JP2013528400A - Method and system for removing undissolved solids prior to extractive fermentation in the production of butanol - Google Patents

Abstract

  Methods and systems are provided for efficiently producing fermentation product alcohols such as butanol using in situ product extraction. Efficiency is obtained by separating the undissolved solids after liquefying a given raw material to produce the raw material and prior to fermentation, for example by centrifugation. Removal of undissolved solids avoids problems associated with having undissolved solids present during in-situ production extraction, thereby increasing the efficiency of alcohol production.

Description

  This application is based on US Provisional Patent Application No. 61 / 356,290, filed Jun. 18, 2010; US Provisional Patent Application No. 61 / 368,451, filed Jul. 28, 2010; 2010. US Provisional Patent Application 61 / 368,436, filed July 28, US Provisional Patent Application 61 / 368,444, filed July 28, 2010; filed July 28, 2010 US Provisional Patent Application No. 61 / 368,429; US Provisional Patent Application No. 61 / 379,546, filed September 2, 2010; and US Provisional Patent, filed February 7, 2011. No. 61 / 440,034; claims the benefit of US Patent Application No. 13 / 160,766, filed June 15, 2011; the entire contents of which are hereby incorporated by reference. Be used in

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

  The present invention relates to a method and system for removing undissolved solids from a fermenter feed stream in the production of fermented alcohols such as butanol.

  Butanol is an important industrial chemical with a variety of uses including as a fuel additive, as a raw chemical in the plastics industry, and as a food grade extractant in the food and fragrance industry. Thus, there is a high demand for butanol, as well as efficient and environmentally friendly production methods.

  The production of butanol using microbial fermentation is one such environmentally friendly production method. Some microorganisms that produce butanol in high yield also have low butanol toxicity thresholds, so that butanol needs to be removed from the fermentor as it is produced. In situ product removal (ISPR) can be used to remove butanol from the fermentor as butanol is produced, thereby allowing the microorganisms to produce butanol in high yield. One method for ISPR described in the art is liquid-liquid extraction (Patent Document 1). Because it is technically and economically feasible, liquid-liquid extraction is the contact of the extractant with the fermentation broth for efficient mass transfer; phase separation of the extractant from the fermentation broth (during and after fermentation) ); And / or efficient recovery and reuse of solvents and extraction agent degradation and / or contamination over long periods of operation.

  When the water stream entering the fermentor contains non-dissolved solids from the raw material, the non-dissolved solids can be used to make liquid-liquid extraction technically and economically feasible by increasing capital and operating costs. This hinders the above requirements. In particular, the presence of undissolved solids during extractive fermentation can reduce the mass transfer coefficient inside the fermenter and interfere with phase separation in the fermentor, such as oil from undissolved solids in the extractant (eg, Corn oil) accumulation, which may lead to a decrease in extraction efficiency over time, and the solvent is trapped in the solid that is finally removed as a soluble matter-containing dry cereal distillation koji (DDGS). Loss of extractant droplets from the fermentation broth, and / or may reduce fermenter volumetric efficiency. Accordingly, there is a continuing need to develop more efficient methods and systems for producing product alcohols such as butanol by extractive fermentation.

US Patent Application Publication No. 2009/030305370

  The present invention provides a method and system for producing product alcohols such as butanol by meeting the above needs and reducing the amount of undissolved solids fed to the fermenter.

  The present invention relates to a method and system for removing undissolved solids from a fermenter feed stream in the production of fermented alcohols such as butanol.

The present invention provides a biomass feedstock slurry comprising a fermentable carbon source, an undissolved solid, and water; and separating at least a portion of the undissolved solid from the slurry, whereby (i) An aqueous solution comprising a fermentable carbon source and (ii) a wet cake byproduct comprising a solid; and adding the aqueous solution to a fermentation broth comprising recombinant microorganisms in the fermentor, thereby producing a fermentation product A process for improving the processing productivity of biomass. In certain embodiments, improved biomass processing productivity includes improved fermentation product and byproduct recoverability compared to fermentation products produced in the presence of undissolved solids. In certain embodiments, improved biomass processing productivity includes one or more of improved process stream reusability, improved fermenter volumetric efficiency, and improved biomass feedstock supply. In certain embodiments, the method further comprises contacting the fermentation broth with an extractant, the extractant having an improved extraction efficiency as compared to a fermentation broth comprising undissolved solids. In some embodiments, the improved extraction efficiency is due to a stabilized partition coefficient of the extractant, improved phase separation of the extractant from the fermentation broth, improved liquid-liquid mass transfer coefficient, improved extractant recovery and reusability. And one or more of the extractants stored for recovery and reuse. In certain embodiments, the extractant is an organic extractant. In certain embodiments, the extraction _agent, fatty _alcohols, fatty acids C 12 _{-C 22} of _C 12 _{~C 22,} C 12 esters of fatty acids _{-C 22,} fatty aldehydes of _{C 12 ~C 22, C 12 ~C} 22 And one or more immiscible organic extractants selected from the group consisting of fatty amides, and mixtures thereof. In certain embodiments, the extraction agent comprises a fatty acid of _C 12 _{-C 22} derived from corn oil. In some embodiments, the undissolved solid is decanter bowl centrifuged, tricanter centrifuged, disk stack centrifuged, filtered centrifuge, decanter centrifuge, filtered, vacuum filtered, belt filter, pressure filtered, sieved Raw slurry by filtration, sieving separation, grading, porous grid, flotation, hydroclone, filter press, screw press, gravity settler, vortex separator, or combinations thereof Separated from. In certain embodiments, the method further comprises the step of liquefying the feedstock to produce a biomass feedstock slurry; the feedstock is crop residues such as corn kernels, corn cobs, corn hulls, corn stover , Grass, corn, wheat, rye, wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugarcane pomace, sorghum, sugarcane, soybeans, ingredients obtained from grinding cereals, cellulosic Selected from materials, lignocellulosic materials, trees, branches, roots, leaves, wood chips, sawdust, shrubs and shrubs, vegetables, fruits, flowers, animal manure, and mixtures thereof. In certain embodiments, the raw material is corn. In certain embodiments, the raw material is fractionated or not fractionated. In certain embodiments, the raw material is wet crushed or dry crushed. In certain embodiments, the method further comprises increasing the reaction temperature during liquefaction. In certain embodiments, the raw slurry includes oils derived from the above raw materials, and the oil is separated from the slurry. In certain embodiments, the wet cake includes a feedstock. In certain embodiments, the wet cake is washed with water to recover the oligosaccharides present in the wet cake. In certain embodiments, the recovered oligosaccharide is added to the fermentor. In certain embodiments, the wet cake is further processed to obtain improved byproducts. In certain embodiments, the by-product is further processed to form an animal feed product. In certain embodiments, the wet cake is washed with a solvent to recover the oil present in the wet cake. In certain embodiments, the solvent is selected from hexane, butanol, isobutanol, isohexane, ethanol, and petroleum distillate. In certain embodiments, the fermentation product is a product alcohol selected from the group consisting of methanol, ethanol, propanol, butanol, pentanol, and isomers thereof. In certain embodiments, the recombinant microorganism comprises an engineered butanol biosynthetic pathway. In certain embodiments, the method, the fermentation broth and products and further comprising the step of at least partially evaporate the CO ₂ optionally wherein the vapor stream is generated, the product is recovered from the vapor stream. In certain embodiments, the method further comprises contacting the vapor stream with an absorbing liquid phase, wherein at least a portion of the vapor stream is absorbed into the absorbing liquid phase; the vapor stream into the absorbing liquid phase. The temperature at the beginning of the absorption of is higher than the temperature at the start of condensation of the vapor stream in the absence of the absorbing liquid phase. In certain embodiments, the evaporation and contacting steps are performed under vacuum conditions. In certain embodiments, separating the majority of undissolved solids from the slurry results in a higher vapor pressure of the fermentation broth compared to the fermentation broth containing undissolved solids. In certain embodiments, a higher vapor pressure results in a more efficient evaporation product recovery. In certain embodiments, more efficient evaporation product recovery results in less capital investment, less evaporation, absorption, compression, and refrigeration equipment, improved mass transfer rate, less energy for evaporation, and lower absorption. Including one or more of the agent flow rates.

  The present invention also relates to a method for producing butanol, the method comprising providing a raw material; and liquefying the raw material to produce a raw slurry containing an oligosaccharide, an oil, and an undissolved solid; Separating the undissolved solids from the raw slurry and producing (i) an aqueous solution comprising oligosaccharides, (ii) a wet cake comprising undissolved solids, and (iii) an oil phase; Contacting the fermentation broth with: fermenting the oligosaccharides in the fermenter to produce butanol; and performing in situ removal of butanol from the fermentation broth as the butanol is produced; Removal of undissolved solids from the raw slurry increases the efficiency of butanol production. In certain embodiments, the feedstock is corn and the oil is corn oil. In certain embodiments, the undissolved solid comprises germ, fiber, and gluten. In certain embodiments, the method further includes the step of dry grinding the feedstock. In certain embodiments, the corn is not fractionated. In certain embodiments, the undissolved solids are decanter bowl centrifuge, tricanter centrifuge, disc stack centrifuge, filtration centrifuge, decanter centrifuge, filtration, vacuum filtration, belt filter, pressure filtration, filtration through a sieve, Separated by sieving separation, grid, porous grid, flotation, hydroclone, filter press, screw press, gravity settling, vortex separator, or combinations thereof. In some embodiments, separating the undissolved solid from the raw slurry includes centrifuging the raw slurry. In an embodiment, the step of centrifuging the raw slurry separates the raw material into a first liquid phase containing an aqueous solution, a solid phase containing a wet cake, and a second liquid phase containing oil. In certain embodiments, the wet cake is washed with water to recover the oligosaccharides present in the wet cake. In certain embodiments, in situ removal includes liquid-liquid extraction. In certain embodiments, the extractant for liquid-liquid extraction is an organic extractant. In certain embodiments, the saccharification of the oligosaccharide in the aqueous solution occurs simultaneously with the fermentation of the oligosaccharide in the fermentation apparatus. In certain embodiments, the method further comprises increasing the reaction temperature during liquefaction. In certain embodiments, the method further comprises saccharifying the oligosaccharide prior to fermenting the oligosaccharide in the fermentation apparatus. In certain embodiments, removing the undissolved solid from the raw slurry includes centrifuging the raw slurry. In an embodiment, the step of centrifuging the raw slurry is performed before saccharifying the sugar. In certain embodiments, the fermentation broth comprises a recombinant microorganism comprising a butanol biosynthetic pathway. In certain embodiments, the butanol is isobutanol. In certain embodiments, the step of removing undissolved solids from the raw slurry increases the efficiency of butanol production by increasing the liquid-liquid mass transfer coefficient of butanol from the fermentation broth to the extractant; the extraction efficiency of butanol by the extractant Increase the efficiency of butanol production by increasing the efficiency of butanol production by increasing the speed of phase separation between the fermentation broth and the extractant; by increasing the recovery and recycling of the extractant, Increase the efficiency of butanol production by increasing the efficiency of butanol production; or by reducing the flow rate of the extractant. The present invention is a liquefaction tank configured to liquefy a raw material to produce a raw slurry: an inlet for receiving the raw material; and an outlet for discharging the raw slurry containing sugar and undissolved solids. A centrifuge configured to remove a non-dissolved solid from a raw slurry to produce a wet cake containing a portion of (i) an aqueous solution containing sugar and (ii) a non-dissolved solid. A centrifuge including an inlet for receiving the raw slurry; a first outlet for discharging the aqueous solution; and a second outlet for discharging the wet cake; to ferment the aqueous solution to produce butanol A first inlet for receiving an aqueous solution; a second inlet for receiving an extractant; a first for discharging an extractant rich in butanol Outlet; and a fermentor containing a second outlet for discharging the fermentation broth also relates to a system for producing butanol. In certain embodiments, the centrifuge further includes a third outlet for discharging the produced oil while removing undissolved solids from the raw slurry. In certain embodiments, the apparatus comprises a saccharification tank configured to saccharify the sugar in the raw slurry: an inlet for receiving the raw slurry; and an outlet for discharging the raw slurry. In addition. In certain embodiments, the apparatus further comprises a saccharification tank configured to saccharify sugars in the aqueous solution: an inlet for receiving the aqueous solution; and an outlet for discharging the aqueous solution. In certain embodiments, the apparatus is a dry pulverizer configured to grind the raw material: an inlet for receiving the raw material; and an outlet for discharging the ground raw material. Further included.

  The present invention comprises 20-35% crude protein, 1-20% crude fat, 0-5% triglyceride, 4-10% fatty acid, and 2-6% fatty acid isobutyl ester. It also relates to a composition. The present invention comprises 25-31 wt% crude protein, 6-10 wt% crude fat, 4-8 wt% triglyceride, 0-2 wt% fatty acid, and 1-3 wt% fatty acid isobutyl ester. It also relates to a composition. The present invention comprises 20-35% crude protein, 1-20% crude fat, 0-5% triglyceride, 4-10% fatty acid, and 2-6% fatty acid isobutyl ester. It also relates to a composition. The present invention comprises 26-34 wt% crude protein, 15-25 wt% crude fat, 12-20 wt% triglyceride, 1-2 wt% fatty acid, 2-4 wt% fatty acid isobutyl ester, It also relates to a composition comprising 2 wt% lysine, 11-23 wt% NDF, and 5-11 wt% ADF.

  In certain embodiments, (a) providing a raw slurry comprising fermentable carbon and undissolved solids from the biomass and water; (b) separating a majority of the undissolved solids from the slurry, thereby ( i) an aqueous solution comprising fermentable carbon and (ii) a wet cake by-product comprising solids; (c) adding the aqueous solution to a fermentation broth comprising recombinant microorganisms in the fermentor, whereby the fermentation product is A process comprising: a process wherein the process productivity of biomass is improved. In certain embodiments, improved biomass process productivity includes improved fermentation product and by-product recoveries compared to fermentation products produced in the presence of undissolved solids. In certain embodiments, improved biomass processing productivity includes one or more of improved process stream reusability, improved fermenter volumetric efficiency, and improved corn loading. In certain embodiments, the improved process stream reusability includes one or more of fermenting recombinant microorganism reuse, water reuse, and energy efficiency.

  In certain embodiments, the method can also include contacting the fermentation broth of (d) (c) with an extractant, the extractant having an improved extraction efficiency as compared to a fermentation broth comprising undissolved solids. In certain embodiments, the improved extraction efficiency includes one or more of a stabilized partition coefficient, improved phase separation, improved mass transfer coefficient, and improved process stream reusability. In certain embodiments, improved extraction efficiency results in a stabilized partition coefficient of the extractant, improved phase separation of the extractant from the fermentation broth, improved liquid-liquid mass transfer coefficient, and improved extractant recovery and reuse. Includes one or more of sex. In certain embodiments, the improved extraction efficiency includes an extractant that is stored for reuse.

  In certain embodiments, the aqueous solution has a viscosity of less than about 20 cps. In certain embodiments, the aqueous solution contains less than about 20 g / L monomeric glucose.

  In certain embodiments, improved product recoverability provides improved recombinant microbial resistance to the product. In certain embodiments, enhanced tolerance is provided by one or more of removal of inhibitors, including undissolved solids, or enhanced liquid-liquid mass transfer coefficient. In certain embodiments, improved extractant efficiency provides improved recombinant microbial resistance. In certain embodiments, improved recombinant microbial resistance is provided by extraction of inhibitors, by-products, and metabolites.

  In certain embodiments, the raw slurry includes oil derived from the raw material, and the oil is separated from the slurry of step (b). In certain embodiments, the wet cake comprises feedstock in an amount less than about 20% of the dry solids of the wet cake.

  In certain embodiments, the majority of the undissolved solids separated from the raw slurry in step (b) is at least about 75% by weight of the undissolved solids. In certain embodiments, the majority of the undissolved solids separated from the raw slurry in step (b) is at least about 90% by weight of the undissolved solids. In certain embodiments, the majority of the undissolved solid separated from the raw slurry in step (b) is at least about 95% by weight of the undissolved solid. In some embodiments, step (b) includes centrifuging the raw slurry. In an embodiment, the step of centrifuging the raw slurry separates the raw material into a first liquid phase containing an aqueous solution and a solid phase containing a wet cake. In certain embodiments, the wet cake is washed with water to recover the sugar or sugar source present in the wet cake. In certain embodiments, the liquid phase comprising the aqueous solution is centrifuged more than once.

In certain embodiments, the extractant is an organic extractant. In certain embodiments, the extraction _agent, fatty _alcohols, fatty acids C 12 _{-C 22} of _C 12 _{~C 22,} C 12 esters of fatty acids _{-C 22,} fatty aldehydes of _{C 12 ~C 22, C 12 ~C} 22 And one or more immiscible organic extractants selected from the group consisting of fatty amides, and mixtures thereof.

  In certain embodiments, the fermentable recombinant microorganism is a bacterium or a yeast cell.

  In certain embodiments, the product is a product alcohol selected from the group consisting of butanol and its isomers.

In certain embodiments, the method also includes the step of (d) at least partially evaporating the fermentation broth and the product of (c) and optionally CO ₂ , wherein a steam stream is generated and generated from the steam stream. Collect items. In certain embodiments, the method also includes contacting the vapor stream with an absorbing liquid phase, wherein at least a portion of the vapor stream is absorbed into the absorbing liquid phase and the vapor stream into the absorbing liquid phase. The temperature at the start of absorption is higher than the temperature at the start of condensation of the vapor stream in the absence of an absorbing liquid phase. In certain embodiments, the evaporation and contacting steps are performed under vacuum conditions. In certain embodiments, separating the majority of undissolved solids from the slurry results in a higher vapor pressure of the fermentation broth compared to the fermentation broth containing undissolved solids. In certain embodiments, a higher vapor pressure results in a more efficient evaporation product recovery. In certain embodiments, more efficient evaporation product recovery results in less capital investment, less evaporation, absorption, compression, and refrigeration equipment, improved mass transfer rate, less energy for evaporation, and lower absorption. Including one or more of the agent flow rates.

  In certain embodiments, the step of separating the majority of the undissolved solid is performed such that starch loss to the undissolved solid is minimized. In certain embodiments, starch loss is one or more optimizations including temperature control, enzyme concentration, pH, ground corn particle size, and reaction time during liquefaction; centrifugation operating conditions; and wet cake washing conditions. Minimized by doing work.

  In certain embodiments, the wet cake is further processed to obtain improved byproducts. In certain embodiments, the by-product is further processed into DDGS. In certain embodiments, DDGS has an improved product profile that includes less feedstock compared to DDGS produced in the presence of undissolved solids. In certain embodiments, DDGS has an improved product profile such that DDGS is produced with minimal contaminating contact with fermentation broths, recombinant microorganisms, fermentation products, and extractants. In certain embodiments, the DDGS produced by the above method meets food labeling requirements for organic animal feed.

  In certain embodiments, the fermentation broth comprises a fermentation product portion and a corn oil portion in a ratio of at least about 4: 1 on a weight basis, wherein the broth is substantially free of undissolved solids. In certain embodiments, the corn oil portion contains at least about 15% by weight free fatty acids. In certain embodiments, the fermentation broth contains no more than about 15% by weight undissolved solids. In certain embodiments, the fermentation broth contains no more than about 10% by weight undissolved solids. In certain embodiments, the fermentation broth contains no more than about 5% by weight of undissolved solids.

  In certain embodiments, the centrifuge product profile comprises a layer of undissolved solids, a corn oil layer, and a supernatant layer comprising fermentable sugars, in the supernatant layer relative to the undissolved solids in the undissolved solid layer. The ratio by weight of fermentable sugar ranges from about 2: 1 to about 5: 1; the ratio by weight of fermentable sugar in the supernatant layer to corn oil in the corn oil layer is about 10: 1. The ratio by weight of undissolved solid in the undissolved solid layer to corn oil in the corn oil layer ranges from about 2: 1 to about 25: 1.

  In certain embodiments, a method for producing butanol comprises: (a) providing a corn feed; and (b) liquefying the corn feed to produce a feed slurry comprising sugar, corn oil, and undissolved solids. (C) removing undissolved solids from the raw slurry, (i) an aqueous solution containing sugar, (ii) a wet cake containing undissolved solids, and (iii) a free corn oil phase (D) contacting the aqueous solution with the broth in the fermentor; (e) fermenting sugar in the fermentor to produce butanol; and (f) butanol being produced. In-situ removal of butanol from the broth, and the removal of non-dissolved solids from the raw slurry increases the efficiency of butanol production. It is In certain embodiments, the undissolved solid comprises germ, fiber, and gluten. In certain embodiments, the method also includes the step of dry grinding the corn raw material. In certain embodiments, the corn is not fractionated.

  In some embodiments, step (c) includes centrifuging the raw slurry. In some embodiments, the step of centrifuging the raw slurry separates the raw slurry into a first liquid phase that includes an aqueous solution, a solid phase that includes a wet cake, and a second liquid phase that includes free corn oil. . In certain embodiments, the wet cake is washed with water to recover the sugars present in the wet cake. In certain embodiments, the liquid phase comprising the aqueous solution is centrifuged more than once. In certain embodiments, at least about 75% by weight of the undissolved solid is removed from the raw slurry in step (c). In certain embodiments, at least about 90% by weight of the undissolved solid is removed from the raw slurry in step (c). In certain embodiments, at least about 95% by weight of the undissolved solid is removed from the raw slurry in step (c).

  In certain embodiments, in situ removal includes liquid-liquid extraction. In certain embodiments, the extractant for liquid-liquid extraction is an organic extractant. In certain embodiments, the organic extractant comprises oleyl alcohol.

  In certain embodiments, the broth comprises a microorganism. In certain embodiments, the microorganism is a bacterium or a yeast cell.

  In certain embodiments, a portion of the broth exits the fermenter, and the method further comprises separating yeast present in the broth portion from the broth and returning the separated yeast to the fermentor. Including. In certain embodiments, a portion of the broth comprises no more than about 25% by weight of undissolved solids present in the raw slurry. In certain embodiments, a portion of the broth comprises no more than about 10% by weight of undissolved solids present in the raw slurry. In certain embodiments, a portion of the broth comprises no more than about 5% by weight of undissolved solids present in the raw slurry.

  In certain embodiments, the saccharification of the sugar in the aqueous solution occurs simultaneously with the fermentation of the sugar in the fermenter. In certain embodiments, the method also includes saccharifying the sugar prior to fermentation of the sugar in the fermenter. In some embodiments, step (c) includes centrifuging the raw slurry. In an embodiment, the step of centrifuging the raw slurry is performed before saccharifying the sugar. In an embodiment, the step of centrifuging the raw slurry is performed after the step of saccharifying the sugar.

  In certain embodiments, the butanol is isobutanol. In certain embodiments, step (c) increases the efficiency of butanol production by increasing the liquid-liquid mass transfer coefficient of butanol from the broth to the extractant. In certain embodiments, step (c) increases the efficiency of butanol production by increasing the extraction efficiency of butanol with an extractant. In certain embodiments, step (c) increases the efficiency of butanol production by increasing the rate of phase separation between the broth and the extractant. In certain embodiments, step (c) increases the efficiency of butanol production by increasing extraction agent recovery and reuse. In certain embodiments, step (c) increases the efficiency of butanol production by reducing the extractant flow rate.

  In certain embodiments, step (c) comprises a stabilized partition coefficient of the extractant, improved fermenter volume frequency, improved corn load supply, improved fermentable recombinant microorganism recycling, improved water. Includes one or more of reuse, improved energy efficiency, improved recombinant microbial resistance to butanol, lower aqueous phase titer, and improved value of DDGS.

  In certain embodiments, the system for producing butanol comprises: (a) a liquefaction tank configured to liquefy the raw material to produce a raw slurry, the inlet for receiving the raw material, sugar and non-dissolving A liquefaction tank including an outlet for discharging a raw material slurry containing solids; (b) a non-dissolved solid is removed from the raw slurry and (i) an aqueous solution containing sugar and (ii) a wet containing a portion of the non-dissolved solid A centrifuge configured to produce a cake, the centrifuge comprising an inlet for receiving a raw slurry, a first outlet for discharging an aqueous solution, and a second outlet for discharging a wet cake A separator; and (c) a fermenter for fermenting an aqueous solution in the fermenter to produce butanol, a first inlet for receiving the aqueous solution, receiving an extractant Second inlet for, and a fermentor containing a second outlet for discharging the first outlet and the fermentation broth for discharging rich butanol extractant. In certain embodiments, the centrifuge further includes a third outlet for discharging the produced oil while removing undissolved solids from the raw slurry. In certain embodiments, the system also includes a saccharification tank configured to saccharify the sugar in the raw slurry, the saccharification tank including an inlet for receiving the raw slurry and an outlet for discharging the raw slurry. . In certain embodiments, the system also includes a saccharification tank configured to saccharify sugars in the aqueous solution, the saccharification tank including an inlet for receiving the aqueous solution and an outlet for discharging the aqueous solution. In certain embodiments, the system also includes a dry grinder configured to grind the raw material, the dry grinder including an inlet for receiving the raw material and an outlet for discharging the ground raw material. Including.

  In certain embodiments, a wet cake formed from a corn mash slurry in a centrifuge comprising undissolved solids comprises at least about 75% by weight undissolved solids present in the corn mash slurry. In certain embodiments, the wet cake comprises at least about 90% by weight of undissolved solids present in the corn mash slurry. In certain embodiments, the wet cake comprises at least about 95% by weight undissolved solids present in the corn mash slurry.

  In certain embodiments, the aqueous solution formed from the corn mash slurry in a centrifuge that includes undissolved solids comprises no more than about 25% by weight of undissolved solids present in the corn mash slurry. In certain embodiments, the aqueous solution comprises up to about 10% by weight of undissolved solids present in the corn mash slurry. In certain embodiments, the aqueous solution comprises no more than about 5% by weight of undissolved solids present in the corn mash slurry.

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

Figure 2 schematically illustrates an exemplary method and system of the present invention where undissolved solids are removed in a centrifuge after liquefaction and prior to fermentation. 1 schematically illustrates an exemplary alternative method and system of the present invention in which raw materials are ground. Fig. 6 schematically illustrates another exemplary alternative method and system of the present invention in which a centrifuge drains an oil stream. Fig. 4 schematically illustrates another exemplary alternative method and system of the present invention in which a saccharification tank is placed between the centrifuge and the fermentation apparatus. FIG. 6 schematically illustrates another exemplary alternative method and system of the present invention in which a saccharification tank is disposed between a liquefaction tank and a centrifuge. Fig. 4 schematically illustrates another exemplary alternative method and system of the present invention in which two centrifuges are used in succession to remove undissolved solids. From an aqueous solution of liquefied corn starch (ie, oligosaccharides) to a dispersion of oleyl alcohol droplets flowing upward through a bubble column when dispensing oleyl alcohol using a nozzle having an inner diameter of 2.03 mm total volume mass transfer coefficient for transfer of i-BuOH, showing the effect of the presence of undissolved corn mash solids for k L _a. From an aqueous solution of liquefied corn starch (i.e., oligosaccharide) to a dispersion of oleyl alcohol droplets flowing upward through a bubble column when dispensing oleyl alcohol using a nozzle having an internal diameter of 0.76 mm total volume mass transfer coefficient for transfer of i-BuOH, showing the effect of the presence of undissolved corn mash solids for k L _a. (Gravity) Indicates the position of the liquid-liquid interface in the fermentation sample tube according to the sedimentation time. Run time: Phase separation data shown for run times of 5.3 hours, 29.3 hours, 53.3 hours, and 70.3 hours. Sample data from extraction-fermentation (2010Y035) where solids were removed from the mash raw material and OA was the solvent. The position of the liquid-liquid interface of the final fermentation broth according to the (gravity) sedimentation time is shown. Data from extraction-fermentation when solids were removed from the mash feed and OA was the solvent (2010Y035). The concentration of glucose in the aqueous phase of the slurry as a function of time for Batch 1 and Batch 2 is shown. The concentration of glucose in the aqueous phase of the slurry as a function of time for Batch 3 and Batch 4 is shown. Figure 5 shows the effect of whether enzyme loading and +/- high temperature steps were applied at some point during liquefaction on starch conversion.

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

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

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

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

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

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

  “Biomass” as used herein refers to corn, sugarcane, wheat, cellulosic or lignocellulosic materials and cellulose, hemicellulose, lignin, starch, oligosaccharides, disaccharides and / or monosaccharides, and their It refers to natural products containing hydrolyzable polysaccharides that provide any saccharides derived from natural resources such as materials including mixtures and fermentable sugars including starch. Biomass can also include additional components such as proteins and / or lipids. The biomass may be derived from one source, or the biomass may include a mixture derived from two or more sources. For example, the biomass may comprise a mixture of corn cobs and corn stover, or a mixture of grass and leaves. Biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from papermaking, garden waste, wood waste and forestry waste. Examples of biomass include, but are not limited to, corn kernels, corn cobs, corn hulls, crop residues such as corn stover, grass, wheat, rye, wheat straw, barley, barley straw , Grass, rice straw, switchgrass, waste paper, sugarcane pomace, sorghum, sugarcane, soy, ingredients obtained from grinding grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and shrubs, vegetables, fruits , Flowers, animal manure, and mixtures thereof. For example, mash, juice, molasses, or hydrolyzate is formed from the biomass by any process known in the art for processing biomass for fermentation, such as by grinding, processing, and / or liquefaction. And contains fermentable sugars and may contain water. For example, cellulosic and / or lignocellulosic biomass can be treated to obtain a hydrolyzate containing fermentable sugars by any method known to those skilled in the art. Low ammonia pretreatment is disclosed in US Patent Application Publication No. 2007/0031918 A1, which is incorporated herein by reference. Enzymatic saccharification of cellulosic and / or lignocellulosic biomass typically involves the degradation of cellulose and hemicellulose to produce hydrolysates containing sugars including glucose, xylose, and arabinose. Is used. (Saccharifying enzymes suitable for cellulosic and / or lignocellulosic biomass are reviewed in Lynd, et al. (Microbiol. Mol. Biol. Rev. 66: 506-577, 2002).

  Soluble-containing dry cereal distillation koji (DDGS) as used herein is a by-product or by-product from fermentation of raw materials or biomass (eg, fermentation of cereals or cereal mixtures to produce product alcohol). Point to. In certain embodiments, DDGS also refers to animal feed produced from a method of making a product alcohol (eg, butanol, isobutanol, etc.).

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

  “Fermentable sugar” as used herein refers to one or more saccharides that can be metabolized by the microorganisms disclosed herein to produce fermented alcohol.

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

As used herein, “fermentation broth” is due to water, sugars (fermentable carbon source), dissolved solids, optionally microorganisms that produce alcohol, product alcohol, and microorganisms present. By means of a mixture of all the other ingredients of the material retained in the fermentor in which the product alcohol is made by reaction of sugars with alcohol, water and carbon dioxide (CO ₂ ). Optionally, the terms “fermentation medium” and “fermented mixture” as used herein can be used interchangeably with “fermentation broth”.

  A “fermentor” as used herein means a tank in which a fermentation reaction takes place, whereby a product alcohol such as butanol is made from sugars. The term “fermenter” can be used synonymously with “fermentor”.

  As used herein, “saccharification tank” means a tank in which saccharification (ie, decomposition of oligosaccharides into monosaccharides) takes place. When fermentation and saccharification are performed simultaneously, the saccharification tank and the fermentation tank may be the same tank.

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

  As used herein, “liquefaction tank” means a tank in which liquefaction is performed. Liquefaction is a process in which oligosaccharides are released from raw materials. In embodiments where the feedstock is corn, the oligosaccharide is released from the corn starch content during liquefaction.

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

  As used herein, “non-dissolved solid” means non-fermentable portions of the raw material that are not dissolved in the liquid phase, such as germs, fibers, and gluten. For example, the non-fermentable portion of the feed includes the portion that remains as a solid of the feed and can absorb liquid from the fermentation broth.

  As used herein, “extractant” means an organic solvent used to extract any butanol isomer.

  As used herein, “in situ product removal (ISPR)” refers to biological, such as fermentation, to control product concentration in a biological process as the product is produced. It means selectively removing a specific fermentation product from the process.

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

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

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

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

  The term “water phase titer” as used herein refers to the concentration of a particular alcohol (eg, butanol) in the fermentation broth.

  As used herein, the term “effective titer” refers to a specific alcohol produced by fermentation (eg, butanol) or an alcohol ester alcohol produced by alcohol esterification per liter of fermentation medium. Refers to the total amount of equivalents.

  The term “water-immiscible” or “insoluble” refers to a chemical component such as an extractant or solvent that cannot be mixed with an aqueous solution, such as a fermentation broth, to form one liquid phase.

  The term “aqueous phase” as used herein refers to the aqueous phase of a two-phase mixture obtained by contacting a fermentation broth with a water-immiscible organic extractant. In one embodiment of the process described herein involving a fermentation extraction, the term “fermentation broth” in this case specifically refers to the aqueous phase in a biphasic fermentation extraction.

  The term “organic phase” as used herein refers to the non-aqueous phase of a two-phase mixture obtained by contacting a fermentation broth with a water-immiscible organic extractant.

  The present invention provides systems and methods for producing fermentation products, such as product alcohols, by fermentation while increasing the process productivity and cost effectiveness of biomass. In certain embodiments, the product alcohol is butanol. The raw material can be liquefied to produce a raw slurry, where the raw slurry includes soluble sugars and undissolved solids. If the raw slurry is fed directly to the fermentor, the undissolved solids can prevent efficient removal and recovery of product alcohol, such as butanol, from the fermentor. In particular, when liquid-liquid extraction is used to extract butanol from the fermentation broth, the presence of undissolved particles reduces the mass transfer rate of butanol to the extractant by preventing contact between the extractant and the fermentation broth. Reducing; producing an emulsion in the fermenter, thereby preventing good phase separation of the extractant and fermentation broth; at least a portion of the extractant and butanol is soluble in dried cereal distillers (DDGS) Reducing the efficiency of recovering and reusing the extractant because it is “trapped” in the final removed solids; because there is solids that occupy volume in the fermenter and also from the fermentation broth This includes lower fermenter volumetric efficiency due to slower extractant withdrawal; and includes shortening of extractant life due to contamination with corn oil. It can lead to inefficiencies limiting system. All these effects increase capital and operating costs. In addition, extractants that are “trapped” by DDGS can deviate from DDGS values and requirements for selling as animal feed. Therefore, to avoid and / or minimize these problems, at least some of the undissolved particles (or solids) are removed from the raw slurry before adding the sugar present in the raw slurry to the fermentor. Is done. When the extraction activity and efficiency of butanol formation is performed in a fermentation broth containing an aqueous solution from which undissolved particles have been removed, compared to an extraction performed in a fermentation broth containing an aqueous solution from which undissolved particles have not been removed Enhanced.

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

  In particular, the raw material 12 can be introduced into the inlet of the liquefaction tank 10. Ingredient 12 can be any suitable biomass material known in the art, including but not limited to rye, wheat, sugarcane, or corn, which contains a fermentable carbon source such as starch.

  The process of liquefying the raw material 12 is a conventional process including hydrolyzing starch in the raw material 12 to form a water-soluble sugar. Any known liquefaction process commonly used in the art, including but not limited to acid processes, acid-enzyme processes, or enzyme processes, as well as corresponding liquefaction tanks may be used. Such processes can be used alone or in combination. In certain embodiments, an enzymatic process can be used and a suitable enzyme 14, such as α-amylase, is introduced into the inlet of the liquefaction tank 10. Water can also be introduced into the liquefaction tank 10.

  The process of liquefying the feedstock 12 produces a feedstock slurry 16 that includes sugars (eg, fermentable carbon) derived from the feedstock or biomass and undissolved solids. The undissolved solid is a non-fermentable part of the raw material 12. In certain embodiments, the feedstock 12 can be corn, such as dry-milled, unfractionated corn kernels, and the undissolved particles can include germ, fiber, and gluten. The raw material slurry 16 can be discharged from the outlet of the liquefaction tank 10. In some embodiments, the raw material 12 is corn or corn seed and the raw slurry 16 is a corn mash slurry.

  The centrifuge 20 configured to remove undissolved solids from the raw slurry 16 has an inlet for receiving the raw slurry 16. The centrifuge 20 agitates or rotates the raw slurry 16 to produce a liquid phase or aqueous solution 22 and a solid phase or wet cake 24.

  The aqueous solution 22 can include, for example, sugars in the form of oligosaccharides, and water. The aqueous solution may comprise at least about 10% by weight oligosaccharide, at least about 20% by weight oligosaccharide, or at least about 30% by weight oligosaccharide. The aqueous solution 22 can be discharged from an outlet located near the top of the centrifuge 20. The aqueous solution can have a viscosity of less than about 20 centipoise. The aqueous solution may contain less than about 20 g / L monomeric glucose, more preferably less than about 10 g / L, or less than about 5 g / L monomeric glucose. Suitable methods for determining the amount of monomeric glucose are well known in the art. Such suitable methods known in the art include HPLC.

  The wet cake 24 may include undissolved solids. The wet cake 24 can be discharged from an outlet located near the bottom of the centrifuge 20. The wet cake 24 may also contain some sugar and water. After the aqueous solution 22 is discharged from the centrifuge 20, the wet cake 24 can be washed with additional water in the centrifuge 20. Alternatively, the wet cake 24 can be washed with additional water in a separate centrifuge. By washing the wet cake 24, sugar or a sugar source (for example, oligosaccharide) present in the wet cake is recovered, and the recovered sugar and water can be reused in the liquefaction tank 10. After washing, the wet cake 24 may be dried by any suitable known method to form a solubilized dry cereal distiller (DDGS). There are several advantages to forming DDGS from the wet cake 24 formed in the centrifuge 20. Since undissolved solids are not sent to the fermenter, extractant and / or butanol are not captured by DDGS, DDGS is not subjected to fermentor conditions, and DDGS does not come into contact with microorganisms present in the fermenter. All these effects provide an advantage for the subsequent treatment and sale of DDGS, for example as animal feed.

  The centrifuge 20 may be any conventional centrifuge used in the industry, including, for example, a decanter bowl centrifuge, a tricanter centrifuge, a disk stack centrifuge, a filtration centrifuge, or a decanter centrifuge. It can be. In certain embodiments, removal of undissolved solids from the raw slurry 16 may include filtration, vacuum filtration, belt filter, pressure filtration, sieving filtration, sieving separation, grate or lattice, porous lattice, This can be done by flotation, hydroclone, filter press, screw press, gravity settler, vortex separator, or any method that can be used to separate solids from liquids. In one embodiment, undissolved solids are removed from the corn mash and compared to two product streams, for example, an aqueous solution of oligosaccharides containing a lower concentration of solids compared to corn mash and corn mash. A wet cake containing a higher concentration of solids can be formed. Furthermore, when a tricanter centrifuge is used to remove solids from corn mash, a third stream containing corn oil can be produced. Thus, several product streams can be produced using various separation techniques or combinations thereof.

  In certain embodiments, the wet cake 24 is a composition formed in the centrifuge 20 from the raw slurry 16, for example, corn mash slurry, wherein the wet cake 24 is at least about present in the raw slurry. 50 wt% undissolved particles, at least about 55 wt% undissolved particles present in the raw slurry, at least about 60 wt% nondissolved particles present in the raw slurry, at least about 65 wt% present in the raw slurry % Undissolved particles, at least about 70 wt.% Undissolved particles present in the raw slurry, at least about 75 wt.% Non-dissolved particles present in the raw slurry, at least about 80 wt.% Present in the raw slurry. Undissolved particles, at least about 85% by weight of undissolved particles, At least about 90 wt.% Of undissolved particles present in Lee, comprising at least about 95 wt.% Of undissolved particles or undissolved particles of about 99 wt% present in the raw material slurry, present in the feed slurry.

  In some embodiments, in the centrifuge 20, the raw slurry 16, eg, an aqueous solution 22 formed from corn mash slurry, is present in the raw slurry at about 50 wt% or less of undissolved particles present in the raw slurry. About 45 wt% or less of undissolved particles, about 40 wt% or less of non-dissolved particles present in the raw slurry, about 35 wt% or less of non-dissolved particles present in the raw slurry, about 30 present in the raw slurry % By weight of non-dissolved particles, about 25% by weight or less of undissolved particles present in the raw slurry, about 20% by weight or less of non-dissolved particles present in the raw slurry, about 15% by weight of the raw slurry The following non-dissolved particles, about 10 wt% or less non-dissolved particles present in the raw slurry, about 5 wt% or less non-dissolved particles present in the raw slurry, Other contains about 1 wt.% Of undissolved particles present in the slurry.

  The fermenter 30 configured to ferment the aqueous solution 22 to produce butanol has an inlet for receiving the aqueous solution 22. The fermenter 30 may include a fermentation broth. A microorganism 32 selected from the group consisting of bacteria, cyanobacteria, filamentous fungi, and yeast is introduced into the fermentation apparatus 30 to be included in the fermentation broth. In certain embodiments, the microorganism 32 may be a bacterium such as E. coli. In certain embodiments, the microorganism 32 is S. cerevisiae. It can be S. cerevisiae. The microorganism 32 consumes the sugar in the aqueous solution 22 and produces butanol. Production of butanol using fermentation by microorganisms, as well as microorganisms that produce high yields of butanol are known, for example, disclosed in US Patent Application Publication No. 2009/030305370, the entire disclosure of which is Which is incorporated herein by reference. In certain embodiments, the microorganism 32 can be a fermentable recombinant microorganism.

  In certain embodiments, the microorganism 32 is modified to include a biosynthetic pathway. In certain embodiments, the biosynthetic pathway is a butanol biosynthetic pathway. In certain embodiments, the biosynthetic pathway converts pyruvate into a fermentation product. In certain embodiments, the biosynthetic pathway comprises at least one heterologous polynucleotide encoding a polypeptide that catalyzes substrate-product conversion of the biosynthetic pathway. In certain embodiments, each substrate-product conversion of the biosynthetic pathway is catalyzed by a polypeptide encoded by a heterologous polynucleotide.

  As butanol is produced by the microorganisms, butanol can be removed from the fermenter 30 using in situ product removal (ISPR), for example, by liquid-liquid extraction. Liquid-liquid extraction is briefly described below and the entire disclosure can be performed according to the methods described in US Patent Application Publication No. 2009/0305370, which is incorporated herein by reference.

Fermentor 30 has an inlet for receiving extractant 34. The extractant 34 is a saturated, monounsaturated, polyunsaturated (and mixtures thereof) C ₁₂ -C ₂₂ fatty alcohol, C ₁₂ -C ₂₂ fatty acid, C ₁₂ -C ₂₂ fatty acid ester, C ₁₂ fatty aldehydes -C _22, may be a C 12 fatty amides _{-C 22,} and an organic extraction agent selected from the group consisting of mixtures thereof. Extractant also saturated, esters of monounsaturated, polyunsaturated (and mixtures _thereof) fatty alcohols of C 4 _{-C 22,} fatty acids C 4 _{-C 28,} fatty acids _{C 4} ~C _28, C 4 fatty aldehydes -C _22, and may be an organic extractant selected from the group consisting of a mixture thereof. Extractant 34 is 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, laurin It can be an organic extractant such as aldehydes, 20-methylundecanal, and mixtures thereof. The extractant 34 comes into contact with the fermentation broth and the butanol present in the fermentation broth is transferred to the extractant 34. A butanol-rich extractant stream 36 is discharged through the outlet of the fermenter 30. The butanol is then separated from the extractant in stream 36 using conventional techniques. A feed stream can be added to the fermenter 30. The fermenter 30 can be any suitable fermenter known in the art.

  In certain embodiments, saccharification and fermentation can occur simultaneously within the fermentation apparatus 30. Any known saccharification process commonly used in the art may be used, including but not limited to acid processes, acid-enzyme processes, or enzyme processes. In certain embodiments, an enzyme 38, such as glucoamylase, may be introduced at the inlet of the fermentation apparatus 30 to decompose the saccharides in the form of oligosaccharides present in the aqueous solution 22 into monosaccharides.

  In certain embodiments, the fermentation broth 40 may be discharged from the outlet of the fermentation apparatus 30. The discharged fermentation broth 40 may include a microorganism 32 such as yeast. The microorganism 32 can be easily separated from the fermentation broth 40 in, for example, a centrifuge (not shown). Thereafter, the microorganisms 32 can be reused in the fermentation apparatus 30. The fermentation apparatus 30 can increase the production rate of butanol over time, thereby increasing the efficiency of production of butanol.

  When a portion of the fermentation broth 40 exits the fermentation apparatus 30, the fermentation broth 40 is about 50 wt% or less non-dissolved particles present in the raw slurry, and about 45 wt% or less non-dissolved particles present in the raw slurry. About 40 wt% or less non-dissolved particles present in the raw slurry, about 35 wt% or less non-dissolved particles present in the raw slurry, about 30 wt% or less non-dissolved particles present in the raw slurry, raw material About 25 wt% or less non-dissolved particles present in the slurry, about 20 wt% or less non-dissolved particles present in the raw slurry, about 15 wt% or less non-dissolved particles present in the raw slurry, in the raw slurry About 10% by weight or less of undissolved particles, about 5% by weight or less of undissolved particles present in the raw slurry, or about 1% by weight or less of undissolved particles present in the raw slurry.

  In certain embodiments, for example, as shown in FIG. 2, the system and method of the present invention may include a grinder 50 configured to dry grind raw material 52. Raw material 52 may be the same as raw material 12 in FIG. 1 and may enter crusher 50 through an inlet. The pulverizer 50 can pulverize or grind the raw material 52. In some embodiments, the raw material 52 may not be fractionated. In certain embodiments, raw material 52 can be unfractionated corn kernels. The grinder 50 can be any suitable known grinder, such as a hammer mill. The dry-pulverized raw material 54 is discharged from the pulverizer 50 through the outlet and enters the liquefaction tank 10. The remaining part of FIG. 2 is identical to FIG. 1 and will not be described again. In other embodiments, the raw material can be fractionated and / or wet milled as is known in the art instead of being fractionated and / or dry milled.

  Wet milling is a multi-step process that separates biomass (eg, corn) into its main components (germ, seed coat fiber, starch, and gluten) to gain value from each by-product separately. This process results in a purified starch stream; however, this process is costly and involves separating the biomass into its non-starch components that are not required for fermentation alcohol production. Fractionation removes fibers and germs that contain most of the lipids present in ground whole corn, resulting in fractionated corn with a higher starch (endosperm) content. Dry fractionation is less expensive than wet milling because it does not separate the germ from the fiber. However, the fraction does not remove the entire fiber or germ and does not completely remove the solid. In addition, some starch is lost during fractionation. While wet grinding of corn is even more expensive than dry fractionation, dry fractioning is more expensive than dry grinding of unfractionated corn.

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

  The raw slurry 16 contains a first liquid phase or an aqueous solution 22 containing fermentable sugar, a wet cake 24 containing a solid phase or an undissolved solid, and an oil 26 containing an oil 26 that can exit the centrifuge 20. It is separated into two liquid phases. In certain embodiments, feedstock 12 is corn and oil 26 is free corn oil. The term free corn oil as used herein refers to corn oil free of corn germ. For example, any suitable conventional centrifuge, such as a tricanter centrifuge, can be used to drain the aqueous solution 22, wet cake 24, and oil 26. In certain embodiments, when the raw material is corn, some of the oil from raw material 12, such as corn oil, remains in wet cake 24. In such a case, the wet cake 24 includes corn oil in an amount less than about 20% by weight of the dry solids of the wet cake 24.

  In certain embodiments, when feedstock 12 (eg, corn) and corn oil 26 are removed from centrifuge 20, the fermentation broth in fermentor 30 contains a reduced amount of corn oil. For example, a fermentation broth that is substantially free of undissolved solids may comprise a product alcohol portion (eg, butanol) and an oil portion (eg, corn oil) in a ratio of at least about 4: 1 by weight. Corn oil may contain at least 15% by weight free fatty acids, for example 16.7% by weight free fatty acids. In certain embodiments, the fermentation broth has no more than about 25% by weight undissolved solids, the fermentation broth has no more than about 15% by weight undissolved solids, and the fermentation broth has no more than about 10% by weight non-dissolved solids. Having dissolved solids, the fermentation broth has no more than about 5 wt% undissolved solids, the fermentation broth has no more than about 1 wt% undissolved solids, or the fermentation broth is about 0.5 wt% % Of undissolved solids.

  In certain embodiments, the centrifuge 20 produces a product profile that includes a layer of undissolved solids, a layer of oil (eg, corn oil), and a supernatant layer that includes fermentable sugars. The ratio of fermentable sugar in the supernatant layer to undissolved solid in the undissolved solid layer on a weight basis can range from about 2: 1 to about 5: 1; to corn oil in the corn oil layer on a weight basis The ratio of fermentable sugars in the supernatant layer can range from about 10: 1 to about 50: 1; and / or the amount of undissolved solids in the undissolved solid layer relative to corn oil in the corn oil layer on a weight basis The ratio can range from about 2: 1 to about 25: 1.

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

  If the oil 26 is not drained separately, it can be removed along with the wet cake 24. When the wet cake 24 is removed via the centrifuge 20, in certain embodiments, if the feed is corn, some of the oil from the feed 12 such as corn oil remains in the wet cake 24. After the aqueous solution 22 has been drained from the centrifuge 20, the wet cake 24 can be washed with additional water in the centrifuge. By washing the wet cake 24, sugar (eg, oligosaccharide) present in the wet cake is recovered, and the recovered sugar and water can be reused in the liquefaction tank 10. After washing, the wet cake 24 can be combined with solubles by any suitable known method and then dried to form a soluble-containing dried cereal distillation koji (DDGS). There are several advantages to forming DDGS from the wet cake 24 formed in the centrifuge 20. Because non-dissolved solids are not sent to the fermentor, DDGS does not capture product alcohol such as extractant and / or butanol, and DDGS is not subjected to fermentor conditions and microorganisms present in the fermentor Do not contact with. All these advantages make it easier to process and sell DDGS as animal feed, for example. In some embodiments, the oil 26 is not drained separately from the wet cake 24, and the oil 26 is included as part of the wet cake 24 and is ultimately present in the DDGS. In such cases, the oil can be separated from DDGS and converted to ISPR extractant for subsequent use in the same or different alcohol fermentation processes.

  Oil 26 may be separated from DDGS using any suitable known method including, for example, a solvent extraction process. In one embodiment of the present invention, DDGS is filled into an extraction tank and washed with a solvent such as hexane to remove oil 26. Other solvents that can be used include, for example, petroleum distillates such as isobutanol, isohexane, ethanol, petroleum ether, or mixtures thereof. After extraction of oil 26, DDGS can be treated to remove any residual solvent. For example, the DDGS can be heated to evaporate any residual solvent using any method known in the art. After removing the solvent, the DDGS can be subjected to a dry process to remove any residual water. Treated DDGS can be used as a supplementary feed for animals such as poultry, livestock, and pets.

  After extraction from DDGS, the resulting oil 26 and solvent mixture can be collected to separate oil 26 from the solvent. In one embodiment, the oil 26 / solvent mixture may be processed by evaporation so that the solvent can be evaporated, collected and reused. The recovered oil can be converted to ISPR extractant for subsequent use in the same or different alcohol fermentation processes.

  Removal of the oil component of the feed is advantageous for butanol production since the oil present in the fermenter can be broken down into fatty acids and glycerol. Glycerin can accumulate in water and reduce the amount of water available for reuse throughout the system. Thus, removal of the feed oil component increases the efficiency of product alcohol production by increasing the amount of water that can be recycled through the system.

  In certain embodiments, for example, as shown in FIGS. 4 and 5, saccharification is performed between the centrifuge 20 and the fermentation apparatus 30 (FIG. 4) or between the liquefaction tank 10 and the centrifuge 20 (FIG. 5). Can be carried out in a separate saccharification tank 60 located in FIGS. 4 and 5 are the same as FIG. 1 except that they include a separate saccharification tank 60 and that the fermenter 30 does not accept the enzyme 38.

  As noted above, any known saccharification process commonly used in the art may be used, including but not limited to acid process, acid-enzyme process, or enzyme process. The saccharification tank 60 may be any suitable known saccharification tank. In certain embodiments, an enzyme 38, such as glucoamylase, may be introduced at the inlet of the saccharification tank 60 to decompose saccharides in the form of oligosaccharides into monosaccharides. For example, in FIG. 4, oligosaccharides present in the water stream 22 discharged from the centrifuge 20 and received in the saccharification tank 60 through the inlet are broken down into monosaccharides. Therefore, the aqueous solution 62 containing monosaccharides is discharged from the saccharification tank 60 through the outlet and is received by the fermentation apparatus 30. Alternatively, as shown in FIG. 5, the oligosaccharides present in the raw slurry 16 that are discharged from the liquefaction tank 10 and received by the saccharification tank 60 through the inlet are decomposed into monosaccharides. Therefore, the raw material slurry 64 containing the monosaccharide is discharged from the saccharification tank 60 through the outlet and is received by the centrifuge 20.

  In certain embodiments, the systems and methods of FIGS. 2 and 3 can be modified to include a separate saccharification tank 60 as described above in connection with the systems and methods of FIGS.

  In certain embodiments, for example, as shown in FIG. 6, the system and method of the present invention may include a series of two or more centrifuges. FIG. 6 is the same as FIG. 1 except that a second centrifuge 20 'has been added and will not be described again in detail.

  The aqueous solution 22 discharged from the centrifuge 20 can be received at the inlet of the centrifuge 20 '. The centrifuge 20 'can be identical to the centrifuge 20 and can be operated in the same way. The centrifuge 20 ′ removes undissolved solids that are not separated from the aqueous solution 22 in the centrifuge 20, and (i) includes the water stream 22 except that it contains a reduced amount of undissolved solids compared to the water stream 22. A similar water stream 22 ′ and (ii) a wet cake 24 ′ similar to the wet cake 24 may be produced. The water stream 22 ′ can then be introduced into the fermentation apparatus 30. In certain embodiments, one or more additional centrifuges may be present after the centrifuge 20 '.

  In certain embodiments, the systems and methods of FIGS. 2-6 can be modified to include additional centrifuges to remove undissolved solids as described above in connection with the systems and methods of FIG.

  In certain embodiments, the fermentation broth 40 can be discharged from the outlet of the fermentation apparatus 30. The absence or minimal of undissolved solids leaving the fermenter 30 with the fermentation broth 40 has several additional advantages. For example, the need for units and operations in downstream processing, such as a beer column or distillation column, can be eliminated, thereby increasing the efficiency of product alcohol production. Also, some or all of the total distillation waste centrifuge can be omitted as a result of less undissolved solids in the final broth exiting the fermenter.

  The processes and systems disclosed in FIGS. 1-6 include removing undissolved solids from the raw slurry 16, resulting in improved biomass processing productivity and cost effectiveness. Improved productivity may include having improved efficiency of butanol production and / or improved extraction activity compared to processes and systems that do not remove undissolved solids prior to fermentation.

  As noted above, undissolved solids can be further processed to produce other by-products such as DDGS or fatty acid esters. For example, fatty acid esters can be recovered to increase the yield of carbohydrate to product alcohol (eg, butanol). This is done by using a solvent, for example, by extracting several fatty acid esters from the by-product formed by combining and mixing several by-product streams and drying the product of the combined mixing process. Can be broken. Such a solvent-based extraction system for recovering corn oil triglycerides from DDGS is described in US 2010/0092603, the teachings of which are incorporated herein by reference. The

  In one embodiment of solvent extraction of fatty acid esters, solids can be separated from the total distillation effluent (“separated solids”), which is the most fatty acid ester in the by-product stream whose streams are not combined. It is because it may contain a proportion. These separated solids can then be fed to an extractor and washed with a solvent. In one embodiment, the separated solid is rotated at least once to ensure that all sides of the separated solid are washed with solvent. After washing, the resulting mixture of lipid and solvent, known as Misella, is collected for separation of the extracted lipid from the solvent. For example, the resulting mixture of lipid and solvent can be deposited in a separator for further processing. During the extraction process, as the solvent washes the separated solids, the solvent not only puts lipids into solution, but also collects solid particulates. These “particulates” are generally undesirable impurities in the micelles, and in one embodiment, the micelles can be discharged from the extractor or separator by a device that separates or scrubs the particulates from the micelles. .

  In order to separate the lipids and solvents contained in the miscella, the miscella can be subjected to a distillation step. In this process, the miscella can be processed, for example, by an evaporator, which is high enough to evaporate the solvent, but not high enough to adversely affect or evaporate the extracted lipids. Heat the miscella to temperature. As the solvent evaporates, the solvent can be collected, for example, in a condenser and reused for future use. Separation of the solvent from the miscella results in the accumulation of crude lipids, which can be further processed to separate water, fatty acid esters (eg, fatty acid isobutyl esters), fatty acids, and triglycerides.

  After lipid extraction, the solid can be sent out of the extractor and subjected to a stripping process to remove residual solvent. Recovery of residual solvent is important for process economy. In one embodiment, the wet solid may be sent to a vapor tight environment for storing and collecting the solvent that temporarily evaporates from the wet solid as the solvent is sent to the desolventizer. As the solid enters the desolventizer, the solid can be heated to evaporate and remove the residual solvent. To heat the solid, the desolvator may include a mechanism for distributing the solid to one or more trays, where the solid is heated directly, such as by direct contact with heated air or steam. Or heated indirectly such as by heating a tray carrying food. To facilitate the movement of solids from one tray to another, the tray carrying the solids can include openings that allow the solids to be transported from one tray to the next. From the desolvator, the solid may optionally be sent to a mixer, where the solid is mixed with other by-products before being sent to the dryer. In this example, the solid is fed to a desolvator where the solid is contacted by steam. In one embodiment, the vapor and solid streams in the desolvator may be retrograde. The solid may then exit the desolvator and be fed to a dryer or optionally a mixer, where various by-products can be mixed. The vapor exiting the desolvator can be condensed, optionally mixed with miscella, and then fed to the decanter. The water-enriched phase leaving the decanter may be fed to a distillation column where hexane is removed from the water-enriched stream. In one embodiment, the hexane-depleted water enriched stream exits the bottom of the distillation column and may be recycled back to the fermentation process, e.g., to slurry ground corn solids. Can be used. In another embodiment, the top and bottom products can be reused in the fermentation process. For example, the lipid-enriched tower bottom can be added to the feed of the hydrolyzer. The top can be condensed, for example, and fed to a decanter. The hexane-enriched stream exiting this decanter can optionally be used as part of the solvent feed to the extractor. The water-enriched phase exiting this decanter can be fed to a column that removes hexane from the water. As those skilled in the art can appreciate, the method of the present invention can be modified in various ways to optimize the fermentation process for the production of product alcohols such as butanol.

  In a further embodiment, by-products (or by-products) are obtained from the mash used in the fermentation process. For example, corn oil can be separated from the mash, and the corn oil can contain triglycerides, free fatty acids, diglycerides, monoglycerides, and phospholipids (see, eg, Example 20). Corn oil may optionally be added to other by-products (or by-products) at various rates, thus creating, for example, the ability to change the amount of triglycerides in the resulting by-products. Thus, the fat content of the resulting by-product is controlled to provide a lower fat, higher protein animal feed that would, for example, better meet the needs of dairy cows compared to higher fat products. obtain.

  In one embodiment, the raw corn oil separated from the mash may be further processed into an edible oil for consumer use, or because of its high triglyceride content, it can be an excellent source of metabolic energy, so animal feed It can also be used as a component of In another embodiment, the crude corn oil can also be used as a feedstock for biodiesel or renewable diesel.

  In one embodiment, the extractant by-product may be used in whole or in part as a component of animal feed by-product, or may be used as a raw material for biodiesel or renewable diesel.

  In further embodiments, the solid may be separated from the mash and may include triglycerides and free fatty acids. These solids (or streams) may be used as animal feed and are recovered either as a drain from centrifugation or after drying. The solid (or wet cake) may be particularly suitable as a ruminant (eg, dairy) feed due to the high content of available lysine and bypass or non-ruminal protein. For example, these solids can be particularly useful in high protein, low fat feeds. In another embodiment, these solids may be used as a base, i.e. other by-products such as syrup are added to this solid to form a product that can be used as animal feed. May be. In another embodiment, various amounts of other by-products may be added to the solid to adjust the properties of the resulting product to meet the needs of a particular animal species.

  The solid composition separated from the total distillation effluent described in Example 21 may include, for example, crude protein, fatty acid, and fatty acid isobutyl ester. In one embodiment, the composition (or by-product) may be used wet or dry as animal feed, in which case, for example, high protein (eg, high lysine), low fat, and high Fiber content is desired. In another embodiment, if a higher fat, low fiber animal feed is desired, fat may be added to the composition, eg, from another byproduct stream. In one embodiment, this higher fat, low fiber animal feed can be used in pigs or poultry. In further embodiments, the non-aqueous composition of Condensed Distillers Soluble (CDS) (see, eg, Example 21) includes, for example, proteins, fatty acids, and fatty acid isobutyl esters and salts and carbohydrates, etc. Other dissolved and suspended solids can be included. The CDS composition may be used, for example, in a wet or dry state as animal feed, in which case a high protein, low fat, high inorganic salt feed component is desired. In one embodiment, the composition may be used as a component of dairy cow feed.

  In another embodiment, oil from the fermentation process can be recovered by evaporation. The non-aqueous composition may include a fatty acid isobutyl ester and a fatty acid (see, eg, Example 20), and the composition (or stream) is passed to a hydrolyzer to recover isobutanol and fatty acid. Can be supplied. In a further embodiment, this stream can be used as a feedstock for biodiesel production.

  The various streams produced by the production of alcohol (eg, butanol) by a fermentation process can be combined in many ways to produce several by-products. For example, if crude corn from mash is used to produce fatty acids that are used as extractants and the lipids are extracted by an evaporator for other purposes, the remaining streams are combined to produce crude protein, crude It can be processed to produce a byproduct composition comprising fat, triglyceride, fatty acid, and fatty acid isobutyl ester. In one embodiment, the composition comprises at least about 20-35% crude protein, at least about 1-20% crude fat, at least about 0-5% triglyceride, at least about 4-10% by weight. Fatty acids, and at least about 2-6% by weight fatty acid isobutyl esters may be included. In one particular embodiment, the byproduct composition comprises about 25% crude protein, about 10% crude fat, about 0.5% triglyceride, about 6% fatty acid, and about 4% by weight. Fatty acid isobutyl ester may be included.

  In another embodiment, the lipid is extracted by an evaporator, the fatty acid is used for other purposes, about 50% by weight of the crude corn from the mash and the remaining stream are combined, processed and obtained. The resulting by-product composition can include proteins, crude fat, triglycerides, fatty acids, and fatty acid isobutyl esters. In one embodiment, the composition comprises at least about 25-31% crude protein, at least about 6-10% crude fat, at least about 4-8% triglyceride, at least about 0-2% by weight. Fatty acids, and at least about 1-3% by weight fatty acid isobutyl esters may be included. In one particular embodiment, the byproduct composition comprises about 28% crude protein, about 8% crude fat, about 6% triglyceride, about 0.7% fatty acid, and about 1% by weight. Fatty acid isobutyl ester may be included.

  In another embodiment, 50% by weight of the corn oil extracted from the mash and solids separated from total distillate waste is combined and the resulting by-product composition is crude protein, crude fat, triglyceride, fatty acid, fatty acid isobutyl Esters, lysines, neutral detergent fibers (NDF), and acidic detergent fibers (ADF) can be included. In one embodiment, the composition comprises at least about 26-34% crude protein, at least about 15-25% crude fat, at least about 12-20% triglyceride, at least about 1-2% by weight. Fatty acids, at least about 2-4% by weight fatty acid isobutyl ester, at least about 1-2% by weight lysine, at least about 11-23% by weight NDF, and at least about 5-11% by weight ADF. In one particular embodiment, the by-product composition comprises about 29% crude protein, about 21% crude fat, about 16% triglyceride, about 1% fatty acid, about 3% fatty acid isobutyl ester. About 1% by weight lysine, about 17% by weight NDF, and about 8% by weight ADF. The high fat, high triglyceride, and high lysine content and lower fiber content of this by-product composition may be desirable for pig and poultry feed.

  As noted above, the various streams generated by the production of alcohol (eg, butanol) by a fermentation process are often used to produce by-product compositions that include crude protein, crude fat, triglycerides, fatty acids, and fatty acid isobutyl esters. Can be combined in the following manner. For example, a composition comprising at least about 6% crude fat and at least about 28% crude protein can be used as an animal feed product for dairy animals. A composition comprising at least about 6% crude fat and at least about 26% crude protein can be used as an animal feed product for feedlot cattle, while at least about 1% crude fat and at least about 27% crude protein. The containing composition can be used as an animal feed product for wintering cattle. Compositions comprising at least about 13% crude fat and at least about 27% crude protein can be used as poultry animal feed products. Compositions comprising at least about 18% crude fat and at least about 22% crude protein can be used as monogastric animal feed products. Thus, the various streams can be combined to customize the feed product for a particular animal species.

  As noted above, the various streams generated by the production of alcohol (eg, butanol) by a fermentation process are often used to produce by-product compositions that include crude protein, crude fat, triglycerides, fatty acids, and fatty acid isobutyl esters. Can be combined. For example, a composition comprising at least about 6% crude fat and at least about 28% crude protein can be used as an animal feed product for dairy animals. A composition comprising at least about 6% crude fat and at least about 26% crude protein can be used as an animal feed product for feedlot cattle, while at least about 1% crude fat and at least about 27% crude protein. The containing composition can be used as an animal feed product for wintering cattle. Compositions comprising at least about 13% crude fat and at least about 27% crude protein can be used as poultry animal feed products. Compositions comprising at least about 18% crude fat and at least about 22% crude protein can be used as monogastric animal feed products. Thus, the various streams can be combined to customize the feed product for a particular animal species.

  In one embodiment, one or more streams produced by the production of alcohol (eg, butanol) by a fermentation process produces a composition comprising at least about 90% COFA that can be used as a fuel source, such as biodiesel. Can be combined in many ways.

  As an example of one embodiment of the method of the present invention, ground cereal (eg, corn treated with a hammer mill) and one or more enzymes are combined to produce a slurryed cereal. This slurried cereal is cooked, liquefied and flushed with flash steam to obtain a cooked mash. The cooked mash is then filtered to remove suspended solids and produce a wet cake and filtrate. Filtration can be performed by several methods, such as centrifugation, sieving, or vacuum filtration, which can remove at least about 80% to at least about 99% suspended solids from the mash.

  The wet cake is reslurried with water and refiltered to remove additional starch and produce a washed filter cake. The reslurry process may be repeated several times, for example 1-5 times. The water used to reslurry the wet cake may be recycled water produced during the fermentation process. The filtrate produced by the reslurry / refiltration process can be returned to the initial mixing step to form a slurry with the milled cereal. The filtrate can be heated or cooled prior to the mixing step.

  The washed filter cake can be reslurried with beer at several stages during the production process. For example, the washed filter cake can be reslurried with beer after the fermenter, before the preflash column, or at the feed point to the cereal dryer of the still. The washed filter cake may be dried separately from other by-products or may be used directly as a wet cake for the production of DDGS.

  The filtrate produced as a result of the initial mixing step can be further processed as described herein. For example, the filtrate can be heated using steam or process to process heat exchange. A saccharification enzyme may be added to the filtrate, and the dissolved starch in the filtrate may be partially or fully saccharified. The saccharified filtrate can be cooled by several means such as interprocess exchange, cooling water exchange, or cold water exchange.

  The cooled filtrate can then be added to a fermentation apparatus as well as a recombinant yeast capable of producing a microorganism suitable for alcohol production, such as butanol. In addition, ammonia and a recycle stream can also be added to the fermenter. The process may include at least one fermenter, at least two fermenters, at least three fermenters, or at least four fermenters. In order to reduce atmospheric emissions (eg, butanol atmospheric emissions) and increase product yield, carbon dioxide produced during fermentation can be vented to a scrubber.

  The solvent may be added to the fermentor via a recycling loop or may be added directly to the fermentor. The solvent can be one or more organic compounds that can dissolve or react with an alcohol (eg, butanol) and can have limited solubility in water. The solvent may be removed from the fermenter in succession as one liquid phase or as two liquid phase materials, or the solvent may be withdrawn batchwise as one or two liquid phase materials.

  The beer can be degassed. The beer can be heated prior to degassing, for example, by inter-process exchange with a hot mash or pre-flash tower overhead. Vapor may be exhausted to a condenser and then to a scrubber. The degassed beer can be further heated, for example, by inter-process heat exchange with other streams in the distillation zone.

  Preheated beer and solvent may enter a preflash tower that can be modified from the beer tower of a conventional dry ground fuel ethanol plant. The column is operated at a pressure below atmospheric pressure and can be driven by water vapor taken from a series of evaporators or from a mash cooking process. The top of the preflash tower can be condensed by heat exchange by several combinations of cooling water and interprocess heat exchange including heat exchange with the preflash tower feed. The liquid condensate can be sent to an alcohol / water decanter (eg, butanol / water decanter).

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

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

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

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

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

  The dilute waste liquor produced from the bottom of the preflash column described above can be sent to a multi-effect evaporator. The evaporator can have two, three, or more stages. The evaporator may have a four body configuration with two effects, similar to the conventional design of a fuel ethanol plant, may have a three bodies with three effects, or other configurations You may have. The dilute waste liquor can enter any of the plurality of utility parts. At least one of the first effect body may be heated by steam from an overpressure alcohol (eg, butanol) tower. Steam can be withdrawn from the lowest pressure utility and heat in the form of water vapor can be provided to preflash and solvent towers at pressures below atmospheric pressure. Syrup from the evaporator may be added to the cereal dryer of the still.

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

  When corn is used as a source of ground cereal, corn oil can be separated from the process stream in any of several ways. For example, a centrifuge may be operated to produce a corn oil stream after filtration of the cooked mash, or a preflush tower water phase centrifuge is operated to produce a corn oil stream. May be. The intermediate concentrated syrup or final syrup can be centrifuged to produce a corn oil stream.

  In another example of an embodiment of the method of the present invention, the material discharged from the fermenter is processed in a separation system including devices such as a centrifuge, a settler, a hydrocyclone, etc., and combinations thereof, A concentrated form of live yeast can be recovered that can be reused for re-use in the next fermentation batch, either directly or after some reconditioning. This separation system can also produce an organic stream containing fatty acid esters (eg, isobutyl fatty acid ester) and alcohol (eg, butanol) produced from fermentation, and an aqueous stream containing a slight amount of immiscible organics. This water stream can be used before or after removing the alcohol (eg, butanol) content to repulp and inject the washed low starch solids separated from the liquefied mash. This has the advantage of avoiding what could otherwise be a long belt driven transport system to move these solids from the liquefaction zone to the grain drying and syrup mixing zone. Furthermore, this total distillation waste obtained after the alcohol (eg butanol) has been removed needs to be separated into a dilute distillation waste and a wet cake fraction using existing or new separation equipment, This dilute distillation liquor will partially form a back flow to combine with cooking water to prepare a new batch of fermentable mash. Another advantage of this embodiment is that a portion of any residual dissolved starch retained in the solid moisture separated from the liquefied mash can be captured and recovered through this backflow. Alternatively, the yeast contained in the solid stream can be considered non-viable and can be redistributed into the water stream, and this combined stream is freed of any alcohol (eg, butanol) content remaining from the fermentation by distillation. Non-viable organics can be further separated for use as nutrients in the propagation process.

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

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

  The methods and systems described above can result in improved extraction activity and / or efficiency in product alcohol production as a result of removal of undissolved solids. For example, extractive fermentation without the presence of undissolved solids results in a higher mass transfer rate of product alcohol from the fermentation broth to the extractant, better phase separation of the extractant from the fermentation inside or outside the fermentor, It can result in a lower hold up of the extractant as a result of the higher extractant drop rise rate. Also, for example, extractant droplets that are retained in the fermentation broth during fermentation will disengage faster and more completely from the fermentation broth, thereby reducing the free extractant in the fermentation broth and during the process. The amount of extractant lost can be reduced. Further, for example, the microorganisms can be reused, and further equipment in downstream processing can be omitted, such as, for example, some or all of the beer tower and / or total distillation waste centrifuge. Furthermore, for example, the possibility of the extractant being lost in DDGS is eliminated. Also, for example, the ability to recycle microorganisms increases the overall rate of product alcohol production, reduces total titer requirements, and / or reduces aqueous titer requirements, thereby healthier microorganisms and higher The production rate can be brought about. In addition, for example, omitting the agitator in the fermenter to reduce capital costs; because the extractant retained is minimized and the volume is used more efficiently because there are no undissolved solids It may be possible to improve the productivity of the fermenter; and / or to use continuous fermentation or a smaller fermenter in an unconstructed plant.

  Examples of improved extraction efficiencies include, for example, a stabilized partition coefficient, improved (eg, faster or more complete) phase separation, improved liquid-liquid mass transfer coefficient, operation at lower titers, improved Process stream recyclability, improved fermentation volumetric efficiency, improved feedstock (eg corn) loading, improved butanol titer tolerance of microorganisms (eg recombinant microorganisms), water reuse, energy reduction, extractant There may be increased reuse and / or microbial reuse.

  For example, the volume occupied by solids in the fermenter is reduced. Therefore, the effective volume of the fermentation apparatus available for fermentation can be increased. In certain embodiments, the volume of fermenter available for fermentation is increased by at least about 10%.

  For example, there can be stabilization of the distribution coefficient. Because the corn oil in the fermenter can be reduced by removing solids from the raw slurry prior to fermentation, the extractant is exposed to less corn oil. Corn oil can be combined with an extractant to reduce the partition coefficient when present in sufficient amounts. Therefore, the distribution coefficient of the extractant phase in the fermenter becomes more stable due to the reduction of corn oil introduced into the fermenter. In certain embodiments, the partition coefficient is reduced by less than about 10% over 10 fermentation cycles.

  For example, there is a higher mass transfer rate of the product alcohol from the fermentation broth to the extractant (eg in the form of a higher mass transfer coefficient), which results in improved efficiency of product alcohol production. There may be an improvement in the extraction efficiency of butanol by the agent. In certain embodiments, the mass transfer coefficient is increased at least 2-fold (see Examples 4 and 5).

  Furthermore, there may be an improved phase separation between the fermentation broth and the extractant, which reduces the likelihood of emission formation, thereby resulting in improved efficiency of product alcohol production. For example, phase separation can occur faster or can be more complete. In certain embodiments, phase separation can occur when no appreciable phase separation has been observed in advance for 24 hours. In certain embodiments, phase separation occurs at least about 2 times faster, at least about 5 times faster, or at least about 10 times faster than phase separation when no solids are removed (Examples 6 and 7). See).

  Furthermore, there may be an increase in extractant recovery and reuse. The extractant is not “trapped” in a solid that can eventually be removed as DDGS, thereby providing improved efficiency of product alcohol production (see Examples 8 and 9). Also, the dilution of the extractant with corn oil can be reduced and the extractant can be less degraded (see Example 10).

  Also, since the flow rate of the extractant can be reduced, operating costs are reduced, thereby providing improved efficiency of product alcohol production.

  Furthermore, retention of the extractant is reduced as a result of the extractant droplets rising at a higher rate, thereby providing improved efficiency of product alcohol production. Reduction in the amount of undissolved solids in the fermenter also results in improved efficiency of product alcohol production.

  Furthermore, since there is no longer a need to suspend undissolved solids, the stirrer can be removed from the fermenter, thereby reducing capital costs and energy and increasing the efficiency of product alcohol production.

  In certain embodiments, the fermentation broth 40 may be discharged from the outlet of the fermentation apparatus 30. The absence or minimal of undissolved solids leaving the fermenter 30 with the fermentation broth 40 has several additional advantages. For example, the need for units and operations in downstream processing, such as beer towers or distillation towers, can be eliminated, thereby increasing the efficiency of product alcohol production. Furthermore, since no undissolved solids are present in the fermentation broth 40 exiting the fermentation apparatus 30, there is no DDGS formed with the “trapped” extractant. Also, some or all of the total distillation waste centrifuge may be omitted as a result of less undissolved solids in the final broth exiting the fermenter.

  As noted above, the method of the present invention provides several advantages that can result in improved productivity (eg, batch or continuous) of product alcohols such as butanol. For example, improved mass transfer allows operation at lower aqueous titers, making microorganisms “healthy”. Better phase separation can lead to improved fermenter volumetric efficiency and the possibility of processing less reactor content through beer towers, distillation towers, and the like. Furthermore, there is less solvent loss through the solid and there is a possibility of cell reuse. The method of the present invention may also provide a high quality of DDGS.

  In addition, the methods described herein provide for removal of pre-fermentation oil (eg, corn oil), which may allow controlled addition of oil to the fermentation. Furthermore, removal of oil prior to fermentation can give some flexibility to the amount of oil present in the DDGS. That is, oil may be added to DDGS in various amounts, producing DDGS with varying fat content depending on the nutritional requirements of a particular animal species.

Recombinant microorganisms and butanol biosynthetic pathways While not wishing to be bound by theory, the methods described herein produce alcohols at any alcohol-producing microorganism, particularly at potencies that exceed their resistance levels. It is considered useful in connection with recombinant microorganisms.

Alcohol-producing microorganisms are known in the art. For example, the fermentation oxidation of methane by a methane-utilizing bacterium (for example, Methylosinus trichosporum) produces methanol, and methanol (C ₁ alkyl alcohol) is esterified with carboxylic acid and methanol together with carboxylic acid. By contacting with a catalyst that can be converted, a methanolic ester of carboxylic acid is formed. Yeast strain CEN. PK113-7D (CBS 8340, the Central Blue room Scheme; van Dijken, et al., Enzyme Microb. Techno. 26: 706-714, 2000) can produce ethanol, which, together with carboxylic acid and ethanol The ethyl ester is formed by contacting the acid with a catalyst capable of esterification (see, eg, Example 36).

  Recombinant microorganisms that produce alcohol are also known in the art (eg, Ohta, et al., Appl. Environ. Microbiol. 57: 893-900, 1991; Underwood, et al., Appl. Environ. Microbiol. 68: 1071-1081, 2002; Shen and Liao, Metab. Eng.10: 312-320, 2008; Hahnai, et al., Appl. Environ.Microbiol.73: 7814-7818, 2007; U.S. Pat. No. 5,712,133; PCT application publication number WO 1995/028476; Feldmann, et al., Appl. Biobiol.Biotechnol.38: 354-361,1992; Zhang, et al., Science 267: 240-243, 1995; U.S. Patent Application Publication No. 2007 / 0031918A1; U.S. Patent No. 7,223,575 U.S. Patent No. 7,741,119; U.S. Patent No. 7,851,188; U.S. Patent Application Publication No. 2009 / 0203099A1; U.S. Patent Application Publication No. 2009 / 0246846A1; and PCT Application Publication No. WO 2010/075241, all of which are incorporated herein by reference).

  Suitable recombinant microorganisms capable of producing butanol are known in the art, and specific suitable microorganisms capable of producing butanol are described herein. Recombinant microorganisms that produce butanol via the biosynthetic pathway include Clostridium, Zymomonas, Escherichia, Salmonella, Serratia, and Erwinia. , Klebsiella genus, Shigella genus, Rhodococcus genus, Pseudomonas genus, Bacillus genus, Lactobacillus genus L , Klebsiella, Paenibatil Genus (Paenibacillus), Arthrobacter, Corynebacterium, Brevibacterium, Schizosaccharomyces, Kryveres, Kluyvermyeso ), Pichia, Candida, Hansenula, Issatchenkia, or Saccharomyces. In one embodiment, the recombinant microorganism is Escherichia coli, Lactobacillus plantarum, Kluyveromyces lactis, Kluyveromyces marciane cerevisiae and Kluyveromyces cerevisiax. Saccharomyces cerevisiae) may be selected. In one embodiment, the recombinant microorganism is yeast. In one embodiment, the recombinant microorganism is selected from the genus Saccharomyces, Zygosaccharomyces, Schizosaccharomyces, Dekkera, Toltopres iss, Torulopsis is, Crabtree positive yeast selected from several species of the genus (Candida). Examples of crabtree positive yeast species include, but are not limited to, Saccharomyces cerevisiae, Saccharomyces kluyveri, Schizosaccharomyces pombe (Schizosaccharomyces pombe).・ Siccharomyces mikatae, Saccharomyces paradoxus, Zygosaccharomyces rouxii, and Candida glabrata ata).

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

  Production of butanol using fermentation by microorganisms, as well as microorganisms that produce butanol, are disclosed, for example, in US Patent Publication No. 2009/0305370, which is incorporated herein by reference. In certain embodiments, the microorganism comprises a butanol biosynthetic pathway. In certain embodiments, at least one, at least two, at least three, or at least four polypeptides that catalyze pathway substrate-product conversion are encoded by heterologous polynucleotides in the microorganism. In certain embodiments, all polypeptides that catalyze pathway substrate-product conversion are encoded by heterologous polynucleotides in the microorganism. In certain embodiments, the microorganism comprises a decrease or disappearance of pyruvate decarboxylase activity. Microorganisms that are substantially free of pyruvate decarboxylase activity are described in US Patent Application Publication No. 2009/030305363, which is incorporated herein by reference. Microorganisms substantially free of enzymes having NAD-dependent glycerol-3-phosphate dehydrogenase activity, such as GPD2, are also described in this document.

  Suitable biosynthetic pathways for the production of butanol are known in the art and certain suitable pathways are described herein. In certain embodiments, the butanol biosynthetic pathway comprises at least one gene that is heterologous to the host cell. In certain embodiments, the butanol biosynthetic pathway comprises two or more genes that are heterologous to the host cell. In certain embodiments, the butanol biosynthetic pathway includes a heterologous gene that encodes a polypeptide corresponding to every step of the biosynthetic pathway.

  Certain suitable proteins having the ability to catalyze specified substrate-product conversion are described herein, and other suitable proteins are provided in the art. For example, U.S. Patent Application Publication Nos. 2008/0261230, 2009/0163376, and 2010/0197519, which are incorporated herein by reference, are acetohydroxy acid isomeroreductases. US Patent Application Publication No. 2010/0081154, incorporated by reference, describes dihydroxy acid dehydratase; US Patent Application Publication, alcohol dehydrogenase, incorporated herein by reference. No. 2009/0269823.

  Many levels of sequence identity are useful for identifying polypeptides from other species, and such polypeptides have the same or similar function or activity and are described herein. It is well understood by those skilled in the art that it is suitable for use with recombinant microorganisms. Useful examples of percent identity include, but are not limited to, 75%, 80%, 85%, 90%, or 95%, or 75%, 76%, 77%, 78%, 79 %, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, Any integer percentage between 75% and 100%, such as 96%, 97%, 98% or 99% may be useful in describing the present invention.

  Suitable strains include the specific applications cited herein and incorporated herein by reference, as well as US Provisional Patent Application No. 61 / 380,563, filed Sep. 7, 2010. What is described is mentioned. Certain suitable strain structures, including those used in the Examples, are provided herein.

Preparation of Saccharomyces cerevisiae strain BP1083 ("NGCI-070"; PNY1504) Strain BP1064 is a CEN. PK 113-7D (CBS 8340; Centalbureau voor Schulmelculures (CBS) Fungal Biodiversity Centre, Netherlands), including deletions of the following genes: URA3, HIS3, PDC1, PDC5, PDC5, PDC5, PDC5, PDC5 BP1064 was transformed with plasmids pYZ090 (SEQ ID NO: 1, described in US Provisional Patent Application No. 61 / 246,844) and pLH468 (SEQ ID NO: 2) to obtain strain NGCI-070 (BP1083, PNY1504). Was generated.

  Homologous recombination with a PCR fragment containing homologous regions upstream and downstream of either the G418 resistance marker for selection of target genes and transformants or the URA3 gene created a deletion that completely removed the entire coding sequence. . The G418 resistance marker flanked by the loxP site was removed using Cre recombinase. The URA3 gene was removed by homologous recombination to create a scarless deletion or, if the loxP site is flanked, removed using Cre recombinase.

  The procedure for scarless deletion is described in Akada, et al. , (Yeast 23: 399-405, 2006). In general, a PCR cassette for each scarless deletion was made by combining the four fragments, ABUC, by overlapping PCR. The PCR cassette, along with a promoter (250 bp upstream of the URA3 gene) and a terminator (150 bp downstream of the URA3 gene), is a native CEN. It contained a selectable / counter-selectable marker, URA3 (fragment U), consisting of the PK 113-7D URA3 gene. Fragments A and C, each 500 bp in length, corresponded to 500 bp (fragment A) immediately upstream of the target gene and 3'500 bp (fragment C) of the target gene. Fragments A and C were used for integration of the cassette into the chromosome by homologous recombination. When a direct repeat of the sequence corresponding to fragment B was generated upon integration of the cassette into the chromosome, fragment B (500 bp in length) corresponds to 500 bp immediately downstream of the target gene, which Used to excise the URA3 marker and fragment C from the chromosome by genetic recombination. Using the PCR product ABUC cassette, the URA3 marker was first integrated into the chromosome and then excised from the chromosome by homologous recombination. Upon initial integration, the gene was deleted except for 3'500 bp. At the time of excision, the 3'500 bp region of the gene was also deleted. In gene integration using this method, the gene to be integrated was contained in the PCR cassette between fragments A and B.

Deletion of URA3 To delete the endogenous URA3 coding region, the ura3 :: loxP-kanMX-loxP cassette was amplified by PCR from pLA54 template DNA (SEQ ID NO: 3). pLA54 is K.I. The loxP site is flanked, containing the K. lactis TEF1 promoter and the kanMX marker, allowing recombination by Cre recombinase and removal of the marker. PCR was performed using Phusion® DNA polymerase (New England BioLabs Inc., Ipswich, Mass.) And primers BK505 and BK506 (SEQ ID NOs: 4 and 5). The URA3 portion of each primer was obtained from the 5 ′ region upstream of the URA3 promoter and the 3 ′ region downstream of the coding region so that incorporation of the loxP-kanMX-loxP marker resulted in substitution of the URA3 coding region. Using standard genetic recombination techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202), the PCR product is CEN. Transformants were selected into YK containing G418 (100 μg / mL) at 30 ° C. transformed into PK 113-7D. Primers LA468 and LA492 (SEQ ID NOs: 6 and 7) and designated CEN. Transformants were screened by PCR using PK 113-7D Δura3 :: kanMX to confirm correct integration.

HIS3 Deletion Four fragments of a PCR cassette for scarless HIS3 deletion were obtained from Phusion® High Fidelity PCR Master Mix (New England BioLabs Inc., Ipswich, Mass.) And Gentra® Puregene®. Trademark: Yeast / Bact, CEN. As a template, prepared using a kit (Qiagen, Valencia, CA). Amplified using PK 113-7D genomic DNA. HIS3 fragment A was amplified using primer oBP452 (SEQ ID NO: 14) and primer oBP453 (SEQ ID NO: 15) containing a 5 ′ tail having homology to the 5 ′ end of HIS3 fragment B. Primer oBP454 (SEQ ID NO: 16) containing a 5 'tail having homology to the 3' end of HIS3 fragment A, and a 5 'tail having homology to the 5' end of HIS3 fragment U Was amplified using a primer oBP455 (SEQ ID NO: 17). Primer oBP456 (SEQ ID NO: 18) containing a 5 'tail having homology to the 3' end of HIS3 fragment B, and HIS3 fragment U, 5 'tail having homology to the 5' end of HIS3 fragment C Was amplified using a primer oBP457 (SEQ ID NO: 19) containing HIS3 fragment C was amplified using primer oBP458 (SEQ ID NO: 20) containing a 5 ′ tail with homology to the 3 ′ end of HIS3 fragment U, and primer oBP459 (SEQ ID NO: 21). The PCR product was purified using a PCR Purification kit (Qiagen, Valencia, CA). HIS3 fragment AB was generated by overlapping PCR by mixing HIS3 fragment A and HIS3 fragment B and amplifying with primers oBP452 (SEQ ID NO: 14) and oBP455 (SEQ ID NO: 17). HIS3 fragment UC was generated by overlapping PCR by mixing HIS3 fragment U and HIS3 fragment C and amplifying with primers oBP456 (SEQ ID NO: 18) and oBP459 (SEQ ID NO: 21). The resulting PCR product was purified on an agarose gel followed by a Gel Extraction kit (Qiagen, Valencia, CA). The HIS3 ABUC cassette was generated by overlapping PCR by mixing HIS3 fragment AB and HIS3 fragment UC and amplifying with primers oBP452 (SEQ ID NO: 14) and oBP459 (SEQ ID NO: 21). The PCR product was purified using a PCR Purification kit (Qiagen, Valencia, CA).

  CEN. Competent cells of PK 113-7D Δura3 :: kanMX were prepared and transformed with HIS3 ABUC PCR cassette using Frozen-EZ Yeast Transformation II ™ kit (Zymo Research Corporation, Irvine, Calif.). The transformation mixture was plated at 30 ° C. in uracil-deficient synthetic complete medium supplemented with 2% glucose. Gentra (R) Puregene (R) Yeast / Bact. Transformants with his3 knockout were screened by PCR with primers oBP460 (SEQ ID NO: 22) and oBP461 (SEQ ID NO: 23) using genomic DNA prepared using the kit (Qiagen, Valencia, CA). Accurate transformants were obtained from strain CEN. Selected as PK 113-7D Δura3 :: kanMX Δhis3 :: URA3.

KanMX marker removal from Δura3 site and URA3 marker removal from Δhis3 site Using the Frozen-EZ Yeast Transformation II ™ kit (Zymo Research Corporation, Irvine, Calif.), PRS423 :: PGAL1-cre (SEQ ID NO: 66, USA) Provisional patent application 61 / 290,639) and CEN. The KanMX marker was removed by transforming PK 113-7D Δura3 :: kanMX Δhis3 :: URA3 and plating in histidine and uracil deficient synthetic complete medium supplemented with 2% glucose at 30 ° C. Transformants were grown in YP supplemented with 1% galactose for about 6 hours at 30 ° C. to induce Cre recombinase and KanMX marker excision and YPD (2% glucose at 30 ° C. for recovery. ) Plated on plate. Isolates grown overnight in YPD and plated at 30 ° C. in synthetic complete medium containing 5-fluoro-orotic acid (5-FOA, 0.1%) to lose URA3 marker Selected. 5-FOA resistant isolates were grown in YPD and plated to remove the pRS423 :: PGAL1-cre plasmid. Isolates were examined for loss of KanMX marker, URA3 marker, and pRS423 :: PGAL1-cre plasmid by assaying growth on YPD + G418 plates, plates of uracil-deficient synthetic complete medium, and plates of histidine-deficient synthetic complete medium. An accurate isolate that was sensitive to G418 and auxotrophic for uracil and histidine was obtained from strain CEN. Selected as PK 113-7D Δura3 :: loxP Δhis3 and represented as BP857. Gentra (R) Puregene (R) Yeast / Bact. Using genomic DNA prepared using the kit (Qiagen, Valencia, CA), primers oBP450 (SEQ ID NO: 24) and oBP451 (SEQ ID NO: 25) for Δura3, and primer oBP460 (SEQ ID NO: 22) for Δhis3 and Deletion and marker removal were confirmed by PCR and sequencing with oBP461 (SEQ ID NO: 23).

PDC6 Deletion Four fragments of a PCR cassette for scarless PDC6 deletion were obtained from Phusion® High Fidelity PCR Master Mix (New England BioLabs Inc., Ipswich, Mass.) And Gentra® Puregene®. Trademark) Yeast / Bact. CEN. As a template prepared using a kit (Qiagen, Valencia, CA). Amplified using PK 113-7D genomic DNA. PDC6 fragment A was amplified using primer oBP440 (SEQ ID NO: 26) and primer oBP441 (SEQ ID NO: 27) containing a 5 ′ tail having homology to the 5 ′ end of PDC6 fragment B. Primer oBP442 (SEQ ID NO: 28) containing a 5 'tail having homology to the 3' end of PDC6 fragment A, and 5 'tail having homology to the 5' end of PDC6 fragment U Amplification was performed using primer oBP443 (SEQ ID NO: 29). Primer oBP444 (SEQ ID NO: 30) containing a 5 'tail having homology to the 3' end of PDC6 fragment B, and 5 'tail having homology to the 5' end of PDC6 fragment C Was amplified using a primer oBP445 (SEQ ID NO: 31) containing PDC6 fragment C was amplified using primer oBP446 (SEQ ID NO: 32) containing a 5 ′ tail with homology to the PDC6 fragment U3 ′ end and primer oBP447 (SEQ ID NO: 33). The PCR product was purified using a PCR Purification kit (Qiagen, Valencia, CA). PDC6 fragment AB was generated by overlapping PCR by mixing PDC6 fragment A and PDC6 fragment B and amplifying with primers oBP440 (SEQ ID NO: 26) and oBP443 (SEQ ID NO: 29). PDC6 fragment UC was generated by overlapping PCR by mixing PDC6 fragment U and PDC6 fragment C and amplifying with primers oBP444 (SEQ ID NO: 30) and oBP447 (SEQ ID NO: 33). The resulting PCR product was purified on an agarose gel followed by a Gel Extraction kit (Qiagen, Valencia, CA). A PDC6 ABUC cassette was generated by overlapping PCR by mixing PDC6 fragment AB and PDC6 fragment UC and amplifying with primers oBP440 (SEQ ID NO: 26) and oBP447 (SEQ ID NO: 33). The PCR product was purified using a PCR Purification kit (Qiagen, Valencia, CA).

  CEN. Competent cells of PK 113-7D Δura3 :: loxP Δhis3 were prepared and transformed with the PDC6 ABUC PCR cassette using the Frozen-EZ Yeast Transformation II ™ kit (Zymo Research Corporation, Irvine, Calif.). The transformation mixture was plated at 30 ° C. in uracil-deficient synthetic complete medium supplemented with 2% glucose. Transformants with pdc6 knockout were obtained from Gentra® Puregene® Yeast / Bact. Screening was performed by PCR with primers oBP448 (SEQ ID NO: 34) and BP449 (SEQ ID NO: 35) using genomic DNA prepared using the kit (Qiagen, Valencia, CA). Accurate transformants were obtained from strain CEN. Selected as PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 :: URA3.

  CEN. PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 :: URA3 was grown overnight in YPD and plated at 30 ° C. in synthetic complete medium containing 5-fluoro-orotic acid (0.1%) Isolates that lost the URA3 marker were selected. Gentra (R) Puregene (R) Yeast / Bact. Deletions and marker removal were confirmed by PCR and sequencing with primers oBP448 (SEQ ID NO: 34) and oBP449 (SEQ ID NO: 35) using genomic DNA prepared using the kit (Qiagen, Valencia, CA). Negative PCR results using primers specific for the coding sequences of PDC6, oBP554 (SEQ ID NO: 36) and oBP555 (SEQ ID NO: 37) demonstrated the absence of the PDC6 gene from the isolate. The correct isolate was obtained as strain CEN. PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 was selected and expressed as BP891.

PDC1 deletion ilvDSm integration The PDC1 gene was deleted and Streptococcus mutans ATCC No. Substitution with the ilvD coding region from 700610. A fragment, followed by the IlvD coding region from Streptococcus mutans of the PCR cassette for PDC1 deletion-ilvDSm integration, the Phusion® High Fidelity PCR Master Mix (New England BioLabs. , Ipswich, Mass.) And Gentra® Puregene® Yeast / Bact. Amplification was performed using NYLA83 genomic DNA as a template prepared using kit (Qiagen, Valencia, CA). NYLA83 is a strain having a PDC1 deletion-ilvDSm integration (incorporated herein by reference in its entirety) as described in US Patent Application Publication No. 2009/030363, which is hereby incorporated by reference in its entirety. Of US Patent Application Publication No. 201110124060). Proliferation of PDC1 fragment A-ilvDSm (SEQ ID NO: 69) with primer oBP513 (SEQ ID NO: 38) and primer oBP515 (SEQ ID NO: 39) containing a 5 ′ tail having homology to the 5 ′ end of PDC1 fragment B did. PDC1 deletion—B, U, and C fragments of the PCR cassette for ilvDSm integration were obtained from Phusion® High Fidelity PCR Master Mix (New England BioLabs Inc., Ipswich, Mass.) And Gentra® Puregene ( (Registered trademark) Yeast / Bact. CEN. As a template prepared using a kit (Qiagen, Valencia, CA). Amplified using PK 113-7D genomic DNA. Primer oBP516 (SEQ ID NO: 40) containing a 5 'tail with homology to the 3' end of PDC1 fragment A-ilvDSm, and PDC1 fragment B with homology to the 5 'end of PDC1 fragment U 'Amplified with primer oBP517 (SEQ ID NO: 41) containing tail. Primer oBP518 (SEQ ID NO: 42) containing a 5 'tail having homology to the 3' end of PDC1 fragment B, and 5 'tail having homology to the 5' end of PDC1 fragment C Was amplified using a primer oBP519 (SEQ ID NO: 43). PDC1 fragment C was amplified using primer oBP520 (SEQ ID NO: 44) containing a 5 ′ tail having homology to the 3 ′ end of PDC1 fragment U, and primer oBP521 (SEQ ID NO: 45). The PCR product was purified using a PCR Purification kit (Qiagen, Valencia, CA). PDC1 fragment A-ilvDSm-B is generated by overlapping PCR by mixing PDC1 fragment A-ilvDSm and PDC1 fragment B and amplifying with primers oBP513 (SEQ ID NO: 38) and oBP517 (SEQ ID NO: 41) did. PDC1 fragment UC was generated by overlapping PCR by mixing PDC1 fragment U and PDC1 fragment C and amplifying with primers oBP518 (SEQ ID NO: 42) and oBP521 (SEQ ID NO: 45). The resulting PCR product was purified on an agarose gel followed by a Gel Extraction kit (Qiagen, Valencia, CA). PDC1 A-ilvDSm-BUC cassette by duplicating PCR by mixing PDC1 fragment A-ilvDSm-B and PDC1 fragment UC and amplifying with primers oBP513 (SEQ ID NO: 38) and oBP521 (SEQ ID NO: 45) (SEQ ID NO: 70) was generated. The PCR product was purified using a PCR Purification kit (Qiagen, Valencia, CA).

  CEN. Competent cells of PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 were made and PDC1 A-UlvDSm cassette with Fzen-EZ Yeast Transformation II ™ kit (Zymo Research Corporation, Irvine, Calif.) Converted. The transformation mixture was plated at 30 ° C. in uracil-deficient synthetic complete medium supplemented with 2% glucose. Transformants with pdc1 knockout ilvDSm integration were obtained from Gentra® Puregene® Yeast / Bact. Screening was performed by PCR with primers oBP511 (SEQ ID NO: 46) and oBP512 (SEQ ID NO: 47) using genomic DNA prepared using the kit (Qiagen, Valencia, CA). Negative PCR results using primers specific for the coding sequences of PDC1, oBP550 (SEQ ID NO: 48) and oBP551 (SEQ ID NO: 49) demonstrated the absence of the PDC1 gene from the isolate. Accurate transformants were obtained from strain CEN. Selected as PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm-URA3.

  CEN. PK 113-7D Δura3 :: loxP Δhis3 Δpdc6: pdc1 :: ilvDSm-URA3 is grown overnight in YPD and plated in synthetic complete medium containing 5-fluoro-orotic acid (0.1%) at 30 ° C. Thus, isolates that lost the URA3 marker were selected. Deletion of PDC1, integration of ilvDSm, and marker removal were performed according to Gentra® Puregene® Yeast / Bact. The genomic DNA prepared using the kit (Qiagen, Valencia, CA) was used to confirm by PCR and sequencing with primers oBP511 (SEQ ID NO: 46) and oBP512 (SEQ ID NO: 47). The correct isolate was obtained as strain CEN. PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm was selected and expressed as BP907.

PDC5 deletion sadB integration The PDC5 gene was deleted and replaced with a sadB coding region derived from Achromobacter xylosoxidans. A segment of the PCR cassette for PDC5 deletion-sadB integration was first cloned into the plasmid pUC19-URA3MCS.

  pUC19-URA3MCS is based on pUC19 and contains the sequence of the URA3 gene derived from Saccharomyces cerevisiae located at the multiple cloning site (MCS). pUC19 contains the pMB1 replicon and a gene encoding β-lactamase for replication and selection in Escherichia coli. In addition to the coding sequence for URA3, upstream and downstream sequences of this gene were included for expression of the URA3 gene in yeast. The vector can be used in the cloning process and can be used as a yeast integration vector.

  Saccharomyces cerevisiae CEN. DNA containing the URA3 coding region along with 250 bp upstream and 150 bp downstream of the URA3 coding region derived from PK 113-7D genomic DNA was added to the Phusion® High Fidelity PCR Master Mix (New England BioLabs Inc., Ipswich, MA). Was amplified using primers oBP438 (SEQ ID NO: 12) containing BamHI, AscI, PmeI, and FseI restriction sites, and oBP439 (SEQ ID NO: 13) containing XbaI, PacI, and NotI restriction sites. Genomic DNA was obtained from Gentra® Puregene® Yeast / Bact. Prepared using kit (Qiagen, Valencia, CA). The PCR product and pUC19 (SEQ ID NO: 71) were ligated with T4 DNA ligase after digestion with BamHI and XbaI to generate 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 sequences of sadB and PDC5 fragment B were cloned into pUC19-URA3MCS to generate the sadB-BU portion of the PDC5 A-sadB-BUC PCR cassette. PLH468-sadB (sequence) as template with primer oBP530 (SEQ ID NO: 50) containing an AscI restriction site and primer oBP531 (SEQ ID NO: 51) containing a 5 'tail having homology to the 5' end of PDC5 fragment B No. 67) was used to amplify the coding sequence of sadB. PDC5 fragment B was amplified using primer oBP532 (SEQ ID NO: 52) containing a 5 'tail with homology to the 3' end of sadB, and primer oBP533 (SEQ ID NO: 53) containing a PmeI restriction site. The PCR product was purified using a PCR Purification kit (Qiagen, Valencia, CA). The sadB-PDC5 fragment B was generated by overlapping the PCR by mixing the sadB and PDC5 fragment B PCR products and amplifying with the primers oBP530 (SEQ ID NO: 50) and oBP533 (SEQ ID NO: 53). The resulting PCR product was digested with AscI and PmeI and, after digestion with the appropriate enzyme, ligated into the corresponding site of pUC19-URA3MCS using T4 DNA ligase. The resulting plasmid was subjected to sadB-fragment B-fragment U using primers oBP536 (SEQ ID NO: 54) and oBP546 (SEQ ID NO: 55) containing a 5 'tail having homology to the 5' end of PDC5 fragment C. Used as a template for amplification of PDC5 fragment C was amplified using primer oBP547 (SEQ ID NO: 56) containing a 5 ′ tail with homology to the 3 ′ end of PDC5 sadB-fragment B-fragment U, and primer oBP539 (SEQ ID NO: 57). . The PCR product was purified using a PCR Purification kit (Qiagen, Valencia, CA). PDC5 sadB-fragment B-fragment U and PDC5 fragment C are mixed by overlapping PCR and amplified with primers oBP536 (SEQ ID NO: 54) and oBP539 (SEQ ID NO: 57) to obtain PDC5 sadB-fragment B- Fragment U-fragment C was generated. The resulting PCR product was purified on an agarose gel followed by a Gel Extraction kit (Qiagen, Valencia, CA). Primer oBP542 (SEQ ID NO: 58) containing a 5 'tail with homology to 50 nucleotides immediately upstream of the native PDC5 coding sequence, and oBP539 (SEQ ID NO: 57) were used to produce PDC5 sadB-fragment B-fragment U- By amplifying fragment C, a PDC5 A-sadB-BUC cassette (SEQ ID NO: 72) was generated. The PCR product was purified using a PCR Purification kit (Qiagen, Valencia, CA).

  CEN. Competent cells of PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm were prepared, and the Frozen-EZ Yeast Transformation II ™ kit (Zymo Research Corporation, Irvine, CA) was used with C-U using PD as C-U5-C). Transform with PCR cassette. The transformation mixture was plated at 30 ° C. in uracil deficient synthetic complete medium supplemented with 1% ethanol (not glucose). Transformants with pdc5 knockout sadB integration were obtained from Gentra® Puregene® Yeast / Bact. Screening was performed by PCR with primers oBP540 (SEQ ID NO: 59) and oBP541 (SEQ ID NO: 60) using genomic DNA prepared by the kit (Qiagen, Valencia, CA). Negative PCR results using primers specific for the coding sequences of PDC5, oBP552 (SEQ ID NO: 61) and oBP553 (SEQ ID NO: 62) demonstrated the absence of the PDC5 gene from the isolate. Accurate transformants were obtained from strain CEN. Selected as PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm Δpdc5 :: sadB-URA3.

  CEN. PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm Δpdc5 :: sadB-URA3 is grown overnight in YPE (1% ethanol), supplemented with ethanol (not glucose) and at 30 ° C. Isolates that lost the URA3 marker were selected by plating in synthetic complete medium containing 5-fluoro-orotic acid (0.1%). Deletion of PDC5, sadB integration, and marker removal were performed as described in Gentra® Puregene® Yeast / Bact. It was confirmed by PCR with primers oBP540 (SEQ ID NO: 59) and oBP541 (SEQ ID NO: 60) using genomic DNA prepared using the kit (Qiagen, Valencia, CA). The correct isolate was obtained as strain CEN. PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm Δpdc5 :: sadB was selected and expressed as BP913.

Deletion of GPD2 To delete the endogenous GPD2 coding region, PCR was performed using gpd2 :: loxP-URA3-loxP cassette (SEQ ID NO: 73) using loxP-URA3-loxP (SEQ ID NO: 68) as template DNA. Amplified with. loxP-URA3-loxP contains a URA3 marker derived from the flanking loxP recombinase site (ATCC No. 77107). PCR was performed using Phusion® DNA polymerase (New England BioLabs Inc., Ipswich, Mass.) And primers LA512 and LA513 (SEQ ID NOs: 8 and 9). The GPD2 portion of each primer was derived from the 5 ′ region upstream of the GPD2 coding region and the 3 ′ region downstream of the coding region, such that incorporation of the loxP-URA3-loxP marker resulted in substitution of the GPD2 coding region. The PCR product was transformed into BP913 and transformants were selected in uracil-deficient synthetic complete medium supplemented with 1% ethanol (not glucose). Transformants were screened to confirm correct integration by PCR using primers oBP582 and AA270 (SEQ ID NOs 63 and 64).

  The URA3 marker was reused by transformation with pRS423 :: PGAL1-cre (SEQ ID NO: 66) and plating in synthetic complete medium lacking histidine supplemented with 1% ethanol at 30 ° C. Transformants are streaked in synthetic complete medium supplemented with 1% ethanol and containing 5-fluoro-orotic acid (0.1%) and cultured at 30 ° C. to lose the URA3 marker Selected isolates. 5-FOA resistant isolates were grown in YPE (1% ethanol) for removal of the pRS423 :: PGAL1-cre plasmid. Deletion and marker removal were confirmed by PCR with primers oBP582 (SEQ ID NO: 63) and oBP591 (SEQ ID NO: 65). The correct isolate was obtained as strain CEN. PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm Δpdc5 :: sadB Δgpd2 :: loxP was selected and expressed as PNY1503 (BP1064).

  BP1064 was transformed with plasmids pYZ090 (SEQ ID NO: 1) and pLH468 (SEQ ID NO: 2) to generate strain NGCI-070 (BP1083; PNY1504).

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

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

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

As used herein, the meanings of the abbreviations used were as follows: “g” means gram, “kg” means kilogram, “L” means liter, “ML” means milliliters, “μL” means microliters, “mL / L” means milliliters / liter, “mL / minute” means milliliters / minute, and “DI” means depleted. Means ionized, “uM” means micrometer, “nm” means nanometer, “w / v” means weight / volume, “OD” means optical density “OD ₆₀₀ ” means optical density at a wavelength of ₆₀₀ nM, “dcw” means dry cell weight, “rpm” means number of revolutions per minute, “° C.” means degree Celsius. , "° C / min" means Celsius temperature / min, “slpm” means standard liters per minute, “ppm” means parts per million, “pdc” means pyruvate decarboxylase enzyme, followed by the enzyme number.

Example 1
Corn Mash Preparation Approximately 100 kg of liquefied corn mash was prepared in 3 equal batches using a 30 L glass coated resin kettle. The kettle was equipped with mechanical agitation, temperature control, and pH control. The protocol used for all three batches was as follows: (a) ground corn was mixed with tap water (30 wt% corn on a dry basis), (b) slurry was agitated (C) the pH of the slurry is adjusted to 5.8 using either NaOH or H ₂ SO ₄ and (d) α-amylase (0.02 wt% on dry corn basis) ), (E) heating the slurry to 85 ° C, (f) adjusting the pH to 5.8, (g) holding the slurry at 85 ° C for 2 hours while maintaining the pH at 5.8, ( h) The slurry was cooled to 25 ° C.

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

Example 2
Solid removal Solids were removed from the mash produced in Example 1 by centrifugation in a large floor centrifuge containing six 1 L bottles. When 73.4 kg of mash was centrifuged at 8000 rpm for 20 minutes at 25 ° C., 44.4 kg of centrifuge and 26.9 kg of wet cake were obtained. It was found that the centrifuge contained less than 1% by weight suspended solids and the wet cake contained approximately 18% by weight suspended solids. This suggests that the original liquefied mash contained about 7% by weight suspended solids. This is consistent with the corn filling and the starch content of the corn used, assuming that most of the starch has been liquefied. If all of the starch was liquefied, the 44.4 kg centrifuge recovered directly from the centrifuge would contain approximately 23% by weight dissolved oligosaccharide (liquefied starch). About 0.6 kg i-BuOH was added to 35.4 kg centrifuge and stored. The resulting 36.0 kg centrifuge containing 1.6 wt% i-BuOH was used as the stock solution.

Example 3
Effect of undissolved solids on the rate of mass transfer To mimic the average composition of the liquid phase for SSF batches, an almost intermediate stage of SSF (simultaneous saccharification and fermentation) fermentation (ie about 50% conversion of oligosaccharides) The following experiment was conducted to determine the effect of undissolved solids on the rate of mass transfer of i-BuOH from the aqueous phase simulating the composition of the fermentation broth obtained from corn mash. The centrifuge liquid from Example 2 mimics the liquid phase composition at the start of SSF. Therefore, a portion of it was diluted with an equal amount of H ₂ O on a mass basis to produce a centrifuge that mimics SSF at about 50% conversion. Additional i-BuOH was added to bring the final concentration of i-BuOH in the diluted centrifuge to 3.0 wt% (about 30 g / L).

  A diluted centrifuge was prepared as follows: 18 kg of the centrifuge stock from Example 2 containing 1.6 wt% i-BuOH was mixed with 18 kg of tap water and 0.82 kg. Of i-BuOH was added. The resulting 36.8 kg solution of diluted centrifuge consisted of about 11 wt% oligosaccharides and about 30 g / L i-BuOH. This solution mimics the liquid phase of corn mash fermentation (SSF) and the aqueous titer of 30 g / L i-BuOH at about 50% conversion of oligosaccharides.

Example 4
Effect of removal of undissolved solids on mass transfer The solution obtained in Example 3 as an aqueous phase that mimics mass transfer performance in broth obtained from liquefied corn mash after most of the undissolved solids have been removed. Was used to conduct a mass transfer test. The purpose of the mass transfer test is to transfer i-BuOH from a simulated broth obtained from liquefied corn mash to a dispersion of solvent (extractant) droplets rising through the simulated broth. Was to determine the effect of undissolved solids on the total mass transfer coefficient (k _L a). The correlation of k _L a with the main design of operating parameters can be used to expand the scale of mass transfer operation. Examples of parameters that should be kept as constant as possible to generate k _L a correlations from smaller scale data that are useful for scaling up are the phase and design parameters that determine droplet size. Both physical properties (eg, nozzle diameter, phase velocity dispersed through the nozzle).

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

  Fresh oleyl alcohol (80/85% grade from Cognis) was sprinkled through the nozzle to the bottom of the column to produce a dispersion of extractant droplets flowing upward through the aqueous phase. After reaching the top of the aqueous phase, the extractant droplets formed a separate organic phase, which then overflowed from the top of the column and collected in a receiver. Typically, 3-5 gallons of OA per test flowed through the column.

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

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

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

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

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

  Mass transfer tests were performed using two different sized nozzles at a nozzle speed ranging from 5 ft / sec to 21 ft / sec using a centrifuge (diluted solids) diluted as an aqueous phase. . Three tests were performed using a nozzle having an inner diameter (ID) of 0.76 mm, and three tests were performed using a nozzle having an inner diameter of 2.03 mm. All tests were performed at 30 ° C. in the above 6 inch diameter column using OA as the solvent. The equilibrium partition coefficient of i-BuOH between OA and the diluted centrifuge obtained from corn mash liquefied by removing solids was about 5. Table 1 shows the results of the mass transfer test using the diluted centrifuge (with the solid removed).

Example 5
Effect of undissolved solids on mass transfer The aqueous phase simulating a fermentation broth from liquefied corn mash (containing undissolved solids) in the middle of SSF was added to the wet cake (liquefied) from Example 2. A portion of (obtained initially by removing solids from corn mash) was synthesized by adding it to a diluted centrifuge (used in the mass transfer test described above in Example 4). Some water was also added to dilute the liquid phase retained in the wet cake. This is because this liquid has the same composition as the concentrated centrifuge liquid. Diluted supernatant 17.8 kg, (containing about 18 wt.% Of undissolved mash solids) wet cake 13.0 kg, _H 2 O of 5.0 kg, and together i-BuOH of 0.83kg When mixed, it contains 36% by weight undissolved solids and a liquid phase consisting of about 13% by weight liquefied starch and about 2.4% by weight i-BuOH (the remainder being H ₂ O). .6 kg of slurry was obtained. This slurry mimics the composition of the fermentation broth in the middle of the corn SSF to i-BuOH at about 30% corn loading. This is because the levels of undissolved solids and oligosaccharides found in these types of broth are about 6-8% and 10-12% by weight, respectively.

  Mass transfer tests were performed using two different sized nozzles at nozzle speeds ranging from 5 feet / second to 22 feet / second using a diluted centrifuge and an undissolved mash solid slurry as the aqueous phase. It was. Three tests were performed using a nozzle having an inner diameter of 0.76 mm, and three tests were performed using a nozzle having an inner diameter of 2.03 mm. All tests were performed at 30 ° C. in the above 6 inch diameter column using OA as the solvent. Table 2 shows the results of a mass transfer test using a diluted centrifuge and a slurry of undissolved mash solids.

FIG. 7 shows the i-BuOH transfer from an aqueous solution of liquefied corn starch (ie, oligosaccharides) to a dispersion of oleyl alcohol droplets flowing upward through a bubble column, relative to the total volume mass transfer coefficient, k _La . The influence of the presence of undissolved corn mash solids is shown. OA was supplied to the column through a nozzle with an inner diameter of 2.03 mm. The ratio of k _{L a} of system solid for k _{L a} of system solids not removed has been removed, it has been discovered that 2 to 5 depending on the nozzle velocity of the nozzle of 2.03 mm.

Figure 8 is a liquefied corn starch (i.e., oligosaccharides) from an aqueous solution of a total volume mass transfer coefficient of the movement of the i-BuOH to dispersion of oleyl alcohol droplets flowing upwardly through the bubble column, for the k L _a The influence of the presence of undissolved corn mash solids is shown. OA was fed to the column through a 0.76 mm ID nozzle. The ratio of k _{L a} of system solid for k _{L a} of system solids not removed has been removed, it has been discovered that 2 to 4 in accordance with the nozzle velocity of the nozzle of 0.76 mm.

Example 6
Effect of removal of undissolved solids on phase separation between aqueous and solvent phases This example was compared to an aqueous solution of oligosaccharides obtained from liquefied corn mash from which undissolved solids were not removed and the same solvent Figure 2 shows improved phase separation between an aqueous solution of oligosaccharides obtained from liquefied corn mash from which undissolved solids have been removed and the solvent phase. Both systems contained i-BuOH. Sufficient separation of the solvent phase from the aqueous phase is important because liquid-liquid extraction is a viable separation method for performing in situ product removal (ISPR).

About 900 g of liquefied corn mash was prepared in a 1 L glass coated resin kettle. The kettle was equipped with mechanical agitation, temperature control, and pH control. The following protocol was used: ground corn was mixed with tap water (26 wt% corn on a dry basis) and the slurry was heated to 55 ° C. with stirring and either NaOH or H ₂ SO _{4 was} added. Use to adjust the pH to 5.8, add α-amylase (0.02 wt% based on dry corn), continue to heat to 85 ° C, adjust the pH to 5.8, and adjust the pH to 5.8 While maintaining, the temperature was maintained at 85 ° C. for 2 hours and cooled to 25 ° C. The corn used was a whole grain yellow corn (3335) from Pioneer. Corn was ground on a hammer mill using a 1 mm sieve. The water content of ground corn was 12% by weight, and the starch content of ground corn was 71.4% by weight on a dry corn basis. The α-amylase enzyme was Liquizyme® SC DS made by Novozymes (Franklinton, NC). The total amount of ingredients used was: 265.9 g ground corn (12% moisture), 634.3 g tap water, and 0.056 g Liquizyme® SC DS. The total amount of recovered liquefied corn mash was 883.5 g.

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

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

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

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

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

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

Example 7
Effect of removal of undissolved solids on phase separation between aqueous phase and solvent phase Similar to Example 6, this example describes the oligosaccharides obtained from liquefied corn mash from which undissolved solids have not been removed. Shows improved phase separation between the aqueous solution of oligosaccharides obtained from liquefied corn mash from which undissolved solids have been removed and the solvent phase compared to an aqueous solution and the same solvent. Both systems contained i-BuOH. Sufficient separation of the solvent phase from the aqueous phase is important because liquid-liquid extraction is a viable separation method for performing in situ product removal (ISPR).

  The same mixture prepared for Example 6 was used in this example. The only difference was that the sample was allowed to stand for several days after completion of the sample preparation described in Example 6 before repeating the phase separation shaking test described in this example. The sample labeled “Contains Solid” consisted of liquefied corn mash, i-BuOH, and oleyl alcohol. The sample labeled “Removed solids” consisted of centrifuge, i-BuOH, and oleyl alcohol produced by removing most of the undissolved solids from the liquefied corn mash. The liquefied mash contained about 7% by weight suspended solids and the centrifuge produced from the mash contained about 0.5% by weight suspended solids. If all of the starch in the ground corn is liquefied, the liquid phase in the liquefied mash and the centrifuge produced from the mash will contain about 20% by weight dissolved oligosaccharide ( Liquefied starch). Both samples contained oleyl alcohol in an amount that was about 1/4 of the phase mass ratio, ie solvent / water phase. In addition, i-BuOH was added to all tests, yielding approximately 25 g / L in the aqueous phase when the phase was in equilibrium.

  The purpose of the phase separation test was to liquefy the solvent phase (containing OA) after aging the multiphase mixture for several days at room temperature to mimic the potential changes in system properties throughout the extraction fermentation. It was to measure the effect of undissolved solids on the degree of phase separation between the aqueous phase obtained from corn mash (with and without solids). Two shaking tests were performed. Both bottles were shaken vigorously for 2 minutes to bring the aqueous and organic phases into close contact. The bottle was left for 1 hour. Pictures were taken at various times (0 min, 2 min, 5 min, 10 min, 20 min, and 60 min) to observe the effect of undissolved solids on phase separation in these systems aged for several days. Time zero (0) corresponds to the time immediately after placing the bottle on the bench.

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

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

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

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

Example 8
Effect of removal of undissolved solids on ISPR extraction solvent loss-Disc stack centrifuge This example uses a semi-continuous disc-stack centrifuge to remove undissolved solids from corn mash prior to fermentation This shows the potential to reduce solvent loss via DDGS produced by the extractive fermentation process.

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

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

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

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

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

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

Example 9
Effect of Removal of Undissolved Solids on ISPR Extraction Solvent Loss-Bottle Rotation Test This example is generated by an extractive fermentation process by removing undissolved solids from corn mash prior to fermentation using a centrifuge. Shows the potential to reduce solvent loss through DDGS.

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

  A liquefied corn mash was made according to the protocol described in Example 6. About 10 mL of mash was placed in a centrifuge tube. Samples were centrifuged at approximately 3000 g's RCF (4400 rpm rotor speed) for a total of 1 minute. Samples were allowed to elapse for about 30-40 seconds at 3000 g's and at a speed of less than 3000 g's for a total of 20-30 seconds with centrifuge acceleration and deceleration. The sample temperature was about 60 ° C.

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

  Table 5 summarizes the mass balance of suspended (non-dissolved) solids in a bottle rotation test under typical conditions of operation of a decanter centrifuge that converts total distillate to dilute distillate in a commercial EtOH process. It is a thing. All values shown in Table 5 are approximate values.

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

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

Example 10
Removal of corn oil by removing undissolved solids This example demonstrates the potential to remove and recover corn oil from corn mash by removing undissolved solids prior to fermentation. The effectiveness of the extraction solvent can be improved when corn oil is removed by removal of undissolved solids. Furthermore, removal of corn oil by removal of undissolved solids can minimize the reduction in solvent partition coefficient and may result in an improved extractive fermentation process.

About 1000 g of liquefied corn mash was prepared in a 1 L glass coated resin kettle. The kettle was equipped with mechanical agitation, temperature control, and pH control. The following protocol was used: ground corn was mixed with tap water (26 wt% corn on a dry basis) and the slurry was heated to 55 ° C. with stirring and either NaOH or H ₂ SO _{4 was} added. Use to adjust the pH to 5.8, add α-amylase (0.02 wt% based on dry corn), continue to heat to 85 ° C, adjust the pH to 5.8, and adjust the pH to 5.8 While maintaining, the temperature was maintained at 85 ° C. for 2 hours and cooled to 25 ° C. The corn used was a whole grain yellow corn (3335) from Pioneer. Corn was ground on a hammer mill using a 1 mm sieve. The moisture content of ground corn was about 11.7% by weight, and the starch content of ground corn was about 71.4% by weight on a dry corn basis. The α-amylase enzyme was Liquizyme® SC DS made by Novozymes (Franklinton, NC). The total amount of ingredients used was: 294.5 g ground corn (11.7% moisture), 705.5 g tap water, and 0.059 g Liquizyme® SC DS. Water (4.3 g) was added to dilute the enzyme and a total of 2.3 g of 20% NaOH solution was added to control the pH. About 952 g of mash was recovered.

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

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

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

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

Example 11
Extraction Fermentation Using Mash and Centrifugation as Sugar Source This example illustrates extraction fermentation performed using corn mash and corn mash centrifuge as a sugar source. A corn mash centrifuge was produced by removing undissolved solids from the corn mash prior to fermentation. Four extractive fermentations were performed in parallel, two using liquefied corn mash as a sugar source (with the solids removed) and two from the liquefied corn mash with the majority of the undissolved solids A liquefied mash centrifuge (oligosaccharide aqueous solution) obtained by removal was used. Oleyl alcohol (OA) was added to two of the fermentations (one containing solids and one with the solids removed) and the product (i-BuOH) was extracted from the broth as the product was formed . Add a mixture of corn oil fatty acids (COFA) to the other two of the fermentations (one containing solids and one removing the solids) and extracting the product from the broth as the product is formed did. COFA was made by hydrolyzing corn oil. The purpose of these fermentations is to test the effect of solid removal on the phase separation between solvent and broth (see Example 11) and to recover non-recovered fermentation broth from which solids have been removed or not removed. It was to measure the amount of residual solvent trapped by the dissolved solid (see Example 12).

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

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

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

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

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

General Methods for Fermentation Seed Flask Growth
Saccharomyces cerevisiae strains modified to produce isobutanol from carbohydrate sources, dpc1 deficient, pdc5 deficient, and pdc6 deficient, were seeded in seed flasks from frozen medium. Growing to .55-1.1 g / L dcw (OD ₆₀₀ 1.3-2.6-Thermo Helios α Thermo Fisher Scientific Inc., Waltham, Massachusetts). The medium was grown at 26 ° C. in an incubator rotating at 300 rpm. The frozen medium was pre-stored at -80 ° C. The composition of the first seed flask medium was as follows:
3.0 g / L dextrose 3.0 g / L absolute ethanol 3.7 g / L ForMedium ™ Synthetic Complete Amino Acid (Kaiser) Drop-Out: no HIS, no URA (reference number DSCK162CK)
6.7 g / L Difco Yeast Nitrogen Base (No. 291920) without amino acids.

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

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

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

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

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

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

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

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

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

Analysis of Analyzed Gas Treated air was analyzed with a Thermo Prima (Thermo Fisher Scientific Inc., Waltham, Massachusetts) mass spectrometer. This was the same treated air that was sterilized and added to each fermentor. The off-gas of each fermentation apparatus was analyzed with the same mass spectrometer. The Thermo Primer dB has a function of executing a calibration check at 6:00 am every Monday. Calibration checks were scheduled by Gas Works v1.0 (Thermo Fisher Scientific Inc., Waltham, Massachusetts) software associated with the mass analyzer. The gas to be calibrated was as follows:

  Carbon dioxide was checked at 5% and 15% during the calibration cycle using other known bottled gases. Oxygen was checked at 15% using other known bottled gases. Based on the off-gas analysis of each fermentor, the amount of stripped isobutanol, consumed oxygen, and carbon dioxide inhaled into the off-gas is analyzed by mass fraction analysis of the mass spectrometer and the gas flow into the fermenter. Measurement was performed using a (mass flow controller). The gas rate per hour was calculated and then integrated during the fermentation.

Biomass Measurement A 0.08% Trypan Blue solution was prepared from a 1: 5 dilution of 0.4% Trypan Blue in NaCl (VWR BDH8721-0) with 1 × PBS. A 1.0 mL sample was withdrawn from the fermentor, placed in a 1.5 mL Eppendorf centrifuge tube, and centrifuged at 14,000 rpm for 5 minutes in Eppendorf, 5415. After centrifugation, the upper solvent layer was removed using an m200 Variable Channel BioHit pipette with a 20-200 μL BioHit pipette tip. Care was taken not to remove the layer between the solvent and the aqueous layer. After removing the solvent layer, the sample was resuspended using Vortex-Genie® set at 2700 rpm.

  A series of dilutions was required to bring the cells to the ideal concentration for hemacytometry. If the OD was 10, a 1:20 dilution could be made to obtain an OD of 0.5 that would allow an ideal amount (20-30) of cells to be factored per square. Multiple dilutions with cut 100-1000 μL BioHit pipette tips were required to reduce dilution inaccuracies due to corn solids. In order to prevent the tip from becoming clogged and widen the opening, about 1 cm was cut from the tip. For a final dilution of 1:20, an initial 1: 1 dilution of the fermentation sample and 0.9% NaCl solution was performed. Next, a 1: 1 dilution of the previous solution and 0.9% NaCl solution, then finally a 1: 5 dilution with the previous solution and Trypan Blue solution. Samples were vortexed between each dilution and the cut tip was rinsed into 0.9% NaCl and Trypan Blue solution.

Coverslip with Hausser Scientific Bright-Line
1492, carefully placed on top of hemacytometer. Using an m20 Variable Channel BioHit pipette with a 2-20 μL BioHit pipette tip, 10 uL was withdrawn from the final Trypan Blue dilution and injected into the hemacytometer. The hemacytometer was placed on a Zeis Axioskop 40 microscope with a 40 × magnification. The center quadrant was divided into 25 squares and the four corners and the center square in both chambers were counted and recorded. After counting both chambers, take the average value, multiply by the dilution factor (20), then multiply by the square number 25 in the middle quarter in the hemacytometer, then count the 4 minutes The volume was divided by 0.0001 mL, which is the volume of 1. The sum of this calculation is the number of cells per mL.

LC analysis of fermentation products in the aqueous phase Samples were refrigerated until ready for processing. Samples were removed from refrigeration for 1 hour and allowed to reach room temperature. Approximately 300 uL of sample is transferred to a 0.2 um centrifuge filter (a modified nylon centrifuge filter from Nanosep MF) using an m1000 Variable Channel BioHit pipette with a 100-1000 μL BioHit pipette tip, and then Centrifugation was performed at 14,000 rpm for 5 minutes using an Eppendorf 5415C. Approximately 200 uL of filtered sample was transferred to a 1.8 autosampler vessel equipped with a 250 uL glass vessel insert with polymer feet. The vessel was capped using a screw cap with a PTFE septum and the sample was vortexed using a Vortex-Genie® set at 2700 rpm.

  The samples were then run on an Agilent 1200 series LC equipped with a binary isocratic pump, vacuum degasser, heated column compartment, sampler cooling system, UV DAD detector and RI detector. The column used was an Aminex HPX-87H, 300 × 7.8 with a Bio-Rad Cation H refill, 30 × 4.6 guard column. The column temperature was 40 ° C. for 40 minutes at a flow rate of 0.6 mL / min using a mobile phase of 0.01 N sulfuric acid. The results are shown in Table 6.

GC analysis of the fermentation product in the solvent phase Samples were refrigerated until ready for processing. Samples were removed from refrigeration for 1 hour and allowed to reach room temperature. Approximately 150 uL of sample was transferred to a 1.8 autosampler vessel equipped with a 250 uL glass vessel insert with a polymer foot using an m1000 Variable Channel BioHit pipette with a 100-1000 μL BioHit pipette tip. The container was capped using a screw cap with a PTFE septum.

  The samples were then run on an Agilent 7890A GC equipped with a 7683B injector and a G2614A autosampler. The column was an HP-InnoWax column (30 m × 0.32 mm inner diameter, 0.25 μm film). The carrier gas is helium at a flow rate of 1.5 mL / min measured at 45 ° C. using a constant top pressure; the injector split is 1:50 at 225 ° C .; the oven temperature is 1.5 For a run time of 45 ° C. per minute for 0 minute at 10 ° C./minute, 45 ° C. to 160 ° C. and then 29 minutes, it was 230 ° C. for 14 minutes at 35 ° C./minute. Flame ionization detection was used at 260 ° C. with 40 mL / min helium makeup gas. The results are shown in Table 7.

  Samples analyzed for fatty acid butyl esters were run on an Agilent 6890 GC equipped with a 7683B injector and a G2614A autosampler. The column was an HP-DB-FFAP column (15 meter x 0.53 mm ID), 1-micron film thickness column (30 m x 0.32 mm ID, 0.25 μm film). The carrier gas is helium at a flow rate of 3.7 mL / min measured at 45 ° C. using a constant top pressure; the injector split is 1:50 at 225 ° C .; the oven temperature is 2.0 100 ° C. per minute, 100 ° C. to 250 ° C. at 10 ° C./min, then for 26 minutes run time was 9 minutes 250 ° C. Flame ionization detection was performed using helium makeup gas at 40 mL / min. Used at <RTIgt; C. </ RTI> The following GC standard (Nu-Chek Prep; Elysian, MN) was used to produce the fatty acid isobutyl ester product: isobutyrate palmitate. , Iso stearic acid - was confirmed attributes of butyl - butyl, iso oleate - butyl, iso linoleic acid - butyl, iso-linolenic acid - butyl, arachidonic acid iso.

Example 11A
The experimental identifier 2010Y033 included: seed flask growth method, initial fermentation device preparation method using corn mash excluding solids, lipase treatment after liquefaction, nutrient addition before inoculation method, fermentation device Inoculation method, operating condition method of fermentation apparatus, and analysis method. Corn oil fatty acids were added to a single batch for 0.1-1.0 hours after inoculation. Butanologen is CEN. PK113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm Δpdc5 :: sadB Δgpd2 :: loxP / pYZ090 + pLH468 (NGCI-070).

Example 11B
The experimental identifier 2010Y034 included: seed flask growth method, initial fermenter preparation method using corn mash containing solids, lipase treatment after liquefaction, nutrient addition before inoculation method, fermenter All of the inoculation method, the fermentation apparatus operating condition method, and the analysis method. Corn oil fatty acids were added to a single batch for 0.1-1.0 hours after inoculation. Butanologen is CEN. PK113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm Δpdc5 :: sadB Δgpd2 :: loxP / pYZ090 + pLH468 (NGCI-070).

Example 11C
Experiment identifier 2010Y035 included: seed flask growth method, initial fermentation device preparation method using corn mash excluding solids, nutrient addition prior to inoculation method, fermenter inoculation method, fermentation device All of the operating condition methods and analysis methods. Oleyl alcohol was added to a single batch for 0.1-1.0 hours after inoculation. Butanologen is CEN. PK113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm Δpdc5 :: sadB Δgpd2 :: loxP / pYZ090 + pLH468 (NGCI-070).

Example 11D
The experimental identifier 2010Y036 included the following: seed flask growth method, initial fermentation device preparation method using solid corn mash, nutrient addition prior to inoculation method, fermenter inoculation method, fermentation device All operating condition methods and analysis methods. Oleyl alcohol was added to a single batch for 0.1-1.0 hours after inoculation. Butanologen is CEN. PK113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm Δpdc5 :: sadB Δgpd2 :: loxP / pYZ090 + pLH468 (NGCI-070).

  The results of Examples 11A to 11D are shown in Table 8.

Example 12
Effect of removing undissolved solids from fermenter feedstock on fermenter volumetric efficiency This example shows the effect of removing undissolved solids from mash prior to fermentation on fermenter volumetric efficiency. Undissolved solids in corn mash occupy at least 5% of the mash volume, depending on corn loading and starch content. By removing solids prior to fermentation, at least 5% more sugar can be charged to the fermenter, thus increasing batch productivity.

  The liquefied corn mash produced in Example 10 assumes that all starch was hydrolyzed to soluble oligosaccharides during liquefaction, the corn loading used (40% on a dry corn basis), It was estimated that it contained about 28% by weight (280 g / L) of liquefied starch, based on the starch content (71.4% on a dry corn basis). When mash is prepared with higher concentrations of oligosaccharides required for fermentation as described in Example 11 and nutrients, inoculum, glucoamylase, and base are added to the initial fermentation broth Dilution was allowed. After adding these ingredients, the mash was diluted by about 10%. Therefore, the expected concentration of liquefied starch in the mash (including solids) at the start of fermentation 2010Y034 and 2010Y036 was about 250 g / L. The actual glucose equivalents input to the 2010Y034 and 2010Y036 fermentations were 239 g / kg and 241 g / kg, respectively (see Table 8). In comparison, the glucose equivalents input to the 2010Y033 and 2010Y035 fermentations were 257 g / kg and 263 g / kg, respectively. Note that the raw material for these fermentations was centrifuge (mash with most of the solids removed). About 1.2 L of sugar source (mash or centrifuge) was added to each fermentation. Therefore, based on this data, about 8.3% more sugar was loaded into the fermentor using the centrifuge (2010Y033 and 2010Y035) compared to the mash (2010Y034 and 2010Y036). These results show that removal of undissolved solids from corn mash prior to fermentation can significantly increase the sugar input per unit volume. This suggests that if a solid is present, it occupies valuable fermenter volume. If the solid is removed from the feed, more sugar can be added to the fermenter (“suitable”) because there is no undissolved solid. This example shows that fermenter volumetric efficiency can be significantly improved by removing undissolved solids from the mash prior to fermentation.

Example 13
Effect of Removal of Undissolved Solids on Phase Separation between Extraction Solvent and Broth—Extracted Fermentation This example illustrates the use of undissolved solids from corn mash prior to fermentation during and after the extractive fermentation process. Shows improved separation between phase and broth phase. Two extractive fermentations are performed in parallel, one using liquefied corn mash as a sugar source (with the solids removed) and one from the liquefied corn mash with most of the undissolved solids The centrifuge liquid (oligosaccharide aqueous solution) produced by the removal was used. Oleyl alcohol (OA) was added to both fermentations and the product (i-BuOH) was extracted from the broth as the product was formed. The fermentation broth referred to in this example when the solid was not removed from the feed (using corn mash) was 2010Y036 as described in Example 10. The fermentation broth mentioned in this example when the solid was removed from the feed (using a centrifuge generated from corn mash) was 2010Y035 as described in Example 10. Oleyl alcohol was the extraction solvent used for both fermentations. The rate and extent of phase separation was measured and compared throughout the fermentation, not just the final fermentation broth. Sufficient phase separation during the extractive fermentation process can lead to minimal loss of microorganisms and minimal solvent loss and lower capital and operating costs of downstream processing.

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

  FIG. 9 is a plot of the position of the liquid-liquid interface in the fermentation sample tube as a function of (gravity) settling time. Data are for samples drawn from extractive fermentation when the centrifuge was supplied as a sugar source (solids removed from corn mash) and OA is the ISPR extraction solvent (of Example 10). Experiment 2010Y035). The phase separation data in this plot is for samples drawn from fermentation 2010Y035 at run times of 5.3 hours, 29.3 hours, 53.3 hours, and 70.3 hours. The interface position is reported as a percentage of the total broth height in the sample tube. For example, the interfacial position of the sample drawn from the 2010Y035 fermentation (centrifugate / OA) at the 5.3 hour run time is 3. from the bottom of the sample tube (no separation) after a 120 minute settling time. Increased to 5 mL. There was about 10 mL of total broth in that particular sample tube. Therefore, the interface position of the sample was reported as 35% in FIG. Similarly, the interfacial position of the sample drawn from the 2010Y035 fermentation (centrifugate / OA) at a run time of 53.3 hours is approximately from the bottom of the sample tube (no separation) after a 125 minute settling time. Increased to 3.9 mL. There was about 10 mL of total broth in that particular sample tube. Therefore, the interface position of the sample was reported as 39% in FIG.

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

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

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

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

Example 14
Effect of Removal of Undissolved Solids on ISPR Extraction Solvent Loss-Extraction Fermentation This example demonstrates the solvent loss for DDGS that exits the final stage of the extractive fermentation process by removing undissolved solids from corn mash prior to fermentation. Indicates the possibility of reduction. Example 10 illustrates two extractive fermentations performed in parallel, one using liquefied corn mash (no removal of 2010Y036-solids) as the sugar source, one of the undissolved solid A liquefied mash centrifuge (2010Y035-oligosaccharide aqueous solution) obtained by removing most from the liquefied corn mash was used. Oleyl alcohol (OA) was added to both fermentations and the product isobutanol (i-BuOH) was extracted from the broth as the product was formed. The amount of residual solvent entrapped in the undissolved solid recovered from the final fermentation broth was measured and compared.

After the fermentations 2010Y035 and 2010Y036 described in Example 10 were complete, the broth was collected and used to perform the phase separation test described in Example 11. Next, undissolved solids (fine particles from corn mash that were not removed prior to fermentation) were recovered from each broth and analyzed for total extractable oil. The oil recovered from each solid lot was analyzed for OA and corn oil. The following protocol was followed for both broths:
-The broth was centrifuged to separate it into an organic phase, an aqueous phase and a solid phase.
The organic and aqueous phases were decanted from the solid, leaving a wet cake at the bottom of the centrifuge bottle.
The wet cake was washed thoroughly with water to remove substantially all of the dissolved solids retained in the cake, such as residual oligosaccharides, glucose, salts, enzymes, etc.
The washed wet cake was dried overnight in a vacuum oven (80 ° C. household vacuum) to remove substantially all of the water in the cake.
A portion of the dry solid was contacted well with hexane in a Soxhlet extractor to remove the oil from the solid.
The oil recovered from the solid was analyzed by GC to determine the relative amount of OA and corn oil present in the oil recovered from the solid.
• The particle size distribution (PSD) for the solids recovered from both fermentation broths was measured.

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

Example 15
Recovery of soluble starch from wet cake produced from the removal of solids from liquefied corn mash by washing the wet cake with water-a two-step process This example involves washing the cake twice with water Shows the recovery of soluble starch from the wet cake, where the cake was produced by centrifuging the liquefied mash. The liquefied corn mash was fed to a continuous decanter centrifuge to produce a centrifuge liquid stream (C-1) and a wet cake (WC-1). The centrifuge liquid is an aqueous solution of soluble starch that does not contain a solid relatively, and the wet cake was concentrated to a solid as compared with the raw material mash. A portion of the wet cake was mixed with hot water to form a slurry (S-1). The slurry was again injected into the decanter centrifuge to produce a wash water centrifuge (C-2) and a washed wet cake (WC-2). C-2 was a dilute aqueous solution of soluble starch that was relatively free of solids. The concentration of soluble starch in C-2 was lower than the concentration of soluble starch in the centrifuge produced from the mash separation. The liquid phase retained in WC-2 was less starchy than the liquid in the wet cake produced from the mash separation. The washed wet cake (WC-2) was mixed with hot water to form a slurry (S-2). The ratio of input water to the amount of soluble starch in the input wet cake was the same in both washing steps. The second wash slurry was re-injected into the decanter centrifuge to produce a second wash water centrifuge (C-3) and wet cake (WC-3) that was washed twice. C-3 was a dilute aqueous solution of soluble starch that was relatively free of solids. The concentration of soluble starch in C-3 is lower than the concentration of soluble starch in the centrifuge produced in the first wash stage (C-2) and thus WC-3 (second washed wet The liquid phase retained in the cake) was thinner than the starch in WC-2 (first washed wet cake). The total soluble starch in the two wash centrifuges (C-2 and C-3) is a starch that can be recovered and reused for liquefaction. The soluble starch in the liquid retained in the final washed wet cake is much less than in the wet cake produced in the original separation of the mash.

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

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

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

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

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

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

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

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

Example 16
The effect of the high temperature stage during liquefaction on the conversion of starch in corn solids to soluble (liquefied) starch This example demonstrates that the operation of liquefaction at a high temperature (or “cooking”) stage at some point during the reaction Figure 5 shows that starch in corn solids can lead to higher conversion to soluble (liquefied) starch. The “cooking” stage shown in this example consists of raising the liquefaction temperature at some point after the start of liquefaction, holding it for a higher period, and then lowering the temperature back to its original value. And completing the liquefaction.

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

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

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

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

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

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

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

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

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

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

Comparison of liquefaction / saccharification results of washed wet cake As described above, the washed wet cake from batches 1 and 2 is reslurried in water to hydrolyze any remaining starch in the solid. In order to convert it to glucose, both very excess α-amylase and glucoamylase were added to the slurry. FIG. 11 shows the concentration of glucose in the aqueous phase of the slurry as a function of time.

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

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

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

Example 17
Effect of high temperature stage during liquefaction on the conversion of starch in corn solids to soluble (liquefied) starch. It was prepared at 85 ° C using Novozymes (α-amylase manufactured by Franklinton, NC). Both batches were run for more than 2 hours at 85 ° C. However, a “cooking” period was added in the middle of batch 4. The temperature distribution in batch 4 was 85 ° C. for about 30 minutes, the temperature was raised from 85 ° C. to 121 ° C., held at 121 ° C. for about 30 minutes, cooled to 85 ° C. and continued to liquefy for an additional 90 minutes. The ground corn used in both batches was the same as in Example 1. A corn loading of 26% by weight (dry corn basis) was used for both batches. The total amount of enzyme used in both runs corresponded to 0.04 wt% (dry corn basis). The pH was controlled at 5.8 during both liquefaction runs. Batch 3 liquefaction was performed in a 1 L glass coated resin kettle and Batch 4 liquefaction was performed in a 200 L stainless steel fermenter. Both reactors were equipped with mechanical stirring, temperature control, and pH control.

  The experimental conditions for this example were similar to those described for Example 14 with the following differences:

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

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

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

  The experimental conditions for this example were similar to those described for Example 14 with the following differences:

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

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

Comparison of the results of liquefaction / saccharification of the washed wet cake As described above, the washed wet cake from batches 3 and 4 is reslurried in water to hydrolyze any remaining starch in the solid. In order to convert it to glucose, both very excess α-amylase and glucoamylase were added to the slurry. FIG. 12 shows the concentration of glucose in the aqueous phase of the slurry as a function of time.

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

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

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

Overview and Comparison of Examples 16 and 17 Liquefaction conditions can affect the conversion of starch in corn solids to soluble (liquefied) starch. Possible liquefaction conditions that may affect the conversion of starch in ground corn to soluble starch are temperature, enzyme (α-amylase) loading, and +/- high temperature (“cooking”) stages being liquefied. Whether it is done at some point. Examples 16 and 17 showed that performing a high temperature (“cooking”) stage at some point during liquefaction resulted in a higher conversion of starch in corn solids to soluble (liquefied) starch. The high temperature stage during liquefaction described in Examples 16 and 17 consists of increasing the liquefaction temperature at some point after liquefaction has started, maintaining it at a higher temperature for a period of time, and then adjusting the temperature to And reducing to the original value again to complete the liquefaction.

  The liquefaction reaction compared in Example 16 was performed with a different enzyme loading than the reaction compared in Example 17. These examples show the effect of two main liquefaction conditions on starch conversion: (1) enzyme loading and (2) +/− whether a high temperature step is applied at some point during liquefaction. .

  The conditions used to prepare the four batches of liquefied corn mash described in Examples 16 and 17 are summarized below and in Table 9.

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

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

  FIG. 13 shows the effect of enzyme loading and whether the +/− hot stage was applied at some point during liquefaction on starch conversion. The level of residual non-hydrolyzed starch in the solid is starch that was not hydrolyzed at the intended liquefaction conditions. FIG. 13 shows that the level of non-hydrolyzed starch in the solid was reduced by almost half by the application of a high temperature (“cooking”) stage at some point during liquefaction. This was shown with two different enzyme loadings. The data in FIG. 13 shows that doubling the enzyme loading, whether or not the high temperature step was applied during liquefaction, almost halved the level of non-hydrolyzed starch remaining in the solid. Show. These examples show that the implementation of liquefaction with higher enzyme (α-amylase) loading and / or the addition of a high temperature (“cooking”) stage at some point during the reaction is present in the liquefied corn mash. It shows that residual unhydrolyzed starch in dissolved solids can be significantly reduced and that starch loss in the process can be reduced when undissolved solids are removed from the mash prior to liquefaction. Any residual starch in the solid after liquefaction has no opportunity to hydrolyze during fermentation in a process where the solid is removed prior to fermentation.

Example 18
Screening Separation of Starch and Insoluble After Enzymatic Digestion at 85 ° C. Mash (301 grams) prepared for each method described in Example 1 is used with drops of NaOH solution if adjustment is required. The pH was maintained at 5.8 and was treated with a manufacturer-specified dose of approximately 0.064 grams of Liquizyme® α-amylase enzyme (Novozyme, Franklinton, NC) and held at 85 ° C. for 5 hours. The product was refrigerated.

  The refrigerated product was warmed to about 50 ° C. and 48 g was poured into a filter assembly containing a 100 mesh screen and connected to a household vacuum source at -15 inches of mercury (in Hg) to -20 inches of mercury. The sieve pan had an exposed sieve surface area of 44 cm 2 and was sealed in a plastic filter housing provided by Nalgene® (Thermo Fisher Scientific, Rochester, NY). When the slurry was filtered, a wet cake was formed on the sieve, and 40.4 g of a yellow turbid filtrate was formed in the receiver bottle. The wet cake was immediately washed with water in place and then interrupted while the vacuum source continued to draw free moisture through the final washed cake. Filtration was stopped when dripping from the bottom of the filter stopped. An additional 28.5 g of wash filtrate was collected over 3 stages, where the final stage filtrate showed the least color and turbidity. The final wet cake mass of 7.6 g was air dried to 2.1 g over 24 hours at room temperature. 2.1 g was found to contain 7.73% water after drying with a heat lamp. The vacuum filtration of this experiment produced a wet cake containing a total of 25% dry solids.

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

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

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

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

Example 19
Screening Separation of Starch and Insoluble After Enzymatic Digestion at 115 ° C. Household deionized water (200 g) is charged into an empty Parr Model 4635 1 liter pressure vessel (Moline, IL) at a temperature of 85 ° C. Until heated. Water was stirred using a magnetic stir bar. Dry ground corn (90 g) prepared as described in Example 1 was added with a spoon. The pH was raised from 5.2 to about 6.0 using a stock solution of aqueous ammonia. About 0.064 grams of Liquizyme® solution was added with a small calibrated pipette. The pressure vessel lid was closed and the vessel was pressurized to 50 psig with household nitrogen. The stirred mixture was heated to 110 ° C. within 6 minutes and held at 106-116 ° C. for a total of 20 minutes. Heating was suppressed, pressure was reduced, and the container was opened. An additional 0.064 g of Liquizyme® was added and the temperature was held at 63-75 ° C. for an additional 142 minutes.

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

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

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

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

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

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

Example 20
Lipid Analysis Lipid analysis was performed by converting various fatty acid-containing compound species to fatty acid methyl esters (“FAME”) by transesterification. Glycerides and phospholipids were transesterified with sodium methoxide in methanol. Glycerides, phospholipids, and free fatty acids were transesterified using acetyl chloride in methanol. The resulting FAME was equipped with a 30-m × 0.25 mm (inner diameter) OMEGAWAX ™ (Supelco, Sigma Aldrich, St. Louis, MO) column after dilution in toluene / hexane (2: 3). Analysis was by gas chromatography using an Agilent 7890 GC. The oven temperature was increased from 160 ° C. to 200 ° C. at 5 ° C./min and then from 200 ° C. to 250 ° C. (held for 10 minutes) at 10 ° C./min. The FAME peaks recorded by GC analysis were identified by their retention times when compared to the retention times of known methyl esters (ME), and the FAME peak area was determined by a known amount of internal standard (C15: 0 Quantification by comparison with the area of triglycerides, taken by the transesterification procedure with the sample). Thus, an approximate amount (mg) of any fatty acid FAME (“mg FAME”) is calculated according to the following formula: (area of FAME peak for a given fatty acid / area of FAME peak at 15: 0) ^* (internal Standard C15: 0 FAME mg). The FAME result can then be corrected to the corresponding fatty acid mg by dividing by an appropriate molecular weight conversion factor of 1.052. All internal standards and reference standards are from Nu-Chek Prep, Inc. Obtained from.

  The fatty acid result obtained from the sample transesterified with sodium methoxide in methanol is converted to the corresponding triglyceride level by multiplying by a molecular weight conversion factor of 1.045. Triglycerides generally account for about 80-90% of the glycerides in the samples examined for this example, with the remainder being diglycerides. Monoglyceride and phospholipid contents are generally negligible. The total fatty acid results obtained for the sample transesterified with acetyl chloride in methanol are corrected for glyceride content by subtracting the fatty acid determined for the same sample using the sodium methoxide procedure. The result is the free fatty acid content of the sample.

  The distribution of glyceride content (monoglyceride, diglyceride, triglyceride, and phospholipid) was determined using thin layer chromatography. A solution of oil dissolved in 6: 1 chloroform / methanol was hung near the bottom of a glass plate pre-coated with silica gel. The spot is then chromatographed using a 70: 30: 1 hexane / diethyl ether / acetic acid solvent system. Next, isolated spots corresponding to monoglycerides, diglycerides, triglycerides, and phospholipids are detected by staining the plate with iodine vapor. The spot is then dropped from the plate and transesterified with acetyl chloride in a methanol procedure and analyzed by gas chromatography. The ratio of the total peak area of each spot to the total peak area of all spots is the distribution of the various glycerides.

Example 21
This example showed the removal of solids from the distillation waste and extraction with a desolvator to recover fatty acids, esters, and triglycerides from the solids. During the fermentation, the solid is separated from the total distillation effluent and fed to the desolventizer where the solid is contacted with 1.1 ton / hour of steam. The total distillation waste liquid wet cake (extractor raw material), the solvent, the extractor miscella, and the flow rate of the discharged solids of the extractor are as shown in Table 10. The unit of the values in the table is short ton / hour.

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

Example 22
This example demonstrates the recovery of by-products from mash. Maize corn oil under the conditions described in Example 10, except that a tricanter centrifuge (Flottweg Z23-4 bowl diameter, 230 mm, length to diameter ratio 4: 1) was used under these conditions. Separate from:
・ Bowl speed: 5000rpm
・ Differential speed: 10rpm
・ Supply speed: 3 gallons / minute ・ Phase separator disk: 138 mm
・ Impeller setting: 144 mm.

  The isolated corn oil has 81% triglycerides, 6% free fatty acids, 4% diglycerides, and a total of 5% phospholipids as measured by the method described in Example 18 and thin layer chromatography. Had monoglycerides.

  The solid separated from the mash under the above conditions has a moisture content of 58% when measured by weight loss on drying and 1.2% when measured by the method described in Example 18. It had triglycerides and 0.27% free fatty acids.

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

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

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

Claims (58)

Providing a biomass feedstock slurry comprising a fermentable carbon source, an undissolved solid, and water;
Separating at least a portion of the undissolved solid from the slurry, thereby producing (i) an aqueous solution comprising a fermentable carbon source and (ii) a wet cake byproduct comprising solids;
Adding the aqueous solution to a fermentation broth containing recombinant microorganisms in a fermentor, thereby producing a fermentation product;
A method for improving the processing productivity of the biomass.   The method of claim 1, wherein the improved biomass processing productivity comprises improved fermentation product and by-product recoveries as compared to fermentation products produced in the presence of undissolved solids.   The improved biomass processing productivity comprises one or more of improved process stream reusability, improved fermenter volumetric efficiency, and improved biomass feedstock loading. Method.   The method of claim 1, further comprising contacting the fermentation broth with an extractant, wherein the extractant has an improved extraction efficiency compared to a fermentation broth comprising undissolved solids.   Improved extraction efficiency results in a stable partition coefficient of the extractant, improved phase separation of the extractant from the fermentation broth, improved liquid-liquid mass transfer coefficient, improved extractant recovery and reusability, and 5. The method of claim 4, comprising one or more of the extractants stored for recovery and reuse.   The method of claim 4, wherein the extractant is an organic extractant. The extracting _agent, C 12 _-C fatty _alcohols, fatty acids C 12 _{-C 22} of _22, esters of fatty acids of C 12 _{-C 22,} fatty aldehydes of C 12 _{-C 22,} fatty amides of C 12 _{-C 22,} 7. The method of claim 6, comprising one or more immiscible organic extractants selected from the group consisting of and mixtures thereof. The extraction agent comprises a fatty acid of _C 12 _{-C 22} derived from corn oil, The method of claim 7.   The undissolved solid is decanter bowl centrifuge, tricanter centrifuge, disc stack centrifuge, filtration centrifuge, decanter centrifuge, filtration, vacuum filtration, belt filter, pressure filtration, filtration using sieve, sieving separation, lattice The method of claim 1, wherein the slurry is separated from the raw slurry by a porous grid, flotation method, hydroclone, filter press, screw press, gravity settler, vortex separator, or combinations thereof.   Further comprising the step of liquefying the raw material to produce a biomass raw material slurry; wherein the raw material is crop residues such as corn kernels, corn cobs, corn hulls, corn stover, grass, corn, wheat, rye , Wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugarcane pomace, sorghum, sugarcane, soybeans, ingredients obtained from grinding grains, cellulosic materials, lignocellulosic materials, trees 2. The method of claim 1, selected from, branches, roots, leaves, wood chips, sawdust, shrubs and shrubs, vegetables, fruits, flowers, animal manure, and mixtures thereof.   The method according to claim 10, wherein the raw material is corn.   The method of claim 10, wherein the raw material is fractionated or not fractionated.   The method according to claim 10, wherein the raw material is wet pulverized or dry pulverized.   The method according to claim 10, further comprising increasing a reaction temperature during liquefaction.   The method of claim 10, wherein the raw slurry includes oil derived from the raw material, and the oil is separated from the slurry.   The method of claim 15, wherein the wet cake comprises a feedstock.   The method of claim 1, wherein the wet cake is washed with water to recover oligosaccharides present in the wet cake.   The method of claim 17, wherein the recovered oligosaccharide is added to the fermentor.   The method of claim 1, wherein the wet cake is further processed to obtain an improved byproduct.   The method of claim 19, wherein the by-product is further processed to form an animal feed product.   The method of claim 1, wherein the wet cake is washed with a solvent to recover oil present in the wet cake.   24. The method of claim 21, wherein the solvent is selected from hexane, butanol, isobutanol, isohexane, ethanol, and petroleum distillate.   The method of claim 1, wherein the fermentation product is a product alcohol selected from the group consisting of methanol, ethanol, propanol, butanol, pentanol, and isomers thereof.   The method of claim 1, wherein the recombinant microorganism comprises a modified butanol biosynthetic pathway. The method of claim 1, further comprising at least partially evaporating the fermentation broth and product and optionally CO ₂ , wherein a steam stream is generated and the product is recovered from the steam stream. . Contacting the vapor stream with an absorbing liquid phase, wherein at least a portion of the vapor stream is absorbed into the absorbing liquid phase;
26. The method of claim 25, wherein the temperature at the beginning of absorption of the vapor stream into the absorbing liquid phase is higher than the temperature at the beginning of condensation of the vapor stream when there is no absorbing liquid phase.   26. The method of claim 25, wherein the evaporation and contacting step is performed under vacuum conditions.   26. The method of claim 25, wherein the step of separating a majority of the undissolved solid from the slurry results in a higher vapor pressure of the fermentation broth compared to a fermentation broth containing undissolved solid.   29. The method of claim 28, wherein the higher vapor pressure results in more efficient evaporation product recovery.   Said more efficient evaporation product recovery is less resource input, less evaporation, absorption, compression, and refrigeration equipment, improved mass transfer rate, less energy for evaporation, and lower absorbent flow rate 30. The method of claim 29, comprising one or more of: A method for producing butanol comprising:
Providing a raw material;
Liquefying the raw material to produce a raw slurry containing oligosaccharides, oil, and undissolved solids;
Separating undissolved solids from the raw slurry to produce (i) an aqueous solution comprising oligosaccharides, (ii) a wet cake comprising undissolved solids, and (iii) an oil phase;
Contacting the aqueous solution with a fermentation broth in a fermentation apparatus;
Fermenting the oligosaccharides in the fermenter to produce butanol;
Performing in situ removal of the butanol from the fermentation broth as the butanol is produced,
A method in which the efficiency of butanol production is increased by removing the non-dissolved solid from the raw slurry.   32. The method of claim 31, wherein the raw material is corn and the oil is corn oil.   34. The method of claim 32, wherein the undissolved solid comprises germ, fiber, and gluten.   34. The method of claim 33, further comprising dry pulverizing the raw material.   35. The method of claim 32, wherein the corn is not fractionated.   The undissolved solid is decanter bowl centrifuge, tricanter centrifuge, disc stack centrifuge, filtration centrifuge, decanter centrifuge, filtration, vacuum filtration, belt filter, pressure filtration, filtration using sieve, sieving separation, lattice 32. The method of claim 31, wherein the separation is by a porous grid, flotation method, hydroclone, filter press, screw press, gravity settler, vortex separator, or combinations thereof.   32. The method of claim 31, wherein the step of separating undissolved solid from the raw slurry comprises centrifuging the raw slurry.   The step of centrifuging the raw slurry separates the raw material into a first liquid phase containing the aqueous solution, a solid phase containing the wet cake, and a second liquid phase containing the oil. 38. The method according to 37.   40. The method of claim 38, wherein the wet cake is washed with water to recover oligosaccharides present in the wet cake.   32. The method of claim 31, wherein the in situ removal comprises liquid-liquid extraction.   41. The method of claim 40, wherein the extractant for liquid-liquid extraction is an organic extractant.   The method according to claim 31, wherein saccharification of the oligosaccharide in the aqueous solution is performed simultaneously with fermentation of the oligosaccharide in the fermentation apparatus.   32. The method of claim 31, further comprising increasing the reaction temperature during liquefaction.   32. The method of claim 31, further comprising saccharifying the oligosaccharide prior to fermenting the oligosaccharide in the fermentation apparatus.   45. The method of claim 44, wherein the step of removing undissolved solids from the raw slurry comprises centrifuging the raw slurry.   The method according to claim 45, wherein the step of centrifuging the raw slurry is performed before saccharifying the sugar.   32. The method of claim 31, wherein the fermentation broth comprises a recombinant microorganism comprising a butanol biosynthetic pathway.   32. The method of claim 31, wherein the butanol is isobutanol.   The step of removing undissolved solids from the raw slurry increases the efficiency of butanol production by increasing the liquid-liquid mass transfer coefficient of the butanol from the fermentation broth to the extractant; Increase the efficiency of butanol production by increasing the extraction efficiency; increase the efficiency of butanol production by increasing the speed of phase separation between the fermentation broth and the extractant; and recover and reuse the extractant 32. The method of claim 31, wherein increasing the butanol production efficiency by increasing; or increasing the butanol production efficiency by decreasing the flow rate of the extractant. A liquefaction tank configured to liquefy a raw material to produce a raw slurry:
An inlet for receiving the raw material; and a liquefaction tank including an outlet for discharging the raw slurry containing sugar and undissolved solids;
A centrifuge configured to remove the undissolved solid from the raw slurry to produce a wet cake comprising (i) an aqueous solution comprising the sugar and (ii) a portion of the undissolved solid:
An inlet for receiving the raw slurry;
A centrifuge including a first outlet for discharging the aqueous solution; and a second outlet for discharging the wet cake;
A fermentation apparatus configured to ferment the aqueous solution to produce butanol:
A first inlet for receiving the aqueous solution;
A second inlet for receiving the extractant;
A system for producing butanol comprising: a first outlet for discharging said extractant rich in butanol; and a fermentation apparatus including a second outlet for discharging fermentation broth.   51. The system of claim 50, wherein the centrifuge further comprises a third outlet for discharging generated oil while removing the undissolved solid from the raw slurry. A saccharification tank configured to saccharify the sugar in the raw slurry:
51. The system of claim 50, further comprising a saccharification tank comprising an inlet for receiving the raw slurry; and an outlet for discharging the raw slurry. A saccharification tank configured to saccharify the sugar in the aqueous solution:
53. The system of claim 52, further comprising a saccharification tank comprising an inlet for receiving the aqueous solution; and an outlet for discharging the aqueous solution. A dry grinder configured to grind the raw material comprising:
51. The system of claim 50, further comprising a dry grinder comprising an inlet for receiving the raw material; and an outlet for discharging ground raw material. 20-35% by weight crude protein,
1-20% by weight crude fat,
0-5% by weight of triglycerides,
A composition comprising 4 to 10% by mass of a fatty acid and 2 to 6% by mass of a fatty acid isobutyl ester. 25-31% by weight crude protein,
6-10% by weight crude fat,
4-8% by weight of triglycerides,
A composition comprising 0-2% by weight of fatty acid and 1-3% by weight of fatty acid isobutyl ester. 20-35% by weight crude protein,
1-20% by weight crude fat,
0-5% by weight of triglycerides,
A composition comprising 4 to 10% by mass of a fatty acid and 2 to 6% by mass of a fatty acid isobutyl ester. 26-34% by weight crude protein,
15-25% by weight crude fat,
12-20% by weight of triglycerides,
1-2% by weight of fatty acids,
2-4% by weight fatty acid isobutyl ester,
1-2% by weight of lysine,
11-23 wt% NDF and 5-11 wt% ADF
A composition comprising

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