids (% FFA) of a mixture of whole maize kernels milled by treatment with lipase at 55 ° C before liquefaction % of FFA Time 0 h 1 h 2 h 4 h 6 h Lipolase® 100L 33 56 74 76 79 Lipex® 100L 34 66 81 83 83 Lipoclean® 2000T 38 55 73 69 65 Lipozyme® CALB L 39 38 37 43 41 Novozyme® CALA L 37 40 44 44 45 Palatase® 20000L 37 49 59 62 66 Without enzymes 38 33 37 41 42 26 Lipase treatment of whole corn kernels milled before simultaneous saccharification and fermentation with extraction of in situ product through the use of oleyl alcohol Three fermentations were performed as described in Examples 7, 8 and 10. For the fermentations performed in Examples 7 and 10, lipase (10 ppm total soluble protein Lipolase®) was added to the suspension of ground corn and heated to 55 ° C for 6 h before liquefaction to produce a liquefied corn paste containing heat-inactivated lipase. No lipase was added to the suspension of ground corn used to prepare the liquefied corn paste for the fermentation described in Example 8, but the suspension was subjected to the same heating step at 55 ° C before liquefaction. The percentage of FFA in the liquefied corn pulp treated with lipase prepared for the fermentations carried out as described in Examples 7 and 10 was 83% and 86%, specifically, compared to 41% without the lipase treatment (Example 8). ). In the course of the fermentations, the concentration of FFA was not reduced in any of the fermentations, including that which contained the heat-inactivated lipase. The percentage of FFA in the OA phase of the fermentation carried out according to Examples 7 and 10 (with heat inactivation of lipase before fermentation) was 97% at 70 hours (end of execution (EOR)), compared with only 49% FFA for the fermentation carried out in accordance with Example 8, in which the whole corn kernels milled had not been treated with lipase prior to liquefaction. The results are shown in Table 10.

Table 10. Lipid and free fatty acid content of fermentations containing oleyl alcohol as ISPR solvent and heat-inactivated lipase (lipase treatment of ground corn suspension before liquefaction) Fermentation Lipase Time (h), Lipids FFA Lipids FFA Lipids +% sample (% by weight) (% by weight) (g) (g) FFA (g) FFÍ Example 7 10 ppm Before the processing 0. 65 0. 22 7.1 2.4 9. 4 25 with lipase / before of the liquuac.

Example 7 10 ppm After processing. 0. 22 0. 65 2.4 7.0 9. 5 74 with lipase / before of the liquuac.

Example 7 10 ppm Liquefied paste 0. 17 0. 79 1.8 8.5 10 .3 83 Example 7 10 ppm 0.3 h, broth 0. 16 0. 79 1.8 8.9 10 .7 83 Example 7 10 ppm 4.8 h, broth 0. 14 0. 31 1.6 3.5 5. 1 69 Example 7 10 ppm 4.8 h, OA 0. 04 0. 68 0.3 5.4 5. 6 95 Example 7 10 ppm 29 h, broth 0. 10 0. 12 1.2 1.3 2. 5 53 Example 7 10 ppm 29 h, OA 0. 03 1. 05 0.2 8.2 8. 4 98 Fermentation Lipase Time (h), Lipids FFA Lipids FFA Lipids +% of sample; by weight) (% by weight) (g) (g) FFA (g) FFA Example 7 10 ppm 53 h, broth Example 7 10 ppm 53 h, OA 0.07 1.14 0.5 9.0 9.5 95 ^ Example 7 10 ppm 70 h, broth 0.11 0.07 1.2 0.8 2.0 39 Example 7 10 ppm 70 h, OA 0.03 1.10 0.2 8.7 8.9 97 Example 8 None Before the trat. 0.62 0.23 6.7 2.5 9.2 27 with lipase / before of the liquuac. 10 Example 8 None After the processing. 0.57 0.26 6.2 2.8 9.0 31 with lipase / before of the liquuac.

Example 8 None Liquefied paste 0.52 0.36 5.6 4.0 9.6 41 Example 8 None 0.3 h, broth 0.50 0.33 5.7 3.8 9.4 40 15 Example 8 None 4.8 h, broth 0.47 0.14 5.3 1.6 6.9 24 Example 8 None 4.8 h, OA 0.12 0.32 1.0 2.9 3.9 73 Example 8 None 29 h, broth 0.30 0.05 3.4 0.6 4.0 16 E ng 8 None 29 h, OA 0.31 0.46 2.7 4.1 6.9 60 Fermentation Lipase Time (h), Lipids FFA Lipids FFA Lipids +% sample% by weight) (% by weight) (g) (g) FFA (g) FFA Example 8 None 53 h, broth Example 8 None 53 h, OA 0. 47 0.50 4.2 4., 4 8.6 51 Example 8 None 70 h, broth 0. 22 0.04 2.5 0. .5 3.0 17 5 Example 8 None 70 h, OA 0., 40 0.39 3.6 3. .5 7.0 49 Example 10 10 ppm Before the processing 0., 67 0.23 7.4 2., 5 9.9 25 with lipase / before of the liquuac.

Example 10 10 ppm After processing 0., 19 0.69 2.1 7. .6 9.7 78 with lipase / before of the liquuac.

Example 10 10 ppm Liquefied paste 0.14 0.85 1.6 9.4 11.0 86 Example 10 10 ppm 0.3 h, broth 0.13 0.82 1.5 9.4 10.9 86 Example 10 10 ppm 4.8 h, broth 0.11 0.29 1.3 3.3 4.6 72 fifteen Example 10 10 ppm 4.8 h, OA 0.04 0.60 0.3 5.2 5.6 94 Example 10 10 ppm 29 h, broth 0.09 0.14 1.0 1.6 2.6 61 Example 10 10 ppm 29 h, OA 0.01 0.96 0.1 8.4 8.5 99 Fermentation Lipase Time (h), Lipids FFA Lipids FFA Lipids +% sample% by weight) (% by weight) (g) (g) FFA (g) FFA Example 10 10 ppm 53 h, broth Example 10 10 ppm 53 h, OA 0.02 0.95 0.2 8. .3 8.4 98 Example 10 10 ppm 70 h, broth 0.09 0.08 1.1 0. .9 1.9 45 Example 10 10 ppm 70 h, OA 0.03 0.99 0.3 8. 7.7 9.0 97 10 Example 27 Lipase treatment of ground whole corn grains or liquefied corn paste for simultaneous saccharification and fermentation with product extraction in situ through the use of corn oil fatty acids (COFA) Five fermentations were performed as described above in Examples 9, 11, 12, 13 and 14. For the fermentations performed as described in Examples 9, 13 and 14, lipase (10 ppm of total soluble protein Lipolase®) was added. ) after the liquefaction and there was no heat inactivation of the lipase. In the fermentations performed as described in Examples 9 and 14, 5 g / 1 of ethanol was added before the inoculation, while in the fermentation performed according to Example 13 no ethanol was added. The fermentations performed as described in Examples 11 and 12 implemented the addition of 10 ppm of total soluble protein Lipolase® in the ground corn suspension prior to liquefaction, which caused the heat inactivation of the lipase during liquefaction. In the fermentation performed as described in Example 11, 5 g / 1 of ethanol was added before inoculation, while in the fermentation performed as described in Example 12 no ethanol was added. The final total grams of isobutanol (i-BuOH) present in the COFA phase of the fermentations containing active lipase were significantly greater than the final total grams of i-BuOH present in the COFA phase of the fermentations containing inactive lipase. The final total grams of isobutanol (i-BuOH) present in the fermentation broths containing active lipase were only slightly lower than the total final grams of i-BuOH present in the fermentation broths containing inactive lipase, so that the total production of i-BuOH (as a combination of free i-BuOH and isobutyl esters of COFA (FABE)) was significantly higher in the presence of active lipase compared to the production obtained in the presence of heat-inactivated lipase. The results are shown in Tables 11 and 12. free and isobutyl esters of COFA (FABE) in fermentations containing corn oil fatty acids (COFA) as ISPR solvent in the presence (Examples 9, 13 and 14) or absence (Examples 11 and 12) of lipase (analysis of the COFA phase) Fermentation g of g of Total g in i-BuOH / FABE / i-BuOH g of FABE / i-BuOH / Fermentation Time (h) kg of kg of kg of kg COFA COFA COFA COFA Example 9 4.5 2.4 0.0 0 2.4 Fermentation g of g of Total g (i-BuOH / FABE / i-BuOH g of FABE / i-BuOR Example 9 28.8 5.4 70.9 16.5 22.0 Example 9 52.4 8.9 199.0 46.4 55.3 Example 9 69.3 4.9 230.9 53.9 69.3 Example 11 6.6 2.3 0.0 0.0 2.3 Example 11 53.5 25.1 2.9 0.6 25.7 Example 11 71.1 24.4 6.3 1.4 25.8 Use 12 6.6 2.3 0.0 0.0 2.3 Example 12 53.5 12.8 1.6 0.4 13.2 Example 12 71.1 12.8 3.0 0.7 13.5 Example 13 6.6 2.3 0.0 0.0 2.3 Example 13 53.5 4.9 72.1 16.0 20.9 Example 13 71.1 4.6 91.4 20.3 24.9 Example 14 6.6 2.1 0.0 0.0 2.1 Example 14 53.5 9.8 197.2 43.8 53.6 Example 14 71.1 4.9 244.5 54.3 59.2 Table 12. Dependence of the production of free isobutanol (i-BuOH) and isobutyl esters of COFA (FABE) in fermentations containing corn oil fatty acids (COFA) as ISPR solvent in the presence ( Examples 9, 13 and 14) or absence (Examples 11 and 12) of active lipase- (analysis of the fermentation broth) Fermentation g of g of g of Total in i-BuOH / FABE / i-BuOH of g of FABE / i-BuOH / Sample Time (h) kg kg of kg kg of broth broth broth broth Example 9 4.5 0.0 0.0 0 0 Example 9 28.8 0.0 12.6 2.9 2.9 Example 9 52.4 0.0 30.3 7.1 7.1 Example 9 69.3 0.0 24.7 5.8 5.8 Example 11 6.6 0.0 0.0 0 0.0 Example 11 53.5 9.8 0.0 0 9.8 Example 11 71.1 9.5 0.0 0 9.5 Example 12 6.6 0.0 0.0 0 0 Example 12 53.5 3.8 0.0 0.0 3.8 Example 12 71.1 5.1 0.0 0.0 5.1 Example 13 6.6 0.0 0.0 0 0 Example 13 53.5 2.1 3.0 0.7 2.8 Example 13 71.1 2.1 7.4 1.6 3.7 Example 14 6.6 0.0 0.0 0.0 Fermentation g of g of g of Total in i-BuOH / FftBE / i-BuOH of g of FABE / i-BuOH / Example 14 53.5 2.9 22.4 5.0 7.9 Example 14 71.1 3.3 19.3 4.3 7.6 While various embodiments of the present invention have been described above, they should be construed as having been presented by way of example only and not in a limiting manner. It will be apparent to those skilled in the art that various changes in shape and detail can be made without departing from the spirit and scope of the invention. Therefore, the breadth and scope of the present invention should not be limited by the illustrative embodiments described above, but should be defined only in accordance with the following claims and their equivalents.

All the publications, patents and patent applications mentioned in this specification indicate the level of knowledge of the persons with experience in the art to which they belong, and are incorporated as reference in the present description to the same extent as if each publication , patent or patent application would have been specifically and individually indicated to be incorporated as a reference.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims

REI INDICATIONS Having described the invention as above, the content of the following claims is claimed as property:

1. A method characterized in that it comprises: (a) providing a fermentation broth comprising a recombinant microorganism that produces an alcohol product from a fermentable carbon source, wherein the recombinant microorganism comprises a reduction or elimination of the pyruvate decarboxylase activity; (b) contacting the fermentation broth with a fermentable carbon source, so that the recombinant microorganism consumes the fermentable carbon source and produces the alcohol product; Y (c) contacting the fermentation broth with fatty acids derived from biomass in a stage of the fermentation process, wherein at least one of (i) the growth rate and (ii) the fermentable carbon consumption of the microorganism is greater in the presence of fatty acids that the rate of growth and / or the fermentable carbon consumption of the recombinant microorganism in the absence of fatty acids.

2. The method according to claim 1, characterized in that the fatty acids are selected from oleic acid, palmitic acid, myristic acid, and mixtures thereof.

3. The method according to claim 1, characterized in that the biomass is derived from corn grains, corn cobs, crop residues such as corn husks, maize stubble, herbs, wheat, rye, wheat straw, barley, straw. of barley, hay, rice straw, needle grass, waste paper, sugar cane bagasse, sorghum, sugar cane, soybeans, components obtained from the grinding of grains, cellulosic material, lignocellulosic material, trees, branches, roots, leaves, wood chips, sawdust, bushes and shrubs, vegetables, fruits, flowers, manure, and mixtures thereof.

4. The method in accordance with the claim 1, characterized in that the alcohol product is butanol.

5. The method according to claim 1, characterized in that steps (b) and (c) occur, practically, simultaneously.

6. The method according to claim 1, characterized in that the fermentable carbon source is derived from biomass.

7. The method according to claim 1, characterized in that the fermentation broth also comprises ethanol.

8. The method according to claim 1, characterized in that the recombinant microorganism has one or more deletions of the pyruvate decarboxylase (PDC) gene.

9. A method for producing an alcohol product, characterized in that it comprises: (a) providing biomass comprising a fermentable carbon source and oil; (b) converting at least a portion of the oil into fatty acids to form a biomass comprising the fatty acids; (c) contacting the biomass with a fermentation broth comprising a recombinant microorganism capable of producing an alcohol product from a fermentable carbon source, and wherein the recombinant microorganism comprises a reduction or elimination of the pyruvate decarboxylase activity; (d) contacting the fatty acids with the fermentation broth, wherein at least one of (i) the growth rate and (ii) the fermentable carbon consumption of the recombinant microorganism is higher in the presence of fatty acids than the growth rate and / or the fermentable carbon consumption of the microorganism in the absence of fatty acids.

10. The method in accordance with the claim 9, characterized in that step (b) of converting at least a portion of the oil into fatty acids comprises contacting the oil with one or more substances capable of hydrolyzing the oil portion into fatty acids.

11. The method in accordance with the claim 10, characterized in that the one or more substances comprise one or more enzymes.

12. The method in accordance with the claim 11, characterized in that the enzyme or enzymes comprise lipase enzymes.

13. The method according to claim 11, characterized in that it also comprises: before step (c), inactivate the enzyme (s) after hydrolysing at least a portion of the oil.

14. The method according to claim 9, characterized in that steps (b), (c) and (d) occur in the fermentation vessel.

15. The method according to claim 9, characterized in that steps (b), (c) and (d) occur, practically, simultaneously.

16. The method according to claim 9, characterized in that the biomass is derived from corn grains, corn cobs, crop residues such as corn husks, maize stubble, herbs, wheat, rye, wheat straw, barley, straw. of barley, hay, rice straw, needle grass, waste paper, sugarcane bagasse, sorghum, sugarcane, soybeans, components obtained from the grinding of grains, cellulosic material, lignocellulosic material, trees, branches, roots, leaves, wood chips, sawdust, bushes and shrubs, vegetables, fruits, flowers, manure and mixtures thereof.

17. The method according to claim 9, characterized in that the fatty acids are selected from oleic acid, palmitic acid, myristic acid and mixtures thereof.

18. The method according to claim 9, characterized in that it also comprises: ferment the fermentable carbon source to produce an alcohol product.

19 The method of compliance with the claim 18, characterized in that the alcohol product is butanol.

20. The method in accordance with the claim 9, characterized in that the fermentation broth further comprises ethanol.

21. The method according to claim 9, characterized in that the recombinant microorganism has one or more deletions of the pyruvate decarboxylase (PDC) gene.

22. The method according to claim 9, characterized in that it also comprises: separating the oil from the biomass before step (b) of converting at least a portion of the oil into fatty acids.

23. The method according to claim 9, characterized in that it also comprises: liquefying the biomass to produce a liquefied biomass, wherein the liquefied biomass comprises oligosaccharides; Y contacting the liquefied biomass with a saccharification enzyme capable of converting the oligosaccharides into fermentable sugar to form a saccharified biomass, and wherein step (c) comprises contacting the saccharified biomass with the fermentation broth comprising a recombinant microorganism.

24. A method for producing an alcohol product, characterized in that it comprises: provide a raw material; liquefy the raw material to create a suspension of raw material; separating the raw material suspension to produce a product comprising (i) an aqueous layer comprising a fermentable carbon source, (ii) an oil layer and (iii) a solids layer; obtaining an oil from the oil layer and converting at least a portion of the oil to fatty acids; introducing the aqueous layer of (c) into a fermentation vessel containing a fermentation broth comprising a recombinant microorganism capable of producing an alcohol product from a fermentable carbon source, wherein the recombinant microorganism comprises a reduction or elimination of the pyruvate decarboxylase activity; ferment the fermentable carbon source of the aqueous layer to produce the alcohol product; Y contacting the fermentation broth with the fatty acids, wherein at least one of (i) the growth rate and (ii) the fermentable carbon consumption of the recombinant microorganism is higher in the presence of the fatty acids than the growth rate and / or the fermentable carbon consumption of the recombinant microorganism in the absence of the fatty acids.

25. The method in accordance with the claim 24, characterized in that step (d) of converting at least a portion of the oil into fatty acids comprises contacting the oil with one or more substances capable of hydrolyzing the oil portion into fatty acids.

26. The method in accordance with the claim 25, characterized in that the substance or substances comprise one or more enzymes.

27. The method in accordance with the claim 26, characterized in that the enzyme or enzymes comprise lipase enzymes.

28. The method according to claim 24, characterized in that the raw material comprises one or more fermentable sugars derived from corn kernels, corn cobs, crop residues such as corn husks, maize stubbles, herbs, wheat, rye, straw wheat, barley, barley straw, hay, rice straw, needle grass, waste paper, bagasse sugar cane, sorghum, sugar cane, soybeans, components obtained from grinding grain, cellulosic material, lignocellulosic material, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, manure and mixtures thereof.

29. The method according to claim 24, characterized in that the fatty acids are selected from oleic acid, palmitic acid, myristic acid, and mixtures thereof.

30. The method according to claim 18, characterized in that the alcohol product is butanol.

31. The method according to claim 24, characterized in that the recombinant microorganism has one or more deletions of the pyruvate decarboxylase (PDC) gene.

32. A composition characterized in that it comprises a recombinant microorganism comprising a reduction or elimination of the pyruvate decarboxylase activity and fatty acids.