CN104919050A - Processes and systems for the production of fermentation products - Google Patents

Abstract

The present invention relates to the production of fermentation products such as alcohols including ethanol and butanol, and processes employing in situ product removal methods wherein an extractant is added to the fermentation broth.

Description

For the production of technique and the system of tunning

This application claims the U.S. Provisional Application 61/699,976 submitted on September 12nd, 2012; The U.S. Provisional Application 61/712,385 that on October 11st, 2012 submits to; The U.S. Patent application 13/828,353 that on March 14th, 2013 submits to; With the rights and interests of the U.S. Patent application 13/836,115 that on March 15th, 2013 submits to; Their each full text is incorporated herein by reference.

Sequence table associated with this application is Electronically submitted to via EFS-Web, and is incorporated to this specification sheets with way of reference in full accordingly.

Technical field

The present invention relates to the production of the tunning of such as alcohol (comprising ethanol and butanols) and adopt the technique of situ product removing method.

Background technology

Multi-chemical and the consumer's goods can utilize fermentation to produce as manufacturing process.Such as, the alcohol of such as ethanol and butanols has multiple industry and science application, such as fuel, reagent and solvent.Butanols is important industrial chemical, and it serves many purposes, and comprises as fuel dope, in plastics industry, is used as chemical feedstocks, and is used as the extraction agent of food grade in food and flavour agent industry.The use of petroleum chemicals can be made to minimize from the material produce butanols of such as plant derived material or butanol isomer, and will the improvement of this area be represented.In addition, utilize the production of the material of plant origin or the chemical of other biomass sources and fuel by provide to petroleum chemicals technique to eco-friendly and continuable replacement scheme.

The technology of such as genetically engineered and metabolic engineering can be used to modified microorganism, to produce some product from the material of plant origin or other biomass sources.Such as, but in the production of butanols, some microorganisms producing butanols with high yield also have low butanols toxicity threshold.Along with it removes by the production of butanols from fermentation, it is a kind of method of these low butanols toxicity thresholds of process.Therefore, exist and split hair in the lasting demand of the effective ways of high produced in yields butanols (although producing the microorganism of butanols have low butanols toxicity threshold) and system.

Situ product removes (ISPR) (be also referred to as extract fermentation) can be used for removing butanols (or other tunning) from producing its fermenting container, thus enables microorganism high productivity produce butanols.A kind of ISPR method for removing fermentable alcohol described in this area is that liquid-liquid extracts (such as, see, U.S. Patent Application Publication 2009/0305370).Generally, with regard to butylic fermentation, such as, the time fermented liquid comprising microorganism being reached before such as toxic level at butanol concentration contacts with extraction agent.Butanols distributes into extraction agent, reduces the butanol concentration in the fermented liquid comprising microorganism, thus limits the exposure of microorganism in the butanols of inhibition.

In order to technology and feasibility economically, liquid-liquid extracts the contact needed between extraction agent and fermented liquid, with make alcohol efficiently mass transfer in extraction agent; Being separated of extraction agent and fermented liquid (between yeast phase and/or afterwards); Reclaim efficiently and recycling extraction agent; And the minimal reduction of the partition ratio of extraction agent after long-time running.Extraction agent can be contaminated along with the time in each circulation, such as, by being used as the accumulation of the lipid existed in the biomass of the raw material fermented, and the reduction that this pollution can cause the partition ratio of extraction agent to accompany with it.

In addition, the existence of extracting undissolved solid in fermenting process adversely can affect the efficiency of the production of alcohol.Such as, the existence of undissolved solid can reduce coefficient of mass transfer, prevention is separated, cause, from the oil accumulation of the undissolved solid in extraction agent, causing extraction efficiency to reduce in time, slow down being separated of extraction agent drop and fermented liquid, cause lower fermenting container volumetric efficiency, and increase the loss of extraction agent, because it finally together with solid is removed as distiller's dried grain and solvend (Dried Distillers Grains with Solubles, DDGS).

Therefore, exist making the fermentation alcohol of such as butanols reduce the toxic effect of microorganism, and the lasting demand of substituting extraction zymotechnique of deterioration of partition ratio of extraction agent can be reduced.Present invention accomplishes demand as herein described, and provide the method for the fermentative production of the alcohol for such as ethanol and butanols, technique and system.

Summary of the invention

The present invention relates to the method from reclaiming tunning for fermented liquid, described method comprises the fermented liquid of providing package containing microorganism, and wherein said microorganism produces tunning in fermentor tank; Described fermented liquid is contacted with at least one extraction agent; And reclaim described tunning.In certain embodiments, the contact of described fermented liquid and at least one extraction agent occur in fermentor tank, external unit or both in.In certain embodiments, described external unit is extractor.In certain embodiments, described extractor is selected from siphon pipe, decantor, whizzer, gravitational settler, phase splitter, mixer-settler, column extractor, centrifugal extractor, stirs extractor, hydrocyclone, spray tower and their combination.In certain embodiments, extraction agent is selected from C ₇-C ₂₂fatty alcohol, C ₇-C ₂₂lipid acid, C ₇-C ₂₂the ester of lipid acid, C ₇-C ₂₂alkanoic, C ₇-C ₂₂fatty amide and their mixture.In certain embodiments, described extraction agent is selected from oleyl alcohol, behenyl alcohol, hexadecanol, lauryl alcohol, tetradecyl alcohol, stearyl alcohol, oleic acid, lauric acid, linolic acid, linolenic acid, tetradecanoic acid, stearic acid, sad, capric acid, undeeanoic acid, Myristicin acid methylester, Witconol 2301,1 nonyl alcohol, 1-decanol, 2-undecyl alcohol, 1-aldehyde C-9,1-undecyl alcohol, the undecyl aldehyde, lauryl aldehyde, the 2-methyl undecyl aldehyde, oleylamide, sub-oleylamide, palmitic amide, stearylamide, 2-ethyl-1-hexanol, 2-hexyl-1-decanol, 2-octyl group-1-lauryl alcohol and their mixture.In certain embodiments, hydrophilic solutes is added into described fermented liquid.In certain embodiments, described hydrophilic solutes is selected from polyol, polycarboxylic acid, polyol compound, ion salt and their mixture.In certain embodiments, the contact of described fermented liquid and at least one extraction agent occurs in two or more external units.In certain embodiments, the contact of described fermented liquid and at least one extraction agent occurs in two or more fermentor tanks.In certain embodiments, described fermentor tank comprises for improving the internals or device that are separated.In certain embodiments, described internals or device are selected from coalescer, baffle plate, perforation plate, hole, tilted plate separator, cone-shaped body or their combination.In certain embodiments, the extraction of measuring in real time and monitoring tunning is used.In certain embodiments, the extraction of tunning is monitored by measuring to be separated in real time.In certain embodiments, the composition by measuring the speed, extraction agent drop size and/or the fermented liquid that are separated is monitored and is separated.In certain embodiments, monitored by conductivity measurement, dielectric medium measurement, viscoelasticity measurement and/or ultrasonic measurement and be separated.In certain embodiments, providing package occurs in two or more fermentor tanks containing the fermented liquid of microorganism.In certain embodiments, described tunning can be product alcohol.In certain embodiments, product alcohol is selected from ethanol, propyl alcohol, butanols, amylalcohol, hexanol and fusel.In certain embodiments, microorganism comprises butanol biosynthetic pathway.In certain embodiments, described butanol biosynthetic pathway is n-butyl alcohol biosynthetic pathway, 2-butanol biosynthetic pathway, isobutanol biosynthetic pathway or 2-butanone approach.In certain embodiments, described microorganism is recombinant microorganism.In certain embodiments, described method is further comprising the steps of: providing package is containing the raw slurry of fermentable carbon source, undissolved solid, oil and water; Described raw slurry is separated, thus forms three kinds of streams: (i) comprises the aqueous solution of fermentable carbon source, and (ii) comprises the wet cake of solid, and (iii) oil; And the described aqueous solution is added into described fermented liquid.In certain embodiments, by described profit solution to form lipid acid.In certain embodiments, described fermented liquid is contacted with lipid acid.In certain embodiments, described oil is by enzymic hydrolysis.In certain embodiments, described enzyme is one or more lipase or Phospholipid hydrolase.In certain embodiments, described raw slurry is generated by the hydrolysis of raw material.In certain embodiments, raw material is selected from rye, wheat, corn, sugarcane, barley, Mierocrystalline cellulose or ligno-cellulosic materials and their combination.In certain embodiments, described raw slurry is separated by following: decanter type bowl formula is centrifugal, three phase centrifugation, dish stacked centrifugal (disk stack centrifugation), filter centrifugation, decanter type are centrifugal, filtration, membrane filtration, micro-filtration, vacuum filtration, belt filter, press filtration, the filtration of use screen cloth, screening, grid filter, the filter of porous grid, flotation, hydrocyclone, pressure filter, helical pressure device, gravitational settler, vortex separator or their combination.In certain embodiments, separate raw materials is single step process.In certain embodiments, will wet cake and aqueous solution.In certain embodiments, described method also comprises the catalyst exposure making the described aqueous solution and the oil in the described aqueous solution is changed into lipid acid.In certain embodiments, the described aqueous solution and lipid acid are added into fermented liquid.In certain embodiments, by described catalyst deactivation.

The invention still further relates to system, described system comprises: one or more fermentor tank, and described fermentor tank comprises: for receiving the entrance of raw slurry; With the outlet for discharging the fermented liquid comprising tunning; With one or more extractor, described extractor comprises: for receiving the first entrance of fermented liquid; For receiving the second entrance of extraction agent; For discharging the first outlet of poor fermented liquid; With the second outlet for discharging rich extraction agent.In certain embodiments, described system also comprises one or more liquefaction unit; One or more tripping device; Optionally, one or more washing system.In certain embodiments, described tripping device be selected from that decanter type bowl formula is centrifugal, three phase centrifugation, dish stacked are centrifugal, filter centrifugation, decanter type are centrifugal, filtration, vacuum filtration, belt filter, press filtration, membrane filtration, micro-filtration, the filtration of use screen cloth, screening, grid filter, the filter of porous grid, flotation, hydrocyclone, pressure filter, helical pressure device, gravitational settler, vortex separator and their combination.In certain embodiments, described system also comprises measuring apparatus on line.In certain embodiments, on line, measuring apparatus is selected from particle-size analyzer, Fourier transform infrared spectroscopy, near infrared spectrum, Raman spectrum, high pressure liquid chromatography, viscometer, specific gravity hydrometer, tonometer, drop size analyser, pH meter, dissolved oxygen probe and their combination.

Accompanying drawing explanation

Be incorporated to herein and the accompanying drawing becoming a part for specification sheets shows the present invention, and be used for further explaining principle of the present invention together with specification sheets and make those skilled in the art to utilize the present invention.

Fig. 1 schematically shows illustrative processes of the present invention and system, and wherein undissolved solid after liquefaction and be removed by separation before fermentation.

Fig. 2 schematically shows illustrative processes of the present invention and system, and wherein ISPR carries out in the downstream of fermentation.

Fig. 3 schematically shows another illustrative processes of the present invention and system, and wherein oily stream is discharged.

Fig. 4 schematically shows another exemplary alternative technique and system of the present invention, and wherein wet cake stands cycles of washing.

Fig. 5 schematically shows another exemplary alternative technique and system of the present invention, and wherein oily stream is discharged and wet cake stands cycles of washing.

Fig. 6 A and 6B schematically shows another exemplary alternative technique and system of the present invention, wherein the aqueous solution and wet cake is mixed and carries out ferment (Fig. 6 A), and the aqueous solution, oil and wet cake mixed and carry out ferment (Fig. 6 B).

Fig. 7 A-7D schematically shows alternative technique of the present invention and system, and the wherein said aqueous solution stands to transform (such as, hydrolysis, transesterify) and/or deactivation.

Fig. 8 schematically shows the exemplary zymotechnique that the present invention includes Downstream processing.

Fig. 9 schematically shows the exemplary zymotechnique that the present invention includes Downstream processing.

Figure 10 A-10M shows the multiple systems that can be used in technique as herein described.

Figure 11 A and 11B schematically shows multipath extraction agent streaming system.

Figure 12 schematically shows on employing line, in line, line is other and/or measure the of the present invention exemplary zymotechnique of monitoring fermenting process in real time.

Figure 13 A and 13B is for reducing the illustrative processes of the present invention of the formation of heterogeneous interlayer.

Figure 14 schematically shows and comprises fermentation, extracts and the illustrative processes of the present invention of distil process.

Figure 15 shows the ratio (aq/org) of fermented liquid to extraction agent to the effect of extraction column efficiency.

Figure 16 A and 16B shows the effect using the ISPR of outside extraction column to compose isobutanol concentration and glucose.

Figure 17 shows and uses the ISPR of mixer-settler to the effect of isopropylcarbinol removal rate.

Figure 18 shows the FTIR spectrum using the starch concentration scope measured in line.

Figure 19 shows the FTIR spectrum of the starch concentration of wet cake in corn mash treating processes.

Figure 20 shows the FTIR spectrum of Semen Maydis oil in corn mash treating processes.

Figure 21 illustrates the real-time measurement of isopropylcarbinol in COFA.

Embodiment

Unless otherwise defined, otherwise the implication of all scientific and technical terminologies used herein and those skilled in the art usually understand the same.If conflict occurs, be as the criterion with present patent application (comprising its definition).In addition, unless needed for context separately has, singular references will comprise plural number and plural term will comprise odd number.For all objects, all publications, patent and other bibliography mentioned herein are incorporated herein by reference all in full.

In order to limit the present invention further, there is provided herein following term and definition.

As used herein, term " comprises ", " comprising ", " having ", " containing " or their other modification any will be understood to mean the integer or integer group that comprise and specifying but not get rid of other integer any or integer group.Such as, comprise the composition of series of elements, mixture, technique, method, goods or equipment and need not be only limitted to those elements, and other element clearly do not listed can be comprised, or the element that such composition, mixture, technique, method, goods or equipment are intrinsic.In addition, unless indicated on the contrary in addition, otherwise "or" refer to inclusive or, instead of refer to exclusive or.Such as, in following any one A or B:A that all satisfy condition be genuine (or existence) and B be false (or non-existent), A is false (or non-existent) and B is genuine (or existence) and A and B is all genuine (or existence).

Equally, the indefinite article of number before element of the present invention or component " " or " one " that relate to element or component example (i.e. number of times) are intended to be nonrestrictive.Therefore, " one " or " one " should be interpreted as and comprise one or at least one, and the word singulative of element or component also comprises plural, obviously represents odd number unless there are numeral.

As used herein, term " invention " or " the present invention " are non-limiting term, and are not intended to mean any independent embodiment of the present invention, but contain as all possible embodiment as described in applying for.

As used herein, the term " about " that the amount of modifying composition of the present invention or reactant uses refers to can by the change of umerical amount such as occurred with under type: for generation of the general measure of enriched material or solution and fluid treatment operation in real world; By unintentional error in these operations; For the preparation of the difference in the manufacture of the composition of composition or manner of execution, source or purity; Deng.Term " about " also comprises owing to producing the different amount from the different equilibrium conditions of the composition of specific starting mixt.No matter whether modified by term " about ", the equal parts of the claim amount of comprising.In one embodiment, term " about " refers in 10% scope of report numerical value, or in 5% scope of report numerical value.

As used herein, " biomass " refer to the natural product comprising hydrolyzable polysaccharide, this hydrolyzable polysaccharide provides fermentable sugars and/or starch, comprise and derive from natural origin, as corn, sugarcane, wheat, Mierocrystalline cellulose or ligno-cellulosic materials and any sugar and starch of material comprising Mierocrystalline cellulose, hemicellulose, xylogen, starch, oligose, disaccharides and/or monose and their mixture.Biomass also can comprise other component, such as protein and/or lipid.Biomass can derive from single source, or biomass can comprise the mixture derived from more than a kind of source.Such as, biomass can comprise the mixture of corn cob and maize straw, or the mixture of grass and blade.Biomass include but not limited to bioenergy crops, agricultural residue, Municipal solid rubbish, industrial solid rubbish, sludge, yard waste, timber and forestry rubbish (such as forest thinning thing) from paper-making industry.The example of biomass includes but not limited to: corn, corn cob, crop residue is as corn husk, maize straw, grass, wheat, rye, wheat stalk, Si Peierte wheat, triticale, barley, Barley straw, oat, hay, rice, rice straw, switchgrass, potato, sweet potato, cassava, jerusalem artichoke (Jerusalem artichoke), bagasse, Chinese sorghum, sugarcane, sugar beet, fodder beet, soybean, palm, coconut, Semen Brassicae campestris, safflower, Sunflower Receptacle, grain, eucalyptus, Chinese silvergrass, the component obtained from the grinding of cereal, tree (such as, branch, root, leaf), wood chip, sawdust, shrub and shrubbery, vegetables, fruit, flower, Animal manure, and their mixture.Such as, by the known in the art any working method for the object processing biological for fermentation, as by grinding and liquefying, wine with dregs, fruit juice, molasses or hydrolysate is formed by biomass.Such as, the hydrolysate of fermentable sugars is comprised with acquisition, such as low ammonia pretreatment disclosed in U.S. Patent Application Publication 2007/0031918 (it is incorporated herein by reference) by any method process Mierocrystalline cellulose well known by persons skilled in the art and/or lignocellulose biomass.The enzymatic saccharification of Mierocrystalline cellulose and/or lignocellulose biomass utilizes enzyme consortium (such as usually, cellulase, zytase, Polyglucosidase, dextranase, lyase) come degraded cellulose and hemicellulose, to produce the hydrolysate comprising sugar, described sugar comprises glucose, wood sugar and pectinose.Be applicable to the saccharifying enzyme of Mierocrystalline cellulose and/or lignocellulose biomass see the people such as Lynd (Microbiol.Mol.Biol.Rev.66:506-577,2002).

As used herein, " fermentable carbon source " or " can ferment carbon substrate " refers to can by the carbon source of microbial metabolism.Suitable fermentable carbon source includes but not limited to monose, such as glucose or fructose; Disaccharides such as lactose or sucrose; Oligose; Polysaccharide, such as starch or Mierocrystalline cellulose; One carbon substrate; And their mixture.

As used herein, " fermentable sugars " refers to can by microbial metabolism disclosed herein to produce one or more sugar of tunning.

As used herein, " raw material " refers to the charging in fermenting process, described charging comprises the fermentable carbon source or do not have with undissolved solid and oil, and if be suitable for, described charging comprised fermentable carbon source before or after being discharged fermentable carbon source by processing further (such as by liquefaction, saccharification or other method) or obtained fermentable carbon source from starch from the degraded of compounding sugar.Raw material comprises maybe can derive from biomass.Suitable raw material includes but not limited to rye, wheat, corn, corn mash, sugarcane, sugarcane wine with dregs, barley, cellulose materials, ligno-cellulosic materials or their mixture.When relating to " stock oil ", be to be understood that this term comprises the oil produced from given raw material.

As used herein, " fermented liquid " refers to water, fermentable carbon source (such as, sugar), the solid dissolved, optionally, produce tunning (such as, product alcohol) microorganism, optionally, the mixture of tunning (such as, product alcohol) and other composition.In certain embodiments, fermented liquid refers to the material remained in fermentor tank, and wherein tunning (such as, product alcohol) is produced by microbial metabolism fermentable carbon source.Sometimes, as used herein, term " fermented liquid " synonymously can use with " fermention medium " or " fermenting mixture ".In certain embodiments, the fermented liquid comprising product alcohol can be called as fermentation beer or beer.

As used herein, " fermentor tank " or " fermenting container " refers to and carries out fermentation reaction wherein, thus produces the unit of tunning (such as, product alcohol, as ethanol or butanols) from fermentable carbon source.Term " fermentor tank " synonymously can use with " fermenting container " herein.

As used herein, " liquefaction unit " refers to the unit carrying out liquefying wherein.Liquefaction is the process discharging oligose wherein from raw material.Be in some embodiments of corn at raw material, during liquefying, from W-Gum content, discharge oligose.

As used herein, " saccharification unit " refers to the unit carrying out saccharification (that is, oligose resolves into monose) wherein.When fermenting and saccharification occurs simultaneously, saccharification unit and fermentor tank can be same unit.

As used herein, " sugar " refers to oligose, disaccharides, monose and/or their mixture.Term " sugar " also comprises carbohydrate, comprises starch, dextran, glycogen, Mierocrystalline cellulose, piperylene and sugar.

As used herein, " saccharifying enzyme " refers to can Polysaccharides and/or oligose, one or more enzymes of the α-Isosorbide-5-Nitrae-glycosidic link of such as glycogen or starch.Saccharifying enzyme also can comprise can the enzyme of hydrocellulose or ligno-cellulosic materials.

What " undissolved solid " referred to raw material as used herein can not fermentation part, such as, and plumule, fiber and seitan and be not dissolved in any other component of water-bearing media.Such as, fermentation part can not the comprising and remain solid and can from the part of the raw material of fermented liquid absorbing fluid of raw material.

" oil " refers to the lipid obtained from plant (biological example matter) or animal as used herein.The example of oil includes but not limited to tallow oil, Semen Maydis oil, canola oil, capric acid/Trivent OCG, Viscotrol C, Oleum Cocois, Oleum Gossypii semen, fish oil, Jojoba oil, lard, linseed oil, neat's-foot oil, oiticica oil, plam oil, peanut oil, rapeseed oil, rice oil, Thistle oil, soya-bean oil, sunflower seed oil, tung oil, curcas oil and vegetables oil blend.

As used herein, " product alcohol " refers to any alcohol that can be produced by microorganism during the fermentation, and described fermenting process utilizes biomass as the source of the carbon substrate that can ferment.Product alcohol includes but not limited to C ₁-C ₈alkyl alcohol.In certain embodiments, product alcohol is C ₂-C ₈alkyl alcohol.In other embodiments, product alcohol is C ₂-C ₅alkyl alcohol.Should be appreciated that C ₁-C ₈alkyl alcohol includes but not limited to methyl alcohol, ethanol, propyl alcohol, butanols, amylalcohol and hexanol.Similarly, C ₂-C ₈alkyl alcohol includes but not limited to ethanol, propyl alcohol, butanols, amylalcohol and hexanol.In certain embodiments, product alcohol also can comprise fusel (or potato spirit) and glycerine." alcohol " is also used in reference to product alcohol in this article.

As used herein, " butanols " refers to the butanol isomer n-butyl alcohol (1-BuOH) of independent or its form of mixtures, 2-butanols (2-BuOH), the trimethyl carbinol (t-BuOH) and/or isopropylcarbinol (iBuOH, i-BuOH, I-BUOH, iB are also referred to as 2-methyl isophthalic acid-propyl alcohol).When relating to butyl alcohol ester, term " butyl ester " and " butyl alcohol ester " are often used interchangeably.

As used herein, " propyl alcohol " refers to propyl alcohol isomer Virahol or 1-propyl alcohol.

As used herein, " amylalcohol " refers to amylalcohol isomer 1-amylalcohol, 3-methyl-1-butanol, 2-methyl-1-butene alcohol, 2,2-dimethyl-1-propyl alcohol, 3-amylalcohol, 2-amylalcohol, 3-methyl-2-butanols or 2-methyl-2-butanols.

As used herein, " situ product removes " (ISPR) refers to that selectivity removes specific product to control the production concentration in bioprocess from the bioprocess such as fermented when product generates.

As used herein, " extraction agent " refers to the solvent for extracting tunning (such as, product alcohol).Sometimes, as used herein, term " extraction agent " synonymously can use with " solvent ".

As used herein, " immiscible with water " refers to that chemical composition such as extraction agent or solvent can not with the aqueous solution as fermented liquid mix to be formed the form of single liquid phase.

As used herein, " carboxylic acid " refers to any organic compound with general chemical formula-COOH, and wherein carbon atom forms carbonyl (-C=O) by double bond and oxygen atoms bond, and by singly-bound and hydroxyl (-OH) bonding.Carboxylic acid can be the form of the form of protonated carboxylic acid, carboxylate form (such as ammonium, sodium or sylvite) or protonated carboxylic acid and carboxylic acid salt mixture.Term carboxylic acid can describe independent chemical substance (such as oleic acid) or the mixture of carboxylic acid, and its fatty acid ester by such as hydrolyzing biomass source or triglyceride level, triglyceride, monoglyceride and phosphatide produce.

As used herein, " lipid acid " refers to have C ₄-C ₂₈carbon atom (is C the most commonly ₁₂-C ₂₄carbon atom) carboxylic acid (such as, aliphatic monocarboxylic acid), described carboxylic acid is saturated or undersaturated.Lipid acid can also be branching or non-branching.Lipid acid can derive from or be contained in the fat of animal or plant, oil or wax with the form of esterification.Lipid acid can glyceride form fat and fatty oil in natural existence, or by hydrolysed fat or by synthesis acquisition.Term lipid acid can describe the mixture of single chemical substance or lipid acid.In addition, term lipid acid can contain free fatty acids.

As used herein, " fatty alcohol " refers to have C ₄-C ₂₂the alcohol of the aliphatic chain of carbon atom, described aliphatic chain is saturated or undersaturated.

As used herein, " alkanoic " refers to have C ₄-C ₂₂the aldehyde of the aliphatic chain of carbon atom, described aliphatic chain is saturated or undersaturated.

As used herein, " fatty amide " refers to have C ₄-C ₂₂the acid amides of the aliphatic chain of carbon atom, described aliphatic chain is saturated or undersaturated.

As used herein, " fatty acid ester " refers to have C ₄-C ₂₂the ester of the aliphatic chain of carbon atom, described aliphatic chain is saturated or undersaturated.

As used herein, " containing aqueous phase " refers to, such as, what comprise the biphase mixture of such as liquid phase and vapor phase contains aqueous phase, refer to comprise two kinds of liquid phases (such as, organic aqeous phase) and vapor phase three-phase mixture containing aqueous phase, refer to wherein containing aqueous phase comprise the two-phase of the suspended solids of a tittle or three-phase mixture containing aqueous phase, or refer to comprise vapor phase, organic phase, four phase mixtures containing aqueous phase and solid phase.In certain embodiments, three-phase mixture can comprise vapor phase, liquid phase and solid phase.In certain embodiments, obtain by making fermented liquid follow organic extraction agent immiscible with water to contact containing aqueous phase.Comprise in an embodiment of the method for fermented extracted described herein, term " fermented liquid " can refer in biphasic fermentation extracts containing aqueous phase.

As used herein, " organic phase " refers to the non-aqueous phase of the mixture (such as, biphase mixture, three-phase mixture, four phase mixtures) obtained by making fermented liquid follow organic extraction agent immiscible with water to contact.As used herein, term " organic phase " synonymously can use with " extraction agent phase " sometimes.

As used herein, " effective titre " refers to the total amount of the specifically fermentation product (such as, product alcohol) that often liter of fermented liquid is produced by fermentation.

As used herein, " part " about process flow refers to any stream part retaining stream composition, comprises any one or the various ingredients of whole stream and stream, comprises all components of stream.

The invention provides the technological process control utilizing fermentative production tunning as product alcohol.Other tunning that technological process control as herein described can be used to produce comprises propylene glycol, butyleneglycol, acetone, acid such as lactic acid, acetic acid, butyric acid and propionic acid; Gas is hydrogen methane and carbon dioxide such as; Amino acid; VITAMIN is as vitamin H, vitamins B ₂(riboflavin), vitamins B ₁₂(such as cobalami), xitix (such as vitamins C), vitamin-E (such as a-tocopherol) and vitamin K (such as vitamin k4); Microbiotic is as erythromycin, penicillin, Streptomycin sulphate and tsiklomitsin; And other product is as citric acid, saccharase, sorbyl alcohol, polygalacturonase and Xylitol.

The invention provides technique and the system of the product alcohol for being produced by zymotechnique by zymotechnique production product alcohol and recovery.As an example of the embodiment of technique described herein, cause fermentation by directly being introduced by raw material in fermentor tank.In certain embodiments, one or more fermentor tank can be used in technique as herein described.Suitable raw material includes but not limited to rye, wheat, corn, corn mash, sugarcane, sugarcane wine with dregs, barley, cellulose materials, ligno-cellulosic materials or their mixture.These raw materials of method process of such as dry grinding or wet-milling can be used.In certain embodiments, before introducing fermentor tank, can by raw material liquefaction to produce raw slurry, described raw slurry can comprise undissolved solid, fermentable carbon source (such as, sugar) and oil.The liquefaction of raw material realizes by any known liquifying method, and described liquifying method includes but not limited to acid system, enzyme process (such as, α-amylase), acid-enzyme process or their combination.In certain embodiments, liquefaction can occur in liquefaction unit.

If raw slurry is fed directly to fermentor tank, undissolved solid and/or oil can hinder effectively removing and reclaiming of product alcohol.Particularly, when utilizing liquid-liquid extraction to extract product alcohol from fermented liquid, the existence of undissolved solid (such as, particle) can cause thrashing, includes but not limited to by hindering contacting between extraction agent with fermented liquid to reduce the quality transfering rate of product alcohol to extraction agent; Produce in fermentor tank or raise emulsion, thus hindering being separated of extraction agent and fermented liquid; The becoming at least partially of extraction agent and product alcohol " being stranded " reduce the recovery of extraction agent and the efficiency of recycle, because can be used as in the solid that distiller's dried grain and solvend (DDGS) be removed; Reduce the volumetric efficiency of fermentor tank, because there is solid to occupy volume in fermentor tank, and because slower being separated of extraction agent and fermented liquid; And shorten the life cycle of extraction agent due to oil pollution.These effects can cause higher fund and running cost.In addition, the extraction agent " be stranded " in DDGS can reduce DDGS value and as the qualification of animal-feed for selling.Therefore, in order to avoid and/or minimize these problems, before adding raw slurry to fermentor tank, from raw slurry, remove undissolved solid at least partially.When extraction be carry out on the removed fermented liquid of undissolved solid time, the active and efficiency of the product alcohol extraction of producing can be enhanced.

With reference to accompanying drawing, this document describes that process raw material is to generate raw slurry and separate raw materials slurries to generate the technique containing aqueous phase and solid phase (such as, wet cake) and system that comprise fermentable carbon source.As shown in Figure 1, in certain embodiments, described system comprises liquefaction 10, and it is configured to liquefaction raw material to produce raw slurry.Such as, raw material 12 can be introduced into liquefaction 10 (such as, by the entrance in liquefaction unit).Raw material 12 can be comprise fermentable carbon source as industrial known any suitable biological material that is sugared and/or starch, includes but not limited to barley, oat, rye, Chinese sorghum, wheat, triticale, Si Peierte wheat, grain, sugarcane, corn or their combination.Also water can be introduced liquefaction 10.

It is water-soluble sugar that the method for liquefaction raw material 12 relates to the Starch Hydrolysis in raw material 12.Any known liquifying method and the liquefaction unit of industrial utilization can be used, include but not limited to acid system, enzyme process or acid-enzyme process.These class methods can be used alone or in combination.In certain embodiments, can enzyme process be utilized and can by suitable enzyme 14, such as α-amylase, introduce liquefaction 10.The example of the α-amylase of technique used in the present invention and system is described in United States Patent (USP) 7,541,026; U.S. Patent Application Publication 2009/0209026; U.S. Patent Application Publication 2009/0238923; U.S. Patent Application Publication 2009/0252828; U.S. Patent Application Publication 2009/0314286; U.S. Patent Application Publication 2010/02278970; U.S. Patent Application Publication 2010/0048446; In U.S. Patent Application Publication 2010/0021587, their each full text is incorporated herein by reference.

In certain embodiments, for liquefy and/or the enzyme of saccharification can be produced by microorganism.The example producing the microorganism of this fermentoid is described in United States Patent (USP) 7,498,159; U.S. Patent Application Publication 2012/0003701; U.S. Patent Application Publication 2012/0129229; PCT international publication WO 2010/096562; And in PCT international publication WO 2011/153516, their each full text is incorporated herein by reference.In certain embodiments, for liquefy and/or the enzyme of saccharification can by the microbial expression of also production product alcohol.In certain embodiments, for liquefy and/or the enzyme of saccharification can by the microbial expression of also expressing butanol biosynthetic pathway.In certain embodiments, described butanol biosynthetic pathway can be n-butyl alcohol biosynthetic pathway, 2-butanol biosynthetic pathway, isobutanol biosynthetic pathway or 2-butanone biosynthetic pathway.

The process of liquefaction raw material 12 creates raw slurry 16 (being also referred to as wine with dregs or thick mash), and it comprises fermentable carbon source (such as, sugar) and undissolved solid.In certain embodiments, raw slurry 16 can comprise fermentable carbon source (such as, sugar), oil and undissolved solid.Undissolved solid can be raw material 12 can not fermentation part.In certain embodiments, raw material 12 can be corn, and such as, through dry grinding, unassorted corn grain, and raw slurry 16 is corn mash slurries.Raw slurry 16 from the outlet drain of liquefaction 10, and can be imported into separation 20.

Separation 20 has the entrance for receiving raw slurry 16, and can be configured to remove undissolved solid from raw slurry 16.Be separated 20 also can be configured to remove oil and/or remove oil and undissolved solid.Be separated and 20 can stir or rotate raw slurry 16, to generate liquid phase or the aqueous solution 22 and solid phase or wet cake 24.

The aqueous solution 22 can comprise sugar (such as, the sugar of oligose form) and water.The aqueous solution 22 can comprise the oligose at least about 10 % by weight, at least about the oligose of 20 % by weight, or at least about 30 % by weight oligose.The aqueous solution 22 discharges from separation 20 by outlet.In certain embodiments, described outlet can be positioned at the near top of separation 20.

Wet cake 24 can comprise undissolved solid.Wet cake 24 discharges from separation 20 by outlet.In certain embodiments, described outlet can be positioned near the bottom of separation 20.Wet cake 24 also can comprise a part for sugar and water.After having discharged the aqueous solution 22 from separation 20, can to wet cake 24 with additional water washing in separation 20.Alternatively, to wet cake 24 with additional water washing by other tripping device.The wet cake 24 of washing will reclaim the sugar (such as oligose) be present in wet cake, and can by the sugar that reclaims and water recycle in liquefaction 10.After washing, process wet cake 24 further by any applicable currently known methods, to form distiller's dried grain and solvend (DDGS).The wet cake 24 formed from separation 20 forms DDGS and has some benefits.Because undissolved solid does not enter fermentor tank, DDGS is without undergoing the condition of fermentor tank.Such as, DDGS does not contact the microorganism be present in fermentor tank maybe may be present in other material any (such as, extraction agent and/or product alcohol) in fermentor tank, and therefore, microorganism and/or other material are not confined in DDGS.These effects are that the follow-up processing of DDGS and sale (such as, as animal-feed) provide benefit.

Be separated any conventional separating device that 20 can be industrial utilization, comprise such as, such as decanter type bowl formula whizzer, three-phase centrifuge, dish stacked whizzer, filtering centrifuge or decanter type whizzer.In certain embodiments, undissolved solid completes from removing of raw slurry 16 by following methods or device: the filtration of filtration, vacuum filtration, belt filter, press filtration, membrane filtration, micro-filtration, use screen cloth, screening, grid or grid filter, the filter of porous grid, flotation, hydrocyclone, pressure filter, helical pressure device, gravitational settler, vortex separator or any method that can be used for from liquid separation solid or device.In certain embodiments, being separated 20 is single step processs.In one embodiment, undissolved solid can be removed to form two kinds of product streams from raw slurry 16, such as, comprise the oligose aqueous solution of lower concentration solid and comprise the wet cake of greater concn solid compared with raw slurry 16 compared with raw slurry 16.In addition, the 3rd stream comprising oil can be generated, such as, if three phase centrifugation is used to remove solid from raw slurry 16.So, by using different isolation technique or their combination to produce multiple product stream.

Three-phase centrifuge can be used to the three phase separation of raw slurry, such as separate raw materials slurries with generate two kinds of liquid phases (such as, current and oil stream) and solid phase (such as, solid or wet cake) (see, such as, Flottweg flottweg AG, Vilsibiburg, Germany).Above-mentioned two kinds of liquid phases can separated and decantation, and such as, by the basin of two blowdown systems from whizzer, to prevent crossed contamination, and solid phase is removed by independent blowdown system.

At use corn as in some embodiments of raw material, three-phase centrifuge can be used to remove solid and Semen Maydis oil from the corn mash of liquefaction simultaneously.Solid may be remaining undissolved solid after starch is hydrolyzed to soluble oligosaccharide in liquefaction process.Semen Maydis oil can milled and/or discharge from the plumule of corn grain in liquefaction process.In certain embodiments, three-phase centrifuge can have an incoming flow and three outlet flow.Incoming flow can be made up of the liquefied corn wine with dregs produced during liquefaction.Wine with dregs can be made up of following: the aqueous solution of oligose (such as, the starch of liquefaction); Comprise the undissolved solid of the undissolved non-starch component from corn; With the Semen Maydis oil be made up of glyceryl ester and free fatty acids.Three outlet flow of three-phase centrifuge can be wet cakes, and it comprises the most undissolved solid from wine with dregs; Comprise the heavy concentration logistics of great majority from the liquefying starch of wine with dregs; With comprise great majority from the light concentration logistics of the Semen Maydis oil of wine with dregs.Heavy concentration logistics can be fed for fermentation.Wet cake useful process recirculated water, evaporator condensation thing as described herein and/or countercurrent washing, for reclaiming Zulkovsky starch from wet cake.Light concentration logistics can be used as byproduct and is sold, is converted to other byproduct or be used to processing, such as, Semen Maydis oil is changed into corn oil fatty acid (COFA).In certain embodiments, COFA can be used as extraction agent.

See Fig. 1, the aqueous solution 22 that is configured to ferment has entrance for receiving the aqueous solution 22 with the fermentation 30 (or fermentor tank 30) of production product alcohol.Fermentation 30 can be any suitable fermentor tank known in the art.Fermentation 30 can comprise fermented liquid.In certain embodiments, synchronous saccharification and fermentation (SSF) can occur in fermentation 30.Any known method for saccharifying of industrial utilization all can use, and includes but not limited to acid system, enzyme process or acid-enzyme process.In certain embodiments, enzyme 38 (such as, glucoamylase) can be introduced into the entrance in fermentation 30, forms monose to make the oligose hydrolysis in the aqueous solution 22.The example of the glucoamylase of system used in the present invention and technique is described in United States Patent (USP) 7,413,887; United States Patent (USP) 7,723,079; U.S. Patent Application Publication 2009/0275080; U.S. Patent Application Publication 2010/0267114; U.S. Patent Application Publication 2011/0014681; With in U.S. Patent Application Publication 2011/0020899, their each full text is incorporated herein by reference in certain embodiments, and glucoamylase can by microbial expression.In certain embodiments, glucoamylase can by the microbial expression of also production product alcohol.In certain embodiments, glucoamylase can by the microbial expression of also expressing butanol biosynthetic pathway.In certain embodiments, described butanol biosynthetic pathway can be n-butyl alcohol biosynthetic pathway, 2-butanol biosynthetic pathway, isobutanol biosynthetic pathway or 2-butanone biosynthetic pathway.

In certain embodiments, the enzyme of such as glucoamylase can be added to liquefaction vessel.The enzyme adding such as glucoamylase can reduce the viscosity of raw slurry or liquefied fermented glutinous rice to liquefaction vessel, and can improve separation efficiency.In certain embodiments, can use can reduce raw slurry concentration any enzyme (such as sigma-Aldrich, St.Louis, MO).The viscosity of raw material measures (such as, viscometer, mobilometer) by any method as known in the art.

Microorganism 32 can be introduced into fermentation 30.In certain embodiments, microorganism 32 can be included in fermented liquid.In certain embodiments, microorganism 32 can breed in independent container or tank (such as, breeding tank).In certain embodiments, the microorganism carrying out self-reproduction tank can be used to inoculate one or more fermentor tank.In certain embodiments, one or more propagation tank can be used in technique as herein described and system.In certain embodiments, described propagation tank can be about 2% of the size of fermentor tank to about 5%.In certain embodiments, described propagation tank can comprise following in one or more: wine with dregs, water, enzyme, nutritive substance, extraction agent and microorganism.In certain embodiments, product alcohol can be produced in propagation tank.

In certain embodiments, microorganism 32 can be bacterium, cyanobacteria, filamentous fungus or yeast.In certain embodiments, the sugar in microorganism 32 metabolic water solution 22, and production product alcohol.In certain embodiments, microorganism 32 can be recombinant microorganism.In certain embodiments, microorganism 32 can be fixed, as passed through absorption, covalent attachment, being cross-linked, retaining and encapsulating.Method for encapsulate cells is as known in the art, and such as, as described in U.S. Patent Application Publication 2011/0306116, the document is incorporated herein by reference.

In certain embodiments, along with product alcohol is produced by microorganism 32, situ product removes (ISPR) can be used to remove product alcohol from fermentation 30.In certain embodiments, liquid-liquid extracts can be used to ISPR.In certain embodiments, fermentation 30 can have the entrance for receiving extraction agent 34.In certain embodiments, extraction agent 34 can be added to fermented liquid in fermentation 30 downstream.Alternative device extraction agent 34 being added to fermentation 30 or fermentation 30 downstreams is represented by dashed line.In certain embodiments, ISPR can carry out in propagation tank.In certain embodiments, ISPR can carry out in fermentor tank and propagation tank.In certain embodiments, ISPR can ferment and/or propagation start time (that is, time 0) carry out.By starting ISPR when fermenting and/or propagation starts, fermentor tank can be maintained at low-level with the concentration of the product alcohol in propagation tank, thus the impact of product alcohol on microorganism is minimized and makes microorganism realize the cell mass increased.In certain embodiments, extraction agent can be added into propagation tank.In certain embodiments, extraction agent can be added before the inoculation of propagation tank.In certain embodiments, extraction agent can be added after the inoculation of propagation tank.In certain embodiments, extraction agent can be added by the multiple time points after the inoculation of propagation tank.In certain embodiments, extraction agent can be added into fermentor tank.In certain embodiments, extraction agent can be added before the inoculation of fermentor tank.In certain embodiments, extraction agent can be added after the inoculation of fermentor tank.In certain embodiments, extraction agent can be added by the multiple time points after the inoculation of fermentor tank.In certain embodiments, extraction agent can be added into fermentor tank and propagation tank.This document describes the example that liquid-liquid extracts.Fermentative production is extracted and the method reclaiming alcohol is described in U.S. Patent Application Publication 2009/0305370 for using from fermented liquid; U.S. Patent Application Publication 2010/0221802; U.S. Patent Application Publication 2011/0097773; U.S. Patent Application Publication 2011/0312044; U.S. Patent Application Publication 2011/0312043; With in PCT international publication WO 2011/159998; Their each full text is incorporated herein by reference.

Extraction agent 34 contacts with fermented liquid, and formation comprises, such as, biphase mixture (such as, have product alcohol rich extraction agent phase and exhausted product alcohol containing aqueous phase) stream 36.In certain embodiments, stream 36 can be four phase mixtures, and it comprises, such as, and vapor phase, organic phase, containing aqueous phase and solid phase.Product alcohol in fermented liquid or its part are transferred to extraction agent 34.In certain embodiments, 36 outlet drain by fermenting in 30 are flowed.Routine techniques can be used from the extraction agent separated product alcohol stream 36.

In certain embodiments, the internals of fermentor tank or device can be used to improve being separated between fermented liquid and extraction agent.Such as, described internals or device can be used as coalescer to promote that being separated and/or improving as physical barriers between fermented liquid and extraction agent is separated.The internals of these fermentor tanks or device also can prevent solid sedimentation in extraction agent phase (or layer), promote to be entrained in the coalescent of the water-containing drop extracted in oxidant layer, and promote waste gas (such as, CO ₂, air) remove, thus make the minimum interference to extraction agent phase and/or liquid-liquid interface.The example that can be used to internals in technique as herein described and system or device includes but not limited to baffle plate, perforation plate, deep hole, tilted plate separator, cone-shaped body etc.In certain embodiments, described perforation plate can be smooth horizontal perforation plate.In certain embodiments, described cone-shaped body can be back taper body or concentric cones.In certain embodiments, described internals can be rotated.In certain embodiments, described internals or device can be positioned at or approximately be positioned at the level place of the liquid-liquid interface of fermented liquid and extraction agent.In certain embodiments, coalescent pad and/or reorientation outlet can be added, to improve containing the coalescent of aqueous phase and to reclaim.

In certain embodiments, before the completing of ISPR and/or fermentation, can from the outlet drain stream 35 fermentation 30.The stream 35 of discharge can comprise microorganism 32.Microorganism 32 can be separated from stream 35, such as, by centrifugal or membrane filtration.In certain embodiments, by removing microorganism before extraction agent is added into fermented liquid, microorganism is not exposed to extraction agent, and is not therefore exposed to any negative impact that extraction agent may have microorganism.In addition, remove microorganism by the upstream at leaching process, (such as, heating or cooling mixture are to promote to be separated, to use higher K for more violent extracting method _dand/or compared with highly selective extraction agent or there is the performance of improvement but there is the extraction agent of lower biocompatibility) can be used to reclaim product alcohol.In certain embodiments, microorganism 32 can be recycled to fermentation 30, and this can improve the productivity of product alcohol, thus causes the production efficiency of product alcohol to improve.

See Fig. 2, in certain embodiments, ISPR can carry out in the downstream of fermentation 30.In certain embodiments, the stream 33 of product alcohol and microorganism 32 can be comprised from the outlet drain fermentation 30, and such as extraction column that it is led to downstream, to reclaim product alcohol.In certain embodiments, before ISPR, stream 33 can be processed by separate microorganism 32.Such as, microorganism 32 completes from removing of stream 33 by following methods or device: the filtration of centrifugal, filtration, vacuum filtration, belt filter, press filtration, membrane filtration, micro-filtration, use screen cloth, screening, grid or grid filter, the filter of porous grid, flotation, hydrocyclone, pressure filter, helical pressure device, gravitational settler, vortex separator or any method that can be used for from liquid separation solid (such as, microorganism) or tripping device.After the removing of microorganism 32, stream 33 can be directed to extraction column, to reclaim product alcohol.

The other embodiment of technique as herein described and system is illustrated in Fig. 3 to 6.Fig. 3 to 6, the extraction (such as, generating stream 33) comprising option for extraction agent being added into fermentor tank (such as, generating stream 36) or carry out in the downstream of fermentor tank, is similar to Fig. 1 and 2 respectively, therefore will no longer describes in detail.

See Fig. 3, system of the present invention and technique can comprise the outlet drain oil 26 from being separated 20.Raw slurry 16 can be separated into the first liquid phase or the aqueous solution 22, the solid phase comprising undissolved solid or wet cake 24 that comprise fermentable sugars and the second liquid phase comprising oil 26 that can leave from separation 20.In certain embodiments, raw slurry 16 can occur to the first liquid phase, second liquid phase in a single step with being separated of solid phase.In certain embodiments, raw material 12 is corns, and oil 26 is Semen Maydis oil.In certain embodiments, oil 26 can import storage tank or be applicable in any unit of oil storage.Any applicable tripping device all can be used to discharge water solution 22, wet cake 24 and oil 26, such as, and three-phase centrifuge.In certain embodiments, when raw material is corn, from the oil of raw material 12, the part as Semen Maydis oil is retained in wet cake 24.In certain embodiments, when removing oily 26 via separation 20 from raw material 12 (such as, corn), the fermented liquid in fermentation 30 comprises the Semen Maydis oil of the amount of minimizing.

As described herein, in certain embodiments, oil is separable can be stored in oily storage element from raw material or raw slurry.Such as, can use for separating of any appropriate method from raw material or raw slurry separating oil, comprise three phase centrifugation or mechanical extraction.In order to improve oil from removing raw material or raw slurry, oil can be utilized to extract auxiliary agent, as tensio-active agent, non-emulsifying agent or flocculation agent and enzyme.Oil extracts the example of auxiliary agent and includes but not limited to non-polymeric, liquid surfactant; Talcum powder; Fine and smooth stone flour; Salt (NaOH); Calcium carbonate; And enzyme, as ultra SP-L, with l (Sigma-Aldrich, St.Louis, MO) and NZ 33095 (Novozymes, Franklinton, NC);

As shown in Figure 4, if do not discharge oil separately, then it can be removed together with wet cake 24.When removing wet cake 24 via separation 20, in certain embodiments, when raw material is corn, the part from the oil (such as Semen Maydis oil) of raw material 12 is retained in wet cake 24.Wet cake 24 can import mixing tank 60 and with water or other solvent, form wet cake mixture 65.In certain embodiments, water can be any water source available in new water, adverse current, the water that boils, process water, lutter water, distilled water or fermentation processing facility or their any combination.Wet cake mixture 65 can import separation 70, produces washing concentrating thing 75, and it comprises the fermentable sugars and wet cake 74 that reclaim from wet cake 24.Washing concentrating thing 75 can be recycled in liquefaction 10.

In certain embodiments, being separated 70 can be can any tripping device of separate solid and liquid, comprise that such as decanter type bowl formula is centrifugal, three phase centrifugation, dish stacked are centrifugal, filter centrifugation, decanter type are centrifugal, filtration, vacuum filtration, belt filter, press filtration, membrane filtration, the filtration of use screen cloth, screening, grid filter, the filter of porous grid, flotation, hydrocyclone, pressure filter, helical pressure device, gravitational settler, vortex separator or their combination.

In certain embodiments, wet cake can stand one or more cycles of washing or washing system.Such as, wet cake 74 is processed further by wet cake 74 is imported the second washing system.In certain embodiments, wet cake 74 can import the second mixing tank 60 ', forms wet cake mixture 65 '.Wet cake mixture 65 ' can import the second separation 70 ', preparing washing enriched material 75 ' and wet cake 74 '.Washing concentrating thing 75 ' can be recycled in liquefaction 10.In certain embodiments, washing concentrating thing 75 ' can mix with washing concentrating thing 75, and is recirculated to liquefaction 10.In certain embodiments, wet cake 74 ' can merge with wet cake 74, for further processing, as described herein.In certain embodiments, being separated 70 ' can be can any tripping device of separate solid and liquid, comprise that such as decanter type bowl formula is centrifugal, three phase centrifugation, dish stacked are centrifugal, filter centrifugation, decanter type are centrifugal, filtration, vacuum filtration, belt filter, press filtration, membrane filtration, micro-filtration, the filtration of use screen cloth, screening, grid filter, the filter of porous grid, flotation, hydrocyclone, pressure filter, helical pressure device, gravitational settler, vortex separator or their combination.In certain embodiments, wet cake can stand one, two, three, four, five, or more cycles of washing or washing system.

Wet cake 74 can merge with syrup, and forms DDGS by any suitable currently known methods drying subsequently.Form DDGS by wet cake 74 and there is multiple benefit.Because undissolved solid does not enter fermentor tank, DDGS does not have by the extraction agent carried secretly and/or product alcohol, and it is without undergoing the condition of fermentor tank, and it does not contact with the microorganism be present in fermentor tank.These benefits make processing DDGS be more prone to, such as, as animal-feed.

In certain embodiments, a part for undissolved solid can be imported into fermentation 30.In certain embodiments, this part of undissolved solid can have less granularity (such as, powder).In certain embodiments, this part of undissolved solid can form full tower and heats up in a steamer thing.In certain embodiments, above-mentioned full tower heats up in a steamer thing can be processed, heats up in a steamer thing and wet cake to form rare tower.In certain embodiments, heat up in a steamer the wet cake of thing from full tower and wet cake 74 and/or 74 ' can be merged, and further processing to produce DDGS.

As shown in Figure 4, oil does not separate with wet cake and discharges, but oil is comprised the part as wet cake, and is finally present in DDGS.If utilize corn as raw material, Semen Maydis oil comprises triglyceride level, triglyceride, monoglyceride, lipid acid and phosphatide, and they provide the metabolizable energy of animal to originate.The oil (such as, Semen Maydis oil) existed in wet cake and final DDGS can provide the animal-feed of expectation, such as high fat content animal-feed.

In certain embodiments, ISPR extraction agent can be changed into from wet cake and DDGS separating oil, follow-up for identical or different alcohol zymotechnique.The method obtaining extraction agent from biomass is described in U.S. Patent Application Publication 2011/0312044; U.S. Patent Application Publication 2011/0312043; With in U.S. Patent Application Publication 2012/0156738; Their each full text is incorporated herein by reference.Any suitable currently known methods can be used from wet cake and DDGS separating oil, comprise such as solvent extraction process.In one embodiment of the invention, wet cake or DDGS may be added to extraction unit, and with the solvent wash of such as hexane, to remove oil.Other solvent available comprises petroleum fractions or their mixture of such as butanols, isohexane, ethanol, such as sherwood oil.After the extraction of oil, wet cake or DDGS can be processed, to remove any residual solvent.Such as, any methods known in the art can be used to heat wet cake or DDGS, to evaporate the solvent of any remnants.After removal of solvents, wet cake or DDGS can stand drying process to remove the water of any remnants.Treated wet cake can be used to generate DDGS.Treated DDGS can be used as feed additive for animal, such as milk cow and beef cattle, poultry, pig, livestock, horse, aquaculture and domestic pets.

In certain embodiments, extraction agent can be used as the mode of the color changing wet cake.Such as, the raw material packet of such as corn is containing the pigment (such as, xenthophylls) that can be used as tinting material in the foodstuff products comprising animal-feed (such as, poultry feed).Be exposed to extraction agent and can change these pigments, cause the wet cake that such as color is more shallow.The more shallow wet cake of color can produce the more shallow DDGS of color, and this can be desirable quality for some animal-feed.

Be used as in some embodiments of raw material at corn, xenthophylls can be separated from corn and/or undissolved solid, and be used as the pigment composition in DDGS or animal-feed, or agent be used for medicine or protective foods purposes as a supplement.Method for separating of xenthophylls include but not limited to chromatography as size exclusion chromatography, solvent extraction as extraction using alcohol and ferment treatment as Sumizyme MP (Alcalase) hydrolysis (see, such as, the people such as Tsui, J.Food Eng.83:590-595,2007; The people Food Science 31:72-77 such as Li, 2010: United States Patent (USP) 5,648,564; United States Patent (USP) 6,169,217; United States Patent (USP) 6,329,557; United States Patent (USP) 8,236,929; Their each full text is incorporated herein by reference).In certain embodiments, can xenthophylls be separated from corn and/or undissolved solid and be added into COFA.In certain embodiments, COFA and/or xenthophylls can be used to food, medicine and protective foods purposes.

After extracting from wet cake or DDGS, the oil of gained and solvent mixture can be collected for separating of oil and solvent.In one embodiment, by evaporation treated oil/solvent mixture, thus evaporation and can collect, recycling solvent.The oil of recovery can be changed into ISPR extraction agent, follow-up for identical or different alcohol zymotechnique.

The production removed for product alcohol of the oil ingredient of raw material is favourable, because the oil existed in fermentor tank can be decomposed into lipid acid and glycerine.Glycerine can gather and reduce the water yield that can be used for whole system recirculation in water.Therefore, the oil ingredient removing raw material can improve by the water yield of system recirculates the efficiency that product alcohol produces by improving.

See Fig. 5, oil can be removed by the different nodes in technological process as herein described.Raw slurry 16 can such as use three-phase centrifuge to be divided into the first liquid phase or the aqueous solution 22, comprise the second liquid phase of oil 26 and solid phase or wet cake 24.Wet cake 24 can be further processed to reclaim fermentable sugars and oil.Wet cake 24 can import mixing tank 60 and with water or other solvent, form wet cake mixture 65.In certain embodiments, water can be adverse current, the water that boils, process water, lutter water, collect other water source any available in self-evaporating water or fermentation processing facility or their any combination.Wet cake mixture 65 can import separation 70 (such as, three-phase centrifuge), produces washing concentrating thing 75, and it comprises fermentable sugars, oil stream 76 and wet cake 74.Washing concentrating thing 75 can be recycled in liquefaction 10.

As described herein, wet cake can stand one or more cycles of washing or washing system.In certain embodiments, wet cake 74 can import the second mixing tank 60 ', forms wet cake mixture 65 '.Wet cake mixture 65 ' can be imported into the second separation 70 ', produces washing concentrating thing 75 ', oil stream 76 ' and wet cake 74 '.Washing concentrating thing 75 ' can be recycled in liquefaction 10.In certain embodiments, washing concentrating thing 75 ' can mix with washing concentrating thing 75, and is recirculated to liquefaction 10.In certain embodiments, wet cake 74 ' can merge with wet cake 74, for further processing, as mentioned below.In certain embodiments, oil stream 76 ' and oil 26 can merged and further processing, and for generating the extraction agent that can be used for fermenting process, or oil stream 76 ' and oily 26 can merged and further processing, for the manufacture of the consumer's goods.

Wet cake 74 can merge with syrup, and utilizes the drying of any suitable method to form DDGS subsequently.Form DDGS by wet cake 74 and there is multiple benefit.Because undissolved solid does not enter fermentor tank, DDGS does not comprise extraction agent and/or product alcohol, and it is without undergoing the condition of fermentor tank, and it does not contact with the microorganism be present in fermentor tank.These benefits make processing DDGS be more prone to, such as, as animal-feed.As described herein, in certain embodiments, wet cake 74,74 ' and heat up in a steamer from Quan Ta the wet cake that thing formed can be merged, and further processing to produce DDGS.

As shown in Figure 6A, the aqueous solution 22 and wet cake 24 can merged, cool and import fermentation 30.Raw slurry 16 can such as use three-phase centrifuge to be divided into the first liquid phase or the aqueous solution 22, comprise the second liquid phase of oil 26 and solid phase or wet cake 24.In certain embodiments, oil 26 can be imported into storage tank or be applicable in any unit of oil storage.It is also again slurried that the aqueous solution 22 and wet cake 24 can be imported into mixing tank 80, forms the aqueous solution/wet cake mixture 82.Mixture 82 can import in water cooler 90, and produce cooling mixture 92, it can be imported in fermentation 30.In certain embodiments, when removing oily 26 via separation 20 from raw slurry 16, mixture 82 and 92 comprises the oil of decrement.

In another embodiment, as shown in Figure 6B, tripping device (such as, three-phase centrifuge) separate raw materials slurries 16 can be used, generating the first liquid phase or the aqueous solution 22, comprise the second liquid phase of oil 26 and solid phase or wet cake 24.The aqueous solution 22, wet cake 24 and oil 26 or their part can be imported into fermentation 30.In certain embodiments, the aqueous solution 22, wet cake 24 and oil 26 or their part can be merged, such as, by mixing, form the aqueous solution, wet cake and oil mixt, and this mixture can be imported into fermentation 30.In certain embodiments, the aqueous solution 22 and wet cake 24 can be merged, form the aqueous solution and wet cake mixture, and then oil 26 can be added into this mixture, form the aqueous solution, wet cake and oil mixt, and this mixture can be imported into fermentation 30.In certain embodiments, the aqueous solution 22 and wet cake 24 can be merged, form the aqueous solution and wet cake mixture, and this mixture and oil 26 or their part can be used as independently stream is imported into fermentation 30.

In the other embodiment of technique as herein described and system, saccharification can occur in independently saccharification system.In certain embodiments, saccharification system or can be separated between 20 and fermentation 30 between liquefaction 10 and separation 20.In certain embodiments, liquefaction and/or saccharification can utilize ative starch enzyme or low temperature hydrolysis enzyme such as Stargen ^tM(Genencor International, Palo Alto, CA) and BPX ^tM(Novozymes, Franklinton, NC) carries out.In certain embodiments, raw slurry can stand ative starch hydrolysis (also referred to as low temperature gelatinization or low temperature hydrolysis).

In certain embodiments, system of the present invention and technique can comprise a series of two or more tripping devices (such as, whizzer) for removing undissolved solid and/or oil.Such as, the entrance of the second separating unit can be conducted to from the aqueous solution of the first separating unit discharge.Described first separating unit and the second separating unit can be identical (such as, two three-phase centrifuges) or can be different (such as, three-phase centrifuge and decanter type whizzers).Be separated and realize by various ways, include but not limited to that decanter type bowl formula is centrifugal, three phase centrifugation, dish stacked are centrifugal, filter centrifugation, decanter type are centrifugal, filtration, vacuum filtration, belt filter, press filtration, membrane filtration, the filtration of use screen cloth, screening, grid filter, the filter of porous grid, flotation, hydrocyclone, pressure filter, helical pressure device, gravitational settler, vortex separator or their combination.

In fermented liquid, undissolved solid does not exist or minimizedly has some benefits.Such as, can be excluded the demand of the operating unit in Downstream processing, thus cause the product alcohol production efficiency of raising.In addition, due to leave fermentor tank fermented liquid in undissolved solid less, some or all heat up in a steamer the centrifugal of thing for the treatment of full tower and can be excluded.Undissolved solid removes from raw slurry the process for producing rate and cost efficiency that can improve biomass.The productivity improved can comprise active relative to the extraction of the product alcohol production efficiency and/or raising of not removing the technique of undissolved solid and the raising of system before fermentation.About the description for being separated the technique of undissolved solid and the other of system from raw slurry see, such as, U.S. Patent Application Publication 2012/0164302 and pct international patent application PCT/US2013/51571, the full text of each document above-mentioned is incorporated herein by reference.

As described herein, use comprises liquid-liquid and is extracted in interior multiple method, can reclaim product alcohol from fermented liquid.In some embodiments of technique as herein described and system, extraction agent can be used to reclaim product alcohol from fermented liquid.Extraction agent used herein can have, such as, one or more in following performance and/or characteristic: the physiologically acceptable of (i) and microorganism, (ii) not miscible with fermented liquid, (iii) with regard to the extraction of product alcohol, high partition ratio (Kp), (iv) with regard to the extraction of nutritive substance and/or water, low partition ratio, v viscosity (u) that () is low, (vi) compared with such as water, to the highly selective of product alcohol, (vii) relative to low density (ρ) or the density different compared with the density of fermented liquid of fermented liquid, (viii) boiling point of the downstream processing to extraction agent and product alcohol is applicable to, (ix) lower than envrionment temperature fusing point, x () be minimal absorptivity in solids, (xi) the low trend forming emulsion with fermented liquid, (xii) in fermenting process stability from start to finish, (xiii) low cost, and (xiv) toxicological harmless.

In certain embodiments, can based on some performance and/or characteristic selective extraction agent as described herein.Such as, the viscosity of extraction agent can the mass transfer performance of influential system, that is, product alcohol can from the efficiency being extracted into extraction agent phase (that is, organic phase) containing aqueous phase.The density of extraction agent can affect and be separated.In certain embodiments, selection rate refers to that product alcohol that extraction agent extracts is to the relative quantity of water.The cost that the product alcohol that can affect boiling point reclaims and method.Such as, when reclaiming butanols by distillation mutually from extraction agent, the boiling point of extraction agent should be enough low, so as to make make any thermal destruction of extraction agent or side reaction or minimized to the demand of high vacuum in still-process while, can separating butanol.

Extraction agent can be compatible with microorganism biological, that is, be nontoxic to described microorganism, or toxicity only reaches and makes described microorganism with the injured level of acceptable degree.In certain embodiments, physiologically acceptable refers to that microorganism utilizes measuring of the ability of fermentable carbon source under the existence of extraction agent.The degree of the biocompatibility of extraction agent can be determined, such as, by the glucose utilization speed of microorganism under the existence of this extraction agent and product alcohol.In certain embodiments, the extraction agent of biocompatible refers to the extraction agent hindering microorganism to utilize fermentable carbon source ability.Such as, the extraction agent of biocompatible does not allow microorganism to utilize glucose with higher than the speed when this extraction agent does not exist by about 25%, high by about 30%, high by about 35%, high by about 40%, high about 45% or high about 50% speed.

Those skilled in the art can select extraction agent, to make performance desired as described herein and/or characteristic maximize, and the recovery of optimized product alcohol.Those skilled in the art can also know, the mixture using extraction agent can be favourable.Such as, extraction agent mixture can be used to the partition ratio improving product alcohol.In addition, extraction agent mixture can be used to the physical property regulating and optimize extraction agent, such as density, boiling point and viscosity.Such as, suitable combination can provide has partition ratio enough with regard to product alcohol and enough can economically for removing the extraction agent of the biocompatibility of product alcohol from fermented liquid.

In certain embodiments, in technique as herein described and system, available extraction agent can be organic solvent.In certain embodiments, in technique as herein described and system, available extraction agent can be the organic solvent miscible with not water.In certain embodiments, extraction agent can be select self-saturating, monounsaturated, polyunsaturated C ₁₂-C ₂₂fatty alcohol, C ₁₂-C ₂₂lipid acid, C ₁₂-C ₂₂the ester of lipid acid, C ₁₂-C ₂₂alkanoic, C ₁₂-C ₂₂organic extraction agent of fatty amide and their mixture.In certain embodiments, extraction agent can also be select self-saturating, monounsaturated, polyunsaturated C ₄-C ₂₂fatty alcohol, C ₄-C ₂₈lipid acid, C ₄-C ₂₈the ester of lipid acid, C ₄-C ₂₂alkanoic, C ₄-C ₂₂organic extraction agent of fatty amide and their mixture.In certain embodiments, described lipid acid can be C ₄-C ₂₄lipid acid, and/or described ester can be C ₄-C ₂₄the ester of lipid acid.In certain embodiments, extraction agent can be select self-saturating, monounsaturated, polyunsaturated C ₁₂-C ₁₈fatty alcohol, C ₁₂-C ₁₈lipid acid, C ₁₂-C ₁₈the ester of lipid acid, C ₁₂-C ₁₈alkanoic, C ₁₂-C ₁₈organic extraction agent of fatty amide and their mixture.In certain embodiments, extraction agent can be select self-saturating, monounsaturated, polyunsaturated C ₁₄-C ₁₈fatty alcohol, C ₁₄-C ₁₈lipid acid, C ₁₄-C ₁₈the ester of lipid acid, C ₁₄-C ₁₈alkanoic, C ₁₄-C ₁₈organic extraction agent of fatty amide and their mixture.In certain embodiments, extraction agent can be select self-saturating, monounsaturated, polyunsaturated C ₁₆-C ₁₈fatty alcohol, C ₁₆-C ₁₈lipid acid, C ₁₆-C ₁₈the ester of lipid acid, C ₁₆-C ₁₈alkanoic, C ₁₆-C ₁₈organic extraction agent of fatty amide and their mixture.In certain embodiments, extraction agent can comprise carboxylic acid.In certain embodiments, the ester of described lipid acid can be the combination (such as, fatty acid ester) of lipid acid and alcohol.In certain embodiments, described alcohol can be product alcohol.In certain embodiments, described ester can be methyl ester, ethyl ester, propyl diester, butyl ester, amyl group ester, polyhexamethylene or glyceryl ester.

In certain embodiments, extraction agent can comprise the first extraction agent, and it is selected from C ₁₂-C ₂₂fatty alcohol, C ₁₂-C ₂₂lipid acid, C ₁₂-C ₂₂the ester of lipid acid, C ₁₂-C ₂₂alkanoic, C ₁₂-C ₂₂fatty amide and their mixture; With the second extraction agent, it is selected from C ₁₂-C ₂₂fatty alcohol, C ₁₂-C ₂₂lipid acid, C ₁₂-C ₂₂the ester of lipid acid, C ₁₂-C ₂₂alkanoic, C ₁₂-C ₂₂fatty amide and their mixture.In certain embodiments, extraction agent can comprise the first extraction agent, and it is selected from C ₁₂-C ₂₂fatty alcohol, C ₁₂-C ₂₂lipid acid, C ₁₂-C ₂₂the ester of lipid acid and their mixture; With the second extraction agent, it is selected from C ₁₂-C ₂₂fatty alcohol, C ₁₂-C ₂₂lipid acid, C ₁₂-C ₂₂the ester of lipid acid and their mixture.In certain embodiments, extraction agent can comprise the first extraction agent, and it is selected from C ₁₂-C ₁₈fatty alcohol, C ₁₂-C ₁₈lipid acid, C ₁₂-C ₁₈the ester of lipid acid and their mixture; With the second extraction agent, it is selected from C ₁₂-C ₁₈fatty alcohol, C ₁₂-C ₁₈lipid acid, C ₁₂-C ₁₈the ester of lipid acid and their mixture.In certain embodiments, extraction agent can comprise the first extraction agent, and it is selected from C ₁₄-C ₁₈fatty alcohol, C ₁₄-C ₁₈lipid acid, C ₁₄-C ₁₈the ester of lipid acid and their mixture; With the second extraction agent, it is selected from C ₁₄-C ₁₈fatty alcohol, C ₁₄-C ₁₈lipid acid, C ₁₄-C ₁₈the ester of lipid acid and their mixture.In certain embodiments, extraction agent can comprise the first extraction agent, and it is selected from C ₁₂-C ₂₂fatty alcohol, C ₁₂-C ₂₂lipid acid, C ₁₂-C ₂₂the ester of lipid acid, C ₁₂-C ₂₂alkanoic, C ₁₂-C ₂₂fatty amide and their mixture; With the second extraction agent, it is selected from C ₇-C ₁₁fatty alcohol, C ₇-C ₁₁lipid acid, C ₇-C ₁₁the ester of lipid acid, C ₇-C ₁₁alkanoic and their mixture.

In certain embodiments, described extraction agent can be organic extraction agent, such as oleyl alcohol, behenyl alcohol, hexadecanol, lauryl alcohol (being also referred to as 1-lauryl alcohol), tetradecyl alcohol, stearyl alcohol, oleic acid, lauric acid, linolic acid, linolenic acid, tetradecanoic acid, palmitinic acid, stearic acid, sad, capric acid, undeeanoic acid, Myristicin acid methylester, Witconol 2301, 1 nonyl alcohol, 1-decanol, 2-undecyl alcohol, 1-aldehyde C-9, 1-undecyl alcohol, the undecyl aldehyde, lauryl aldehyde, the 2-methyl undecyl aldehyde, oleylamide, sub-oleylamide, palmitic amide, stearylamide, 2-ethyl-1-hexanol, 2-hexyl-1-decanol, 2-octyl group-1-lauryl alcohol, and their mixture.In certain embodiments, extraction agent can comprise following in one or more: oleic acid, lauric acid, linolic acid, linolenic acid, tetradecanoic acid, palmitinic acid, stearic acid, sad, capric acid and undeeanoic acid.In certain embodiments, extraction agent can comprise following in one or more: oleic acid, linolic acid, linolenic acid, tetradecanoic acid, palmitinic acid and stearic acid.In certain embodiments, extraction agent can comprise following in one or more: oleic acid, linolic acid, palmitinic acid and stearic acid.In certain embodiments, extraction agent can comprise following in one or more: oleic acid, lauric acid, linolic acid, linolenic acid, tetradecanoic acid, palmitinic acid, stearic acid, sad, capric acid and undeeanoic acid, and one or more esters of oleic acid, lauric acid, linolic acid, linolenic acid, tetradecanoic acid, palmitinic acid, stearic acid, sad, capric acid and undeeanoic acid.In certain embodiments, extraction agent can comprise following in one or more: oleic acid, linolic acid, linolenic acid, tetradecanoic acid, palmitinic acid and stearic acid, and oleic acid, linolic acid, linolenic acid, tetradecanoic acid, palmitinic acid and one or more esters stearic.In certain embodiments, extraction agent can comprise following in one or more: oleic acid, linolic acid, palmitinic acid and stearic acid, and oleic acid, linolic acid, palmitinic acid and one or more esters stearic.In certain embodiments, extraction agent can comprise following in one or more: oleyl alcohol, behenyl alcohol, hexadecanol, lauryl alcohol, tetradecyl alcohol, stearyl alcohol.In certain embodiments, extraction agent can comprise following in one or more: 1 nonyl alcohol, 1-decanol, 2-undecyl alcohol, 1-aldehyde C-9,1-undecyl alcohol, the undecyl aldehyde, 2-ethyl-1-hexanol, 2-hexyl-1-decanol, 2-octyl group-1-lauryl alcohol.

In certain embodiments, extraction agent can be biocompatible and the mixture of the extraction agent of biocompatible.The mixture example of the extraction agent of biocompatible extraction agent and biocompatible includes but not limited to oleyl alcohol and nonyl alcohol, oleyl alcohol and 1-undecyl alcohol, oleyl alcohol and 2-undecyl alcohol, oleyl alcohol and 1-aldehyde C-9, oleyl alcohol and decyl alcohol and oleyl alcohol and lauryl alcohol.The other example of the extraction agent of biocompatible extraction agent and biocompatible is described in U.S. Patent Application Publication 2009/0305370 and U.S. Patent Application Publication 2011/0097773; Their each full text is incorporated herein by reference.In certain embodiments, biocompatible extraction agent can have high atmospheric boiling point.Such as, biocompatible extraction agent can have the atmospheric boiling point higher than the atmospheric boiling point of water.

In certain embodiments, hydrophilic solutes can be added in the fermented liquid contacted with extraction agent.Hydrophilic solutes is separated can improve containing the existence in aqueous phase, and can improve the mark of the product alcohol be dispensed in organic phase.The example of hydrophilic solutes can include but not limited to polyol, multi-carboxylic acid compounds, polyol compound and free ion salt.The sugar of such as glucose, fructose, sucrose, maltose and oligose can be used as hydrophilic solutes.Other polyol can comprise glycerine, ethylene glycol, propylene glycol, Polyglycerine and hydroxylation soccerballene.Multi-carboxylic acid compounds can comprise citric acid, tartrate, toxilic acid, succsinic acid, polyacrylic acid, and their sodium, potassium or ammonium salt.The ion salt that can be used as the hydrophilic solutes in fermented liquid comprises positively charged ion, and it comprises sodium, potassium, ammonium, magnesium, calcium and zinc; And negatively charged ion, it comprises sulfate radical, phosphate radical, chlorion and nitrate radical.The amount of the hydrophilic solutes in fermented liquid can be selected by those skilled in the art, to make product alcohol from containing aqueous phase (such as, fermented liquid) to organic phase (such as, extraction agent) transfer maximize, not there is negative impact to the microbial growth of production product alcohol and/or throughput simultaneously.High-caliber hydrophilic solutes can bring osmotic stress and/or toxicity to microorganism.Those skilled in the art can use the currently known methods of any amount to determine the amount of the best of hydrophilic solutes, to make to minimize the osmotic stress of microorganism and/or the effect of toxicity.

In some embodiments of butanols in product alcohol, can for attracting the moieties of butanols and carrying out selective extraction agent for providing hardly to the avidity of water.Do not provide and optionally will absorb alcohol with the extraction agent of the hydrogen bonding of such as water.In certain embodiments, extraction agent can comprise aromatic substance.In certain embodiments, extraction agent can comprise the benzene that alkyl replaces, and it includes but not limited to isopropyl benzene, p-Methylisopropylbenzene (being also referred to as 1-methyl-4-(1-methylethyl) benzene), a cymene (being also referred to as 1-methyl-3-(1-methylethyl) benzene), a diisopropyl benzene, to diisopropyl benzene, triethylbenzene, ethyl-butyl benzene and t-butyl styrene.An advantage of the benzene using alkyl to replace is the butanols avidity relatively high relative to other hydrocarbon.In addition, sec.-propyl replace or benzene that isobutyl-replaces can provide in butanols avidity especially higher than the advantage of benzene that other replaces.Additional advantage is lower viscosity, lower surface tension, higher thermostability and higher chemical stability, and it contributes to being separated property and long-term recycling.In certain embodiments, attract the extraction agent of moieties of butanols can mix with another extraction agent of the avidity of the hydrogen bonding form of the hydroxylic moiety provided such as butanols, make this mixture provide optimum balance between selectivity and the distribution on water.In certain embodiments, the extraction agent comprising butanols can be separated with fermented liquid, and is distilled in the tower worked under vacuo.Above-mentioned distillation can be carried out with backflow, to keep the distillate of the high purity butanols comprising few extraction agent.Bottom can comprise a part for the butanols comprised in distillation charging, makes the boiling temperature again under vacuum be applicable to indirectly transmitting heat from available steam.Distillation can be carried out with partial condenser, wherein only withdrawing fluid is condensed, and the steam overhead product being substantially butanols composition can be imported into the bottom of rectifying tower, rectifying tower is distillated from the beer column tower top of condensation the butanols stream that steam drains by simultaneously charging.An advantage of such distillation is, by the heat integration from the steam out produced by butanols stripping from extraction agent, eliminates the demand of the reboiler to the butanols stream drained for purifying.

In certain embodiments, extraction agent can produce from raw material.Such as, the oil being present in the such as Semen Maydis oil in raw material can be used to generate the extraction agent for extracting fermentation.Glyceryl ester in oil can be changed into reaction product by chemistry or enzymatic, and such as lipid acid and/or fatty acid ester (such as, ethyl ester, butyl ester, fusel ester), it can be used as extraction agent, for the recovery of product alcohol.Use Semen Maydis oil as an example, Semen Maydis oil triglyceride level can with alkali as ammonium hydroxide reacts, to obtain fatty amide and glycerine.In certain embodiments, the oil in raw material can catalyzed dose of hydrolysis, to generate lipid acid.In certain embodiments, by making oil and catalyst exposure, what can make the acyl glyceride in oil is hydrolyzed to carboxylic acid at least partially.In certain embodiments, the acid/oil compositions obtained comprises monoglyceride from the partial hydrolysis of the acyl glyceride in described oil and/or triglyceride.In certain embodiments, the acid/oil compositions obtained comprises the by-product glycerin of acylglycerol Ester hydrolysis.In certain embodiments, the acid/oil compositions obtained comprises the lysophospholipid of the partial hydrolysis from the phosphatide in oil.The method obtaining extraction agent from biomass is described in U.S. Patent Application Publication 2011/0312044; U.S. Patent Application Publication 2011/0312043; With in U.S. Patent Application Publication 2012/0156738, their each all full text is incorporated herein by reference.

In certain embodiments, by catalyzer is added into fermentor tank, the conversion of the oil in raw material or raw slurry (such as, be hydrolyzed, transesterify) can occur in fermentor tank.Such as, the catalyzer of such as lipase can be added into fermentor tank, and the oil existed in raw material or raw slurry is changed into lipid acid and/or fatty acid ester.In certain embodiments, the conversion of the oil in raw material or raw slurry can occur in independently unit.Such as, raw material or raw slurry can be imported into a certain unit, and the catalyzer of such as lipase can be added into this unit, makes the oil existed in raw material or raw slurry change into lipid acid.And for example, raw material or raw slurry can be imported into a certain unit, and the catalyzer of such as lipase and alcohol (such as, ethanol, butanols, fusel) can be added into this unit, make the oil existed in raw material or raw slurry change into fatty acid ester.In certain embodiments, lipid acid and/or fatty acid ester can be added into fermentor tank, and can be used as extraction agent, for the recovery of product alcohol.In certain embodiments, lipid acid and/or fatty acid ester can be added into outside extractor or extraction agent tower, and can be used as extraction agent, for the recovery of product alcohol.

In certain embodiments, oil can be separated from raw slurry, and described oil can be imported into a certain unit, and the catalyzer of such as lipase can be added into this unit, generates fatty acid stream.Fatty acid stream can be heated with deactivation lipase, and then described fatty acid stream can be imported into outside extractor or storage tank.Lipid acid from storage tank can be imported into outside extractor, for from broth extraction product alcohol.In certain embodiments, the oil be separated from raw slurry can be stored in storage tank.The catalyzer of such as lipase can be added into storage tank, generates fatty acid stream.Fatty acid stream can be heated with deactivation lipase, cooling, is then imported into outside extractor, for from broth extraction product alcohol.In certain embodiments, the oil be separated from raw slurry can be imported into a certain unit, and the catalyzer of such as lipase can be added into this unit, generates fatty acid stream.Fatty acid stream can be heated with deactivation lipase, cooling, and then described fatty acid stream can be imported into fermentor tank.

In certain embodiments, one or more catalyzer described can be one or more enzymes, such as, and lytic enzyme.In certain embodiments, one or more catalyzer described can be one or more enzymes, such as, and lipase.Lipase can derive from any source, comprises the such as mould genus of pears head (Absidia), achromobacter (Achromobacter), Aeromonas (Aeromonas), Alcaligenes (Alcaligenes), Alternaria (Alternaria), Aspergillus (Aspergillus), achromobacter (Achromobacter), aureobasidium genus (Aureobasidium), bacillus (Bacillus), Beauveria (Beauveria), circumfili Pseudomonas (Brochothrix), mycocandida (Candida), chromobacterium (Chromobacter), Coprinus (Coprinus), fusarium (Fusarium), Geotrichum (Geotricum), Hansenula (Hansenula), Humicola (Humicola), Hyphozyma, Bacterium lacticum (Lactobacillus), Metarhizium (Metarhizium), Mucor (Mucor), Nectria (Nectria), Neurospora (Neurospora), paecilomyces (Paecilomyces), Penicillium (Penicillium), Rhodopseudomonas (Pseudomonas), Rhizoctonia (Rhizoctonia), Rhizomucor (Rhizomucor), head mold (Rhizopus), Rhodosporidium (Rhodosporidium), Rhodotorula (Rhodotorula), yeast belong (Saccharomyces), pig belongs to (Sus.), Sporobolomyces (Sporobolomyces), thermophilic fungus belongs to (Thermomyces), Thiarosporella, Trichoderma (Trichoderma), Verticillium (Verticillium) and/or Ye Shi yeast belong (Yarrowia).In certain embodiments, the source of lipase can be selected from Bu Shi colter mould (Absidia blakesleena), absidia corymbifera (Absidia corymbifera), Achromobacter iophagus (Achromobacter iophagus), Alcaligenes (Alcaligenes sp.), the raw rod method (Alternaria brassiciola) of rape, flavus (Aspergillus flavus), aspergillus niger (Aspergillus niger), Tabin aspergillus (Aspergillus tubingensis), Aureobasidium pullulans (Aureobasidium pullulans), Bacillus coagulans (Bacillus coagulans), bacillus pumilus (Bacillus pumilus), bacstearothermophilus (Bacillus strearothermophilus), subtilis (Bacillus subtilis), heat kill rope silk bacterium (Brochothrix thermosohata), Burkholderia cepacia (burkholderia cepacia), Candida cylindracea (Candida cylindracea) (fold candida (Candida rugosa)), secondary Candida lipolytica (Candida paralipolytica), antarctic candida (Candida Antarctica) lipase A, antarctic candida (Candida Antarctica) lipase B, Ou Nuo is than candiyeast (Candida ernobii), deformity candiyeast (Candida deformans), fold candida (Candida rugosa), Candida parapsilosis (Candida parapsilosis), Chromobacter viscosum (Chromobacter viscosum), Coprinus cinereus (Coprinus cinerius), different spore Fusariumsp (Fusarium heterosporum), Fusarium oxysporum (Fusarium oxysporum), eggplant fusarium (Fusarium solani), pea differentiated eggplant fusarium (Fusarium solani pisi), the yellow Fusariumsp (Fusarium roseum culmorum) of wheat, geotrichum candidum (Geotrichum candidum), geotrichum penicillatum (Geotricum penicillatum), Hansenula anomala (Hansenula anomala), short and small humicola lanuginosa (Humicola brevispora), short and small humicola lanuginosa high temperature mutation (Humicola brevis var.thermoidea), Humicola insolens (Humicola insolens), lactobacillus curvatus (Lactobacillus curvatus), Rhizopus oryzae (Rhizopus oryzae), mucor javanicus (Mucor javanicus), Neuraspora crassa (Neurospora crassa), the red shell of red sphere bundle (Nectria haematococca), penicillium cyclopium (Penicillium cyclopium), skin falls mould (Penicillium crustosum), Penicilllum expansum (Penicillium expansum), penicillum requeforti (Penicillium roqueforti), penicillium cammenberti (Penicillium camembertii), Penicillium I (Penicillium sp.I), Penicillium II (Penicillium sp.II), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Pseudomonas alcaligenes (Pseudomonas alcaligenes), pseudomonas cepacia (Pseudomonas cepacia) (that is, Burkholderia (Burkholderia cepacia)), Pseudomonas fluorescens (Pseudomonas fluorescens), Pseudomonas fragi (Pseudomonas fragi), Pseudomonas Maltophilia (Pseudomonas maltophilia), pseudomonas mendocina (Pseudomonas mendocina), separate fat Pseudomonas mephitica (Pseudomonas mephitica lipolytica), Pseudomonas alcaligenes (Pseudomonas alcaligenes), Pseudomonas plantarii (Pseudomonas plantari), pseudomonas pseudoalcaligenes (Pseudomonas pseudoalcaligenes), pseudomonas putida (Pseudomonas putida), pseudomonas stanieri (Pseudomonas stutzeri), with Wisconsin pseudomonas (Pseudomonas wisconsinensis), dry thread Pyrenomycetes (Rhizoctonia solani), rhizomucor miehei (Rhizomucor miehei), rhizopus arrhizus (Rhizopus arrhizus), De Shi head mold (rhizopus delemar), Japan's head mold (Rhizopus japonicus), Rhizopus microsporus (Rhizopus microsporus), knotter head mold (Rhizopus nodosus), Rhizopus oryzae (Rhizopus oryzae), the red winter spore yeast (Rhodosporidium toruloides) of circle, rhodotorula glutinis (Rhodotorula glutinis), yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), sesame paddy shadow yeast (Sporobolomyces shibatanus), wild boar (Sus scrofa), dredge the thermophilic hyphomycete of cotton like (Thermomyces lanuginosus) (being once named as Humicola lanuginosa (Humicola lanuginose)), Thiarosporella phaseolina, trichoderma harziarum (Trichoderma harzianum), Trichodermareesei (Trichoderma reesei) and Yarrowia lipolytica (Yarrowia lipolytica).

In certain embodiments, lytic enzyme and/or lipase can by microbial expressions.In certain embodiments, described microorganism can by through engineering approaches, to express lytic enzyme and/or the lipase of homology or allos.In certain embodiments, lytic enzyme and/or lipase can by the microbial expressions also producing product alcohol.In certain embodiments, lytic enzyme and/or lipase can by the microbial expressions of also expressing butanol biosynthetic pathway.In certain embodiments, described butanol biosynthetic pathway can be n-butyl alcohol biosynthetic pathway, 2-butanol biosynthetic pathway, isobutanol biosynthetic pathway or 2-butanone biosynthetic pathway.

The commercial lipases preparation being suitable as catalyzer includes but not limited to 100L, 100L, 2000T, cALB L, cALA L and Palatase 20000L, can purchased from Novozymes (Franklinton, NC), or from Pseudomonas fluorescens (Pseudomonas fluorescens), pseudomonas cepacia (Pseudomonas cepacia), rice black wool mould (Mucor miehei), pig pancreas, Candida cylindracea (Candida cylindracea), fold candida (Candida rugosa), Rhizopus niveus (Rhizopus niveus), antarctic candida (Candida antarctica), the lipase of rhizopus arrhizus (Rhizopus arrhizus) or Aspergillus (Aspergillus), can purchased from Sigma Aldrich (St.Louis, MO).In certain embodiments, lipase can be heat-staple and/or heat-resisting and/or solvent tolerance.

In certain embodiments, one or more catalyzer described can be Phospholipid hydrolases.Phospholipid hydrolase available in the present invention can derive from various biological source, such as but not limited to the filamentous fungus strain in Fusarium (Fusarium), the such as bacterial strain of yellow Fusariumsp (Fusarium culmorum), different spore Fusariumsp (Fusarium heterosporum), eggplant fusarium (Fusarium solani) or Fusarium oxysporum (Fusarium oxysporum); Or the filamentous fungus strain in Aspergillus (Aspergillus), the such as bacterial strain of Aspergillus awamori (Aspergillus awamori), smelly aspergillus (Aspergillus foetidus), aspergillus japonicus (Aspergillus japonicus), aspergillus niger (Aspergillus niger) or aspergillus oryzae (Aspergillus oryzae).Equally usefully the thermophilic hyphomycete of cotton like (Thermomyces lanuginosus) Phospholipid hydrolase variant, such as commodity are dredged in the present invention ultra (Novozymes A ' S, Denmark).One or more Phospholipid hydrolases can be used as lyophilized powder, immobilized or be employed in aqueous.

In certain embodiments, Phospholipid hydrolase can by microbial expression.In certain embodiments, described microorganism can by through engineering approaches, to express the Phospholipid hydrolase of homology or allos.In certain embodiments, Phospholipid hydrolase can by the microbial expression of also production product alcohol.In certain embodiments, Phospholipid hydrolase can by the microbial expression of also expressing butanol biosynthetic pathway.In certain embodiments, described butanol biosynthetic pathway can be n-butyl alcohol biosynthetic pathway, 2-butanol biosynthetic pathway, isobutanol biosynthetic pathway or 2-butanone biosynthetic pathway.

The by product of fermentation, such as isopropylformic acid, phenylethyl alcohol, 3-methyl-1-butanol, 2-methyl-1-butene alcohol, isobutyric aldehyde, acetic acid, ketoisovaleric acid, pyruvic acid and dihydroxyisovaleric acid, can be inhibited to microorganism.In certain embodiments, these by products are modified by esterification.Such as, described by product can by carboxylic acid, alcohol, lipid acid or other by product esterification.In certain embodiments, these esterifications can by lipase or Phospholipase enzymes catalyze.Such as, the esterification of the by product that the lipase existed in fermented liquid can produce in catalystic, fermentative process.The esterification of these by products can make them minimize the restraining effect of microorganism.

See Fig. 7 A, raw material 12 can processed described in Fig. 1 to 6, therefore will no longer describe in detail.The aqueous solution 22 can be further processed subsequently, to remove the oil of any remnants.In certain embodiments, the aqueous solution 22 can stand centrifugal, decantation or can be used for removing any other method of oil.In certain embodiments, the aqueous solution 22 can be imported into unit 25 (or container), and catalyzer 23 (such as, lipase) can be added into unit 25, makes the oil existed in the aqueous solution 22 change into lipase, generates stream 27.Then stream 27 can be imported fermentation 30, and microorganism 32 also can be added into fermentation 30, with production product alcohol.After fermentation 30, the stream 31 comprising product alcohol and lipid acid can be imported into external unit, and such as, outside extractor or outside extraction ring, for reclaiming product alcohol.

See Fig. 7 B, in certain embodiments, catalyzer 23 can be inactivated, such as, by heating.In certain embodiments, before being added into fermentation 30, the stream 27 comprising catalyzer 23 can be heated (q), to make catalyzer 23 deactivation.See Fig. 7 C, in certain embodiments, deactivation can be carried out in independently unit (such as, deactivation unit).In certain embodiments, flow 27 and can be imported into deactivation 28.After deactivation, stream 27 ' can be imported fermentation 30, and microorganism 32 also can be added into fermentation 30, with production product alcohol.

Due to more effective fermentation, because the energy requirement of the removal of oil and the pollution of less equipment, reduction (such as, dry energy needed for vinasse) and the operation of improvement of vaporizer or evaporation series, change into lipid acid by making oil and can carry out the saving of energy for production equipment belt except deoiling from the aqueous solution 22.In addition, the production removed for product alcohol of the oil ingredient of raw material is favourable, because the oil existed in fermentor tank can be decomposed into lipid acid and glycerine.Glycerine can gather and reduce the water yield that can be used for whole system recirculation in water.Therefore, the oil ingredient of raw material is removed by improving the efficiency that improve product alcohol production by the water yield of system recirculates.In addition, by removing oil, comparatively stable emulsion can not be formed.In some embodiments of the invention, when forming emulsion, by mechanical treatment, adding protonic solvent or by other ordinary method, easily can destroy emulsion.

In another embodiment, see Fig. 7 D, the aqueous solution 22 can be imported into fermentation 30, and catalyzer 23 (such as, lipase) can be added into fermentation 30, makes the oil existed in the aqueous solution 22 change into lipid acid and/or fatty acid ester.In certain embodiments, fatty acid ester can derive from the combination of lipid acid and alcohol.In certain embodiments, described alcohol can be any alcohol in fermentation 30, comprises product alcohol.In certain embodiments, the amount changing into the oil in the aqueous solution 22 of lipid acid and/or fatty acid ester can be at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 95%.In certain embodiments, the fatty acid ester generated by the conversion of oil and the ratio of lipid acid can be about 75: 25.In certain embodiments, the ratio of fatty acid ester and lipid acid can be about 80: 20.In certain embodiments, catalyzer 23 can be added into fermentation 30 with the amount maintaining certain oily transformation efficiency.

After fermentation 30, the stream 31 comprising product alcohol, lipid acid and fatty acid ester can be further processed, to reclaim product alcohol.Such as, stream 31 can be imported into external unit, and such as, outside extractor or outside extraction ring, for reclaiming product alcohol.In certain embodiments, the lipid acid in stream 31 and fatty acid ester can be used as extraction agent.In certain embodiments, described external unit can comprise extraction agent.In certain embodiments, described extraction agent can comprise the ester of lipid acid and/or lipid acid.

Present invention also offers the technique for reclaiming the product alcohol produced by fermenting process and system.That liquid-liquid extracts for reclaiming these class methods of one of product alcohol.Make the net present value (NPV) of the capital investment needed for this technology of enforcement maximized liquid-liquid extracting method, contribute to liquid-liquid to extract most to be used as ISPR technology.The maximized aspect of the net present value (NPV) of liquid-liquid extracting method is made to be that the large capital avoiding being associated with from separation of fermentative broth extraction agent and operation cost are paid.

In an embodiment of liquid-liquid extracting method, extraction agent can be added directly to fermentor tank, and fermented liquid and extraction agent can to make mass transfer onset (such as, product alcohol is from fermented liquid to the transfer of extraction agent) and the mode that can proceed to high effective product alcohol titre that makes to ferment is mixed together.In these class methods, if mixing is too fierce or violent, fermented liquid and extraction agent may have to utilize the tripping device of such as whizzer to be separated.If mixing is not too violent, be separated the gravity settling realization caused by the density difference between extraction agent and fermented liquid.In any one situation, other fermentor tank may be needed, to overcome the loss of the fermenter volume that the extraction agent that is added into fermentor tank occupies.Regardless of being separated in fermentor tank, extraction agent to the direct interpolation in fermentor tank can with in batches, partly in batch or continuous print pattern carry out.If employing continuous mode, and the gravity separation of fermented liquid and extraction agent is impossible, then product alcohol may need the tripping device of such as whizzer from the separation of extraction agent.If the separation method being used to remove from extraction agent product alcohol makes the microorganism existed fermented liquid be alive in sepn process, then can not need separate fermentation liquid and product alcohol/extraction agent.

Another embodiment of liquid-liquid extracting method can comprise outside extractor or extraction column.Such as, can be imported into from the fermented liquid of fermentor tank the outside extractor that fermented liquid mixes with extraction agent.Then, the mixture of fermented liquid and extraction agent can be separated, generates the fermentation broth stream of poor product alcohol and the extraction agent stream compared with by-pass product alcohol.Poor fermentation broth stream can be back to fermentor tank.Richer extraction agent stream can be further processed, with from extraction agent separated product alcohol at least partially, in order to reclaim product alcohol.In certain embodiments, flow back to from extraction agent the speed that the speed receiving product alcohol can be arranged on the production maintaining facility.In certain embodiments, liquid-liquid extracting method can comprise one or more outer liquid-liquid extractor.

In certain embodiments, fermentation can occur in fermentor tank and outside extractor.The additional volume of the fermented liquid existed in outside extractor can play the effect increasing overall fermenter volume, and therefore can improve the overall manufacturing of product alcohol.

Outside extractor can be depending on removing the performance in product alcohol the physical properties of surface-area, fermented liquid and the extraction agent that can be used for face Contact, the relative quantity of the two kinds of phases (such as ferment liquid phase and extraction agent phase) be present in outside extractor and fermented liquid and extraction agent mutually between concentration drive power difference.By reducing the drop size (such as, by designs of nozzles, internal components designs and/or stirring) of the disperse phase in outside extractor, the maximization of the efficiency of outside extractor with regard to given product alcohol concentration can be realized.In certain embodiments, the design and running of outside extractor can provide enough mixing to shift to realize product alcohol suitable between fermented liquid and extraction agent phase, to maintain the needs of product alcohol throughput.

In some cases, the CO from fermentation can be generated in outside fermentor tank ₂, cause the formation that can hinder the drop be separated.Such as, the drop of fermented liquid can be attached to the CO risen mutually by extraction agent ₂.In certain embodiments, extraction agent can be maintained external phase, mutually to improve the coalescent of drop.In certain embodiments, outside extraction agent can comprise for CO ₂internals or outlet.Such as, coalescent pad can be added into outside extractor and/or can be provided with outlet, to improve the coalescent of fermentation liquid phase and to reclaim.

May be harmful to the microorganism be present in fermented liquid from the condition of separation of fermentative broth product alcohol.In certain embodiments, can make fermented liquid to contact with extraction agent before from separation of fermentative broth microorganism.In certain embodiments, can the mixture of fermented liquid and extraction agent be separated (or process) before from this mixture separation microorganism.All can use from any separation method of the mixture separation microorganism of fermented liquid or fermented liquid and extraction agent, comprise such as centrifugal.By make fermented liquid to contact with extraction agent before separate microorganism, likely use more violent extraction conditions, such as higher temperature and/or the extraction agent of biocompatible.If employ not to the separation method of harmful microorganism, then can not need separate fermentation liquid and extraction agent before the removing of product alcohol.

If extraction agent and fermented liquid do not have separated, then extraction agent can be included in evaporator series charging, thus becomes the component of the syrup formed in evaporative process, and may be impregnated in animal-feed.In certain embodiments, any separation method (comprising, such as, centrifugal) can be used from the agent of syrup separation and Extraction.The biocompatible extraction agent of lower boiling (such as, being equivalent to water) can not need this type of to be separated, because described extraction agent and water can be recycled, for the production of in process.

By the water of recycle production unit, make the water of circulation in evaporator series, distill to remove other undissolved solid of salt and beer, the balance of water can be maintained in typical corn to product alcohol production unit.The syrup from evaporator series obtained can with undissolved solids mixing, this mixture can be dried and sell as animal-feed.Process undissolved solid for the technique of animal-feed and system description in, such as, U.S. Patent Application Publication 2012/0164302; U.S. Patent Application Publication 2011/0315541; U.S. Patent Application Publication 2013/0164795; With in pct international patent application PCT/US2013/51571, each entirety above-mentioned is incorporated herein by reference.

As described herein, before raw material is added into fermentation, undissolved solid can be removed from raw material (or raw slurry).If do not remove undissolved solid in the upstream of fermentation, then may be necessary that centrifugal beer is to remove undissolved solid, thus avoid the pollution of vaporizer.Such as, dry grind in production unit in corn to the product alcohol of business, the undissolved solid in evaporator series charging can run with about 3% suspended solids total amount, and can up to 3.5-4% suspended solids total amount.Removing the upstream process that enough solids are equal to or less than these percent value with the per-cent maintaining suspended solids total amount can get rid of centrifugal demand, such as, before beer being imported vaporizer (or evaporation series).This centrifugal discharge will cause the saving of the fund of reequiping needed for dry milled corn to product alcohol production unit.

Be present in undissolved solid in raw slurry at least partially by removing before fermentation, the fermented liquid in outside extractor and extraction agent mutually between face between surface area be clearly separated between fermented liquid and extraction agent by amount, the transfer promoting product alcohol between fermented liquid and extraction agent and providing reducing the undissolved solid of interface and be increased.Clearly be separated the demand also can got rid of other separating step (such as, centrifugal), thus spending of economizing on the use of funds.

The fermented liquid leaving outside extractor can be affected by the design and running of the gas content in the size-grade distribution of the solids content in fermented liquid and solids component, fermented liquid and the physical property (including but not limited to viscosity, density and surface tension) of bubble size distribution, fermented liquid and extraction agent and the design and running of outside extractor and fermentor tank with being separated of extraction agent.These characteristics can determine the demand of the separating device (such as, whizzer) to the fermented liquid and extraction agent in order to leave outside extractor or fermentor tank.Run eliminating under to the condition of the demand of separating device, the capital outlay that enforcement liquid-liquid can be made to extract ISPR minimizes.In addition, for given one group of fermented liquid and extraction agent physical property, made the minimized in size of extractor by the interfacial area maximized between fermented liquid and extraction agent phase, the ability be separated of fermented liquid and extraction agent low cost can be maintained.By getting rid of capital and the running cost of the separating device of such as whizzer, the net present value (NPV) of dry milled corn to product alcohol production unit that employing liquid-liquid extracts ISPR can be improved.

In another embodiment of technique as herein described and system, the extractor design comprising the ability of being separated can be modulated to the physical property adapting to fermented liquid and extraction agent.If if too low less than the product alcohol concentration removed undissolved solid or fermented liquid from raw slurry, adopt the extractor not comprising phase-separating device possibly cannot remove enough product alcohol to maintain the throughput of equipment.Therefore, the invention provides the technique removing and utilize the product alcohol of outside extractor to reclaim and system that comprise solid, wherein said extractor has been designed to improvement and has been separated ability, maximizes to make the recovery of product alcohol.

Illustrative methods of the present invention is described in Fig. 8.Use and some techniques in the same names that uses in Fig. 1-7 and Digital ID Fig. 8 and stream, and to have represented and the same or similar technique described in Fig. 1-7 and stream.

Can be as described herein, with reference to figure 1-7 process raw material 12 also separate solid (100).In brief, raw material 12 can be liquefied, to generate the raw slurry of the oil comprising undissolved solid, fermentable sugars (or fermentable carbon source) and depend on raw material.Then, raw slurry can be made to stand separation method, to remove suspended solids, generate wet cake, the aqueous solution 22 (or concentrated solution) comprising the fermentable sugars of dissolving and optional oil stream.The separation of solid realizes by various ways, include but not limited to that decanter type bowl formula is centrifugal, three phase centrifugation, dish stacked are centrifugal, filter centrifugation, decanter type are centrifugal, filtration, vacuum filtration, belt filter, press filtration, membrane filtration, micro-filtration, the filtration of use screen cloth, screening, grid filter, the filter of porous grid, flotation, hydrocyclone, pressure filter, helical pressure device, gravitational settler, vortex separator or their combination.

Can add the aqueous solution 22 and microorganism 32 to fermentation 30, wherein microorganism 32 ferments fermentable sugars to produce the stream 105 comprising product alcohol.In certain embodiments, during the fermentation, a part for stream 105 can be transferred to extractor 120 (or extracting 120), flows 105 wherein and contacts with extraction agent 124.In certain embodiments, extraction agent can be stored in extraction agent storage tank or unit.In certain embodiments, when the concentration of product alcohol and/or other meta-bolites reaches predetermined concentration, stream 105 can be removed from fermentation 30.In certain embodiments, described predetermined concentration can be the product alcohol of the metabolism adversely affecting microorganism and/or the concentration of other meta-bolites.In certain embodiments, stream 105 can be removed from fermentation 30 when fermenting and starting.In certain embodiments, stream 105 can be removed from fermentation 30 to minimize to make the impact of product alcohol on microorganism 32.In certain embodiments, ferment 30 can comprise one, two, three, four, five, six, seven, eight, or more fermentor tank.

In certain embodiments, extraction agent can be added into fermentation 30.In certain embodiments, the part comprising the fermented liquid of extraction agent can be transferred to extractor 120, and in certain embodiments, can reclaim extraction agent from the fermented liquid comprising extraction agent.By extraction agent is added into fermentation 30, ISPR can be started in fermentation 30.

Product alcohol or its part are transferred to extraction agent 124 from stream 105, and the stream 122 comprising the extraction agent of by-pass product alcohol can be imported into separation 130.The stream 127 comprising the fermented liquid of poor product alcohol can be back to fermentation 30.Separation 130 removes a part for product alcohol from stream 122, and the stream 125 comprising poor extraction agent can be back to extractor 120.In certain embodiments, extractor 120 can in the outside of fermentation 30.In certain embodiments, fermentation 30 can comprise extractor.In certain embodiments, extraction agent, fermented liquid or both can be immiscible at least in part.Stream 135 can be conducted to downstream for processing further (such as, distillation), comprises recovery product alcohol.

In the process extracted, the loss of a part for extraction agent or extraction agent may be there is.In certain embodiments, by extraction agent being added into extractor 120 or extraction agent storage unit, extraction agent 124 can be supplemented.In certain embodiments, such as, extraction agent can derive from raw material or raw slurry, can supplement extraction agent 124 by the oil in raw material or raw slurry is changed into extraction agent.Such as, catalyzer can be added into fermentation 30, make the oil in the aqueous solution 22 change into lipid acid and/or fatty acid ester (see, such as, Fig. 7 D), and the part comprising the stream 105 of product alcohol, lipid acid and/or fatty acid ester can be transferred to extractor 120, flowing 105 wherein can contact with extraction agent 124.The stream 122 comprising the extraction agent of by-pass product alcohol, lipid acid and/or fatty acid ester can be imported into separation 130, generates the stream 125 comprising poor extraction agent, lipid acid and/or fatty acid ester.In certain embodiments, flow 125 can be further processed before it is returned to extractor 120.Such as, the fatty acid ester be present in stream 125 can stand hydrolysis, generates the stream comprising product alcohol and lipid acid.The stream comprising product alcohol and lipid acid can be imported into extractor 120, or this stream can merge with stream 122, and the stream merged can be imported into separation 130.In another embodiment, comprise product alcohol and can be imported into this stream of lipid acid and be separated 130, or this stream can be imported into another separating unit, generate product alcohol stream and fatty acid stream.Fatty acid stream can be imported into extractor 120, and product alcohol stream can merge with stream 135, and is further processed, to reclaim product alcohol.

In certain embodiments, fermented liquid after extractor and being separated of extraction agent may be insufficient, the extraction agent of the dispersion of unacceptable level is remained in be back in the fermented liquid of fermentor tank, and/or the fermented liquid drop of unacceptable level remains in and advances in the extraction agent of distillation.In certain embodiments, leave the top of extractor or the heterogeneous mixture of bottom by one or more hydrocyclone or similar swirling device process, being separated of fermented liquid and extraction agent can be promoted.In certain embodiments, static mixer can be used to replace extractor, fermented liquid and extraction agent are contacted with each other, and the heterogeneous mixture formed is pumped through one or more hydrocyclone or similar swirling device, to realize containing aqueous phase (such as, fermented liquid) and the separation of organic phase (such as, extraction agent).In certain embodiments, one or more hydrocyclone or similar swirling device can be used to remove liquid or drop from gas stream.In certain embodiments, gas stream can from fermentor tank.In certain embodiments, gas stream can from de-gassing vessel.

In batches or in semi-batch zymotechnique, when a part for fermentable sugars is by microorganism 32 metabolism, the stream 103 comprising beer can be imported into the separation 140 in downstream, with from beer separated product alcohol.The stream 145 comprising product alcohol can be conducted to downstream for processing further (such as, distillation), comprises recovery product alcohol.In continuous fermentation process, the stream 103 comprising beer can be imported into the separation 140 in downstream, with from beer separated product alcohol.Comprise the stream 142 that full tower heats up in a steamer thing and can be conducted to downstream for further processing, comprise removing solid and generating rare tower and heat up in a steamer thing.

In certain embodiments, fermentation 30 can comprise two or more fermentor tanks, and flows the multiple streams from two or more fermentor tanks that 105 can comprise merging.In certain embodiments, multiple streams of merging can be imported into extractor 120.In certain embodiments, stream 127 can be shunted, and the part flowing 127 can be back to multiple fermentor tank.In certain embodiments, extractor 120 can be a series of unit that are in parallel or that be connected in series.

In certain embodiments, extraction can carry out regular hour section.Extraction can proceed to, such as, until the concentration of product alcohol in fermentation 30 is low to moderate do not need separation 140.In certain embodiments, the time period can carrying out extending is extracted.

In some embodiments of technique as herein described and system, decantor can be used to be separated.In certain embodiments, decantor can use in combination with extraction agent.In certain embodiments, the surface of decantor can be modified, is separated to improve.Such as, by adding the surface of hydrophilic and/or hydrophobic surfaction decantor.

In certain embodiments, oxygen, air and/or nutritive substance can be added into stream 125 and/or stream 127.In certain embodiments, nutritive substance can be solvable in extraction agent.In certain embodiments, the concentration of oxygen can be measured in various stream, and be used as a part for control loop, for changing the flow of the oxygen entering described process.In certain embodiments, wine with dregs can be added into extractor 120, to allow higher effective titre.In certain embodiments, being separated 130 and 140 can be extractor.In certain embodiments, these extractors can use water to extract product alcohol from extraction agent, and subsequently can from containing aqueous phase separation product alcohol.In certain embodiments, extraction agent can be combined with and strengthen its solute from the ability of broth extraction product alcohol.In certain embodiments, surge tank can at extractor 120 and be separated between 130, as the mode of the product alcohol concentration in the forward horizontal stand extraction agent being separated (such as, distilling).

In certain embodiments, for the object of mixed fermentation liquid and extraction agent, extractor 120 can be designed to utilize the CO generated in fermenting process ₂.In certain embodiments, extractor 120 can be designed to allow the CO in fermented liquid ₂be easy to depart from.This design will be conducive to by the CO risen by extractor 120 ₂the level of foam control mixing.In certain embodiments, fermented liquid can be removed from fermentation 30, to make CO in stream 105 ₂concentration minimize.In certain embodiments, the design of extractor disengagement zone can comprise the surface be separated that promotes between fermented liquid and extraction agent.In certain embodiments, wetting ability and/or hydrophobic surface can be installed in described disengagement zone, are separated to improve.In certain embodiments, outside extraction agent can comprise for CO ₂internals or outlet.Such as, coalescent pad can be added into outside extractor.

In certain embodiments, for making CO ₂minimize, extractor can be designed to have little diameter in the bottom of extractor, becomes the large diameter (such as, conical by its shape) at extractor top gradually.In certain embodiments, extractor can be designed to the step increase with diameter.Such as, extractor can comprise the first area of constant diameter, is then that the step of diameter increases, until the second area of constant diameter.In certain embodiments, the second-order that extractor also can comprise diameter increases in a leap and adds, until the 3rd region of constant diameter.In certain embodiments, extractor can comprise a place or the increase of many places step of diameter.In certain embodiments, extractor can comprise the region of a place or many places constant diameter.

Process by fermentation, gas content (such as, the CO of fermented liquid ₂) change, and by utilizing gas stripper to remove these gas from fermented liquid.By changing through the flow of gas stripper and/or the pressure of gas stripper, the amount of the adjustable gas from fermented liquid stripping.In certain embodiments, CO in fermented liquid ₂amount can be lowered before fermented liquid is transferred to extractor.Such as, use gas stripper or any method known to those skilled in the art, CO can be peeled off from fermented liquid ₂.In certain embodiments, CO ₂removal can carry out under the condition being equal to or less than environmental stress.In certain embodiments, fermentation can continue in extractor, and CO ₂can be produced by microorganism.In certain embodiments, in order to make the CO in extractor ₂minimize, the residence time of fermented liquid in extractor can be reduced.In certain embodiments, the height by changing extractor reduces the residence time.In certain embodiments, the height of extractor can be reduced.The height reducing extractor can make theoretical stage number of extracting reduce.In certain embodiments, extracting stage number to remain theoretical, extractor can be replaced with the extractor of two or more height reduction.In certain embodiments, two or more extractors described can be series connection.In certain embodiments, two or more extractors described can be connect.In certain embodiments, two or more extractors described can connect in the mode making adverse current be kept.In certain embodiments, degassed column plate can be added into one or more extraction column plate.

See Fig. 8, in certain embodiments, the size of the dispersed phase drop in extractor 120 can measured and adjustment in several ways, to improve the speed of mass transfer.Such as, such as focused beam reflection measurement can be used or particulate is observed and is measured drop size is measured in the sreen analysis of technology (Mettler-Toledo, LLC, Columbus OH).In certain embodiments, fermented liquid can be disperse phase and extraction agent can be external phase, and under these conditions, the solid be present in fermented liquid can interact with lower degree and extraction agent.In certain embodiments, the condition being separated 130 can be controlled, to make to minimize the oxidation of extraction agent and thermolability effect.

In certain embodiments, the quality of extraction agent can be monitored, and produce necessary frequency with the success of product alcohol and supplement extraction agent.In certain embodiments, extraction agent can be heated up in a steamer thing solid by full tower and takes away.Full tower heats up in a steamer thing can be separated into liquid (such as, rare tower heats up in a steamer thing) and efflux of solids, and solid can be washed, to reclaim extraction agent.In certain embodiments, the temperature of extractor 120 can be conditioned, to improve the efficiency of overall craft.In certain embodiments, fermented liquid and extraction agent can be following current or adverse current to the stream of extractor 120.In certain embodiments, film can be used to the mixing of fermented liquid and extraction agent is minimized.In certain embodiments, extraction agent can be the polymer beads of absorption product alcohol or inorganic globule.In certain embodiments, described polymer beads or inorganic globule can preferentially absorption product alcohol.

In certain embodiments, measure (on such as line, in line, line is other and/or measure in real time) and the concentration measuring product alcohol and/or metabolic by-prods in various stream can be used to.These measurements can be used as a part for control loop, with the flow changed between various unit or container (such as, ferment 30, extractor 120, be separated 130 and 140 etc.) with improve overall craft.

Another illustrative methods of the present invention is described in Fig. 9.Use and some techniques in the same names that uses in Fig. 1-8 and Digital ID Fig. 9 and stream, and to have represented and the same or similar technique described in Fig. 1-8 and stream.

Can be as described herein, with reference to figure 1-7 process raw material 12 also separate solid 100.In certain embodiments, raw material 12 can be mixed with the water of the recirculation produced by evaporation 160 (such as, flowing 162).As described herein, raw slurry can stand separation method to remove suspended solids, generates wet cake 24, comprises the aqueous solution 22 (or concentrated solution) of the fermentable sugars of dissolving, depends on the oil of raw material.Wet cake 24 can be dried in moisture eliminator 170, and for the production of DDGS.In certain embodiments, wet cake 24 can be repulped by water (such as, the water/stream 162 of recirculation), and stand to be separated, and to remove extra fermentable sugars, generates wet cake through washing (such as, 74,74 ', as described in Figure 4 and 5).In certain embodiments, wet cake stream 24,74 and 74 ' can be merged, and the wet cake stream merged can be dried in moisture eliminator 170, and for the production of DDGS.

Can add the aqueous solution 22 and microorganism 32 to fermentation 30, wherein microorganism 32 metabolism fermentable sugars is to produce the stream 105 comprising product alcohol.In certain embodiments, enzyme can be added into fermentation 30.Stream 105 can be imported into extractor 120, and can contact with extraction agent 124.The stream 127 comprising the fermented liquid of poor product alcohol can be back to fermentation 30, and the stream 122 comprising the extraction agent of by-pass product alcohol can be imported into separation 130.In certain embodiments, extractor 120 can run in the mode making stream 122 comprise MIN cell mass and MIN substrate.Be separated 130 and can damage microorganism 32 or substrate, cause the reduction of fermentation rate.Run extractor 120 with MIN cell mass and MIN substrate can make to minimize by being separated the 130 any possible infringements caused.The stream 125 comprising poor extraction agent can be back to extractor 120.The stream 135 carrying out self-separation 130 can be imported into purifying 150, for further processing, comprises recovery product alcohol.In certain embodiments, extraction agent can be added into fermentation 30.In certain embodiments, the part comprising the fermented liquid of extraction agent can be transferred to extractor 120, and in certain embodiments, can reclaim extraction agent from the fermented liquid comprising extraction agent.In certain embodiments, fermented liquid and extraction agent can be changed to the flow rate of extractor, are separated to improve.Such as, in the early stage of fermentation or late period, the lower overall flow rate entering extractor can improve being separated of fermented liquid and extraction agent.

As described herein, after batch process, or as the stable effluent stream in continuous fermentation process, the stream 103 comprising beer can be imported into the separation 140 in downstream, to heat up in a steamer thing 142 separated product alcohol from Quan Ta.Employing upstream solid removing method can reduce the undissolved solids content in rare mash, thus centrifugal full tower can not be needed to heat up in a steamer thing 142 to remove solid.Therefore, full tower heats up in a steamer thing 142 and can be imported directly evaporation 160.The syrup 165 generated by evaporation 160 can mix with the wet cake 24,74,74 ' in moisture eliminator 170, to form DDGS.

In certain embodiments, comprise the adverse current of heating up in a steamer the total suspended solid (TSS) of thing from full tower can be used (or recirculation), for the preparation of raw slurry.In certain embodiments, full tower heats up in a steamer thing or full tower, and to heat up in a steamer the part of thing processed by solid separation system (including but not limited to that the turbine before evaporation filters (turbo filtration) or ultracentrifugation), or full tower heats up in a steamer the part that thing or full tower heat up in a steamer thing and can be processed for the purifying of itself clean water.

In some embodiments that grain solid is removed by the wine with dregs from liquefaction, the full tower produced heats up in a steamer thing can comprise trickle solid and undissolved microbial chips, and turbine can be used to filter the solid removing these dispersions.Turbine filters to comprise and makes feed suspension stand centrifugal motion, by retaining the strainer of fine solid.These fine solid can comprise when forming wet cake be attracted to trickle cereal-granules surface and hole in some extraction agents.In some cases, wash wet cake with water to be not enough to reclaim extraction agent from described wet cake.In certain embodiments, concentrated product alcohol stream reclaims extraction agent as organic phase can be used to heat up in a steamer the thing cake that wets from Quan Ta.In certain embodiments, this organic phase can be formed in decantor.In certain embodiments, use the washed wet cake of product alcohol can subsequently by water washing, to reclaim product alcohol from described wet cake.

In certain embodiments, technique as herein described and system can comprise extraction agent reservoir (or tank or container).Extraction agent can be added into extraction agent reservoir, and said extracted agent can be circulated to extractor.In certain embodiments, extraction agent can be imported into extractor, and can be back to extraction agent reservoir from the stream of described extractor.In certain embodiments, the extraction agent from extraction agent reservoir can be circulated to extractor and/or fermentor tank.In certain embodiments, extraction agent stream can at extraction agent reservoir, be recycled between extractor and fermentor tank.In certain embodiments, when fermentation completes, the content of extraction agent reservoir, extractor and/or fermentor tank can be further processed, to reclaim product alcohol.

Use methods known in the art, include but not limited to realize the division mutually etc. that siphon, decantation, centrifugal, gravity settling, film are auxiliary being separated from extraction agent or extracting product alcohol.In certain embodiments, extraction can use such as mixer-settler to carry out.Mixer-settler is the extractor of column plate type, and has the multiple element for mixing, such as pump, agitator, static mixer, mixing tee, jet apparatus, circulation screen cloth or rain bucket.The example of mixer-settler is presented in Figure 10 A-10H.Such as, Figure 10 A shows the mixer-settler using pump as mixed source.Figure 10 B shows the mixer-settler using mixing tank as mixed source.Figure 10 C shows the mixer-settler using static mixer as mixed source.Figure 10 D shows the mixer-settler using mixing tee as mixed source.Figure 10 E shows the mixer-settler using jet mixer as mixed source.Figure 10 F shows the mixer-settler using rain bucket or mesh screen as mixed source.Figure 10 G shows the mixer-settler using whizzer as settling vessel.Figure 10 H shows the mixer-settler using hydrocyclone or vortex separator as settling vessel.In certain embodiments, one or more mixing device can be used in technique as described herein and system.

In certain embodiments, mixing tank can comprise agitator, such as, and flat blade, tiltedly leaf turbine or bending water screw.The drop size produced by stirring mixer can be controlled by agitator design, tank size, agitator speed and operational mode.With regard to static mixer, drop size can control by the diameter of mixing tank and flow rate.Such as, can by change by fermentation process by the flow control drop size of mixing tank.In certain embodiments, gas and mixing tank can be used to mix object.

In certain embodiments, one or more mixer-settler can be used in technique as described herein and system.In certain embodiments, described one or more mixer-settler can be connected or be arranged, as shown in Figure 10 I and 10J with counter-flow pattern.In certain embodiments, mixer-settler can be stacked in pillar decoration form, provides multiple mixing and settling zone.In certain embodiments, settling vessel can comprise hydrophilic or hydrophobic surface, to promote to be separated.

In another embodiment, column extractor or centrifugal extractor can be used in technique as described herein and system.Column extractor is to provide the difference extractor of the condition for mass transfer of the length along them, has the concentration distribution of steady change.Dissimilar difference extractor can be classified as non-mechanical, pulse is stirred with Stirring.Centrifugal extractor is another kind of difference extractor, centrifuge contactor is the one of this type.

In certain embodiments, non-mechanical spray tower can be used in technique as herein described and system.An example of non-mechanical spray tower comprises the non-mechanical spray tower without pillar internals.Multiple nozzle and nozzle diameter can be used to determine drop size.In certain embodiments, described spray tower can have internals.In certain embodiments, spray tower can comprise spirrillum pipeline.Spirrillum pipeline can allow additional the mixing of the rising of drop and fermented liquid and extraction agent.In certain embodiments, the non-mechanical extractor of such as filling type tower, sieve tray and baffle pen can be used in technique as herein described and system.The example of these extractors is shown in Figure 10 K.In certain embodiments, the filling mode of this type of extractor can be random or structurized.

In certain embodiments, the extractor that pulse is stirred can be used in technique as described herein and system.The extractor that pulse is stirred has different designs, and comprises reciprocal tower tray or oscillating plate, and described tower tray moves in a vertical manner wherein.Whole filling type and/or sieve-tray tower tray-type column also can vibrate in a vertical manner, to promote less dispersed phase drop and more mass transfer.The example of these extractors is shown in Figure 10 L.In certain embodiments, Stirring or rotating disc contactor can be used in technique as described herein and system.The example of these extractors is shown in Figure 10 M.

In certain embodiments, stir extractor can be used in technique as described herein and system.Such as, the stirring extractor with whizzer can provide high quality transfering rate and clearly be separated.In certain embodiments, the tower that band stirs can be used in technique as described herein and system.Such as, the tower that the band with internals stirs can provide high quality transfering rate.

An aspect of liquid-liquid extracting method is the successful operation condition for extractor of the fermenting process determined through constantly changing.Such as, typical corn adopts the fermented liquid initial kind of bacterium of microorganism (or cell mass) being added into certain volume in fermentor tank to product alcohol batch fermentation, then further fermentor tank is filled to the volume of specifying.Fermentation is allowed to carry out, until the fermentable carbon source of predetermined amount (such as, sugar) is consumed.Through the process of batch fermentation, the concentration of cell mass, reaction intermediate, byproduct of reaction and substrate component changes in time, and the physical property comprising the fermented liquid of viscosity, density and surface tension is as the same.In order to improve the performance perameter of fermentation, such as, the yield parameter of speed, titre and production, and the equipment economy of such as sales volume, investment repayment and profit, extractor can run in a variable manner, to compensate the change of fermented liquid.In addition, the characteristic of dynamic fermentation can affect the limitation of size of extractor.By the mathematical model of operation, to the correct integration of the operation of extractor and fermentor tank can be useful (see, such as, Daugulis and Kollerup, Biotechnology and Bioengineering 27:1345-1356,1985).Expand above-mentioned mathematical model, such as, arranging key model parameter by experimental data, is also valuable.Consider for the yield improving speed, titre and fermenting process, comprise the maximum total flux to extractor of the cross-sectional area by extractor tower for difference extractor design variable, and with given fermented liquid, extractor height needed for enough product alcohol is removed to extraction agent ratio.In batch fermentation process, change per unit area peak flow and extractor height may be necessary.Another consideration for difference extractor is the drop size of disperse phase.Suitable drop size can be little of enough providing sufficient mass transfer but the large balance to enough allowing to leave between being clearly separated of extractor.In column plate type extractor, the corresponding time needed for the mixture strength needed for effective mass transfer, sedimentation and/or the energy needed for separation are mutually the other factors that will consider.In column plate type or differentiated two kinds any one type extractor in, the fermented liquid being fed to extractor works in the determination of extractor size to the ratio of extraction agent.

In certain embodiments, if employ the extractor of fixed measure, and due to the physical property of fermented liquid and the change of concentration, process by fermentation, avoid can changing from low value to high level (such as, from 1/3 to 2/3 of the maximum value with regard to the design of given extractor) by permissible flow to the maximum of overflow of extractor, then the flow to extractor can change, be no more than peak flow, still within the rational time, complete fermentation simultaneously.In certain embodiments, if extractor is stirred, the speed of stirring can along with the change in process of fermentation, to compensate the change of fermented liquid.Drop size can be measured in fermentor tank, and at the controllable speed all the time fermented to maintain fixing drop size, to compensate the change of fermented liquid.By measure the concentration of product alcohol in entrance and exit stream and adjusting condition (such as, flow, throughput ratio, stirring) with the process control mass transfer along with fermentation, the amount of the mass transfer putting generation at any time can be assessed.

In certain embodiments, multiple extractors of different size can be used, and regulate the condition (such as, flow, throughput ratio, stirring) in each extractor, to provide the control of the improvement to fermenting process.In certain embodiments, fermented liquid can be modified the ratio of extraction agent, improving extraction efficiency, improves the flow by extractor needed for the concentration (being equivalent to raise the efficiency) of product alcohol in extraction agent and reduction.

In the other embodiment of technique as herein described and system, two or more fermented liquids or aqueous stream can be had.The extraction agent having absorbed product alcohol from the first aqueous stream can be made to follow the second aqueous stream comprising less product alcohol compared with the first aqueous stream or fermented liquid to contact mutually, make product alcohol can from the phase transition to the second of rich extraction agent containing aqueous phase.In certain embodiments, the contact of rich extraction agent and rare aqueous stream can occur in stage-wise contactless or in the static mixer of following by settling vessel.In certain embodiments, occur in the same device that the contact of rich extraction agent and rare aqueous stream can contact with fermented liquid at poor extraction agent.The extractor with perforated retainer will allow fermented liquid and rare aqueous stream to dirty in independently compartment, and the extraction agent of poor product alcohol can form the external phase throughout all compartments simultaneously.An advantage of this configuration is, if extraction agent keeps being limited in the closed volume of extractor, will need the extraction agent of the amount reduced.Another advantage of this configuration be extraction agent without undergoing deterioration possible in still-process, longer work-ing life can be shown.By product alcohol is transferred to uniform aqueous stream, consider tower capacity and heat integration, by product alcohol being imported more than one stripping tower to the distribution of rare aqueous stream.When in the process from broth extraction product alcohol or after immediately product alcohol is extracted in water-bearing media, the clean demand being exposed to the equipment of extraction agent can be reduced.

In certain embodiments, the barrier of the transmitting selective for product alcohol can be crossed over, product alcohol is transferred to the second aqueous stream or extraction agent from fermented liquid.In certain embodiments, above-mentioned barrier can be provided by mould material.Mould material can be organic or inorganic.The example of mould material comprises polymkeric substance and pottery.In certain embodiments, hydrogel can be used from separation of fermentative broth product alcohol.In certain embodiments, hydrogel can comprise the interactional functional imperative promoted with product alcohol, such as but not limited to the character, network size etc. of hydroxy functionality, hydrocarbon.In certain embodiments, hydrogel can have polymer network structure or polymer formulations.The example of polymer formulations include but not limited to following in one or more: vinylformic acid, sodium acrylate, hydroxy ethyl methacrylate, methacrylic ester, hydroxyl butyl propyleneglycol acid esters, butyl propyleneglycol acid esters, vinylation polyethylene oxide, vinylation poly(propylene oxide), vinylation polycyclic butane oxidation thing (vinylated polytetratmethylene oxide), the acrylate of polyoxyethylene glycol and diacrylate, the polyvinyl alcohol of polyvinyl alcohol and hydrocarbon derivatize, and vinylbenzene and styrene derivatives.In certain embodiments, hydrogel can comprise hydroxy ethyl methacrylate and methacrylic ester, hydroxyl butyl propyleneglycol acid esters and methacrylic ester or butyl propyleneglycol acid esters and methacrylic ester.

In other embodiment of technique as herein described and system, fermented liquid can be removed higher than normal atmosphere from the bottom of fermentor tank, and make it pass through the first flash drum run with normal atmosphere, to discharge dissolved gases as CO ₂.Above-mentioned first flash drum can be degassed cyclonic separator, and can merge with the steam from fermentor tank from the steam of the first flash drum, and is imported into scrubber.In certain embodiments, fermented liquid from the first flash drum can be made by lower than the second flash drum of running of normal atmosphere ground, to discharge more such as CO ₂dissolved gases.Above-mentioned second flash drum can be degassed cyclonic separator, and is merging with the steam from fermentor tank from the steam of the second flash drum and can be compressed to normal atmosphere, cooling partly concentrating before being imported into scrubber.The fermented liquid leaving the second flash drum can be pumped to higher than the extraction column that runs of normal atmosphere ground, make any remnants' or the formation that will not cause vapor phase in extraction column of the new dissolved gases formed.

In another embodiment of technique as herein described and system, fermented liquid can be imported into extractor and contact with extraction agent, generates aqueous stream and comprises the organic stream of extraction agent and product alcohol.Above-mentioned organic stream can be imported into flash drum (such as, vacuum flash), for from extraction agent separated product alcohol.In certain embodiments, the extraction agent stream from flash drum can be recirculated to extractor and/or fermentor tank.In certain embodiments, organic stream can be imported into the second extractor before flash drum.Second extractor can be used to remove, such as, and the water of any remnants in organic stream.Extractor can be siphon pipe, decantor, whizzer, gravitational settler, mixer-settler or their combination.In certain embodiments, extraction agent can be oil, such as but not limited to tallow oil, Semen Maydis oil, canola oil, capric acid/sad Witepsol W-S 55, Viscotrol C, Oleum Cocois, Oleum Gossypii semen, fish oil, Jojoba oil, lard, linseed oil, neat's-foot oil, oiticica oil, plam oil, peanut oil, rapeseed oil, rice oil, Thistle oil, soya-bean oil, sunflower seed oil, tung oil, curcas oil and vegetable oil mixt, or derive from its lipid acid.

In some embodiments of technique as herein described and system, self acting self-cleaning filters and can be used in these techniques and system.Fermented liquid can be removed from fermentor tank, and make it cool with water cooler (such as, existing water cooler in fermentative production facility) before entering self acting self-cleaning strainer.When the wine with dregs clarified is through strainer, some particles can be retained in the screen media of strainer.Other strainer can experience backwash simultaneously, and a part for the wine with dregs wherein clarified is carried particle and flowed counterflow through screen cloth with it, the efflux of solids that discharge is concentrated.In certain embodiments, a part for the wine with dregs of clarification can enter the top of extractor, and extraction agent is fed to the bottom of extractor simultaneously.Clarification wine with dregs and extraction agent by density variation passively or by the normally used method in this area under the help of mechanical movement (such as, tower) contact.In certain embodiments, the organic liquid stream comprising the extraction agent of product alcohol occurs from the top of extractor, and has exhausted that the aqueous liquid stream of the fermented liquid of product alcohol occurs from the bottom of extractor at least in part relative to the wine with dregs of clarification.Aqueous liquid stream and concentrated efflux of solids can be merged, and are back to fermentor tank.The extraction agent stream of by-pass product alcohol can be heated in interchanger, interchanger poor product alcohol extraction agent stream and can between the extraction agent stream and the extraction agent stream deriving from extractor bottoms of poor product alcohol transmit heat.After some heat of release, poor extraction agent can cool further with water in interchanger, to reach the temperature being suitable for fermenting.The circulation of fermented liquid can comprise the approach by heat-transfer arrangement and quality transfer device, makes it possible to every one way and removes heat and product alcohol through external cooling circuit.In addition, in certain embodiments, by regulating the flow by the circular flow of external cooling circuit, the flow regulating cooling fluid in interchanger and/or adjustment extraction agent, make the removal speed of heat and product alcohol between yeast phase and the generation rate equation of heat and product alcohol.

In some embodiments of technique as herein described and system, temperature and/or pH by changing described process promote being separated of extraction agent from fermented liquid.Such as, described process can be run with the temperature of the temperature and/or pH that are different from fermentor tank and/or pH.In certain embodiments, described process can be run with the pH reduced compared to fermentor tank.In certain embodiments, described process can be run with the temperature higher compared to fermentor tank.In certain embodiments, described process can be run with the pH reduced compared to fermentor tank and higher temperature.Higher temperature can improve product alcohol in the kinetics containing mass transfer between aqueous phase and organic phase, and can improve and be dispersed in containing the coalescent of the extraction agent drop in aqueous phase and the coalescent kinetics being dispersed in the water-containing drop in organic phase.In certain embodiments, entered fermented liquid and/or the extraction agent of extractor by heating, the temperature of the extractor inside comprising fermented liquid and extraction agent can be improved.Directly or by interchanger indirectly fermented liquid is heated by injection water vapour or steam.In certain embodiments, the extraction agent being fed to extractor can derive from distillation, and its temperature may be raised in distillation.In certain embodiments, extraction agent can be cooled to the temperature higher than leavening temperature.

In certain embodiments, the pH of reduction can make the solubleness of extraction agent in moisture fermentation liquid phase and dispersiveness minimize.In certain embodiments, extraction agent can be the lipid acid with known relevant pKa value.In certain embodiments, the pH of fermented liquid can be reduced to the pKa lower than extraction agent, makes the hydroxy-acid group of lipid acid be protonated in fact.In certain embodiments, by by CO ₂gas is introduced in fermented liquid, or by the liquid acid of a small amount of such as sulfuric acid or other organic or inorganic acid any is injected fermented liquid, can reduce pH.In certain embodiments, be separated with extraction agent after the pH of fermented liquid can be adjusted to the pH of fermentation.

In some embodiments of external phase mutually at extraction agent, containing aqueous phase can distribute or be dispersed in extraction agent mutually in.Such as, the fermented liquid comprising product alcohol is imported into extractor (such as, outside extractor) by divider or diverting device.In certain embodiments, described divider or diverting device can be the nozzles of such as fog nozzle.In certain embodiments, described divider or diverting device can be spray towers.Such as, the drop of fermented liquid can be made by extraction agent, and product alcohol is transferred to extraction agent.The drop of fermented liquid is coalescent in the bottom of extractor, and can be returned to fermentor tank.The extraction agent comprising product alcohol can be further processed, to reclaim product alcohol, as described herein.In addition, when fermentation completes, product alcohol remaining in fermentor tank also can be further processed, to reclaim product alcohol.In certain embodiments, extraction agent can be adverse current mutually.

Extraction agent be external phase mutually and containing aqueous phase be in some embodiments of disperse phase, by use electrostatic spraying make containing aqueous phase be dispersed in extraction agent mutually in, can quality transfering rate be improved.In certain embodiments, one or more fog nozzle can be used to electrostatic spraying.In certain embodiments, one or more fog nozzle can be anode.In certain embodiments, one or more fog nozzle can be negative electrode.

In certain embodiments, extractor effluent can be used to promotion and is separated.Such as, the part from the rich extraction agent (that is, the extraction agent of by-pass product alcohol) at the top of extractor can be used as the top that backflow is back to extractor, and the rich extraction agent of remnants can be further processed, to reclaim product alcohol.In addition, the part from the poor fermented liquid of the bottom of extractor can be used as the bottom that backflow is returned to extractor, and the poor fermented liquid of remnants can be returned to fermentor tank.In another embodiment, rich extraction agent can leave the top of extractor, enters decantor, and is separated into heavy phase and light phase.Heavy phase from decantor can be imported into the top of extractor, to promote to be separated.Light phase from decantor can be further processed, to reclaim product alcohol.

In some embodiments of technique as herein described and system, multichannel extraction agent stream can be used to the recovery of product alcohol.Such as, can use multiple fermentor tank and extractor, the fermentation cycle of each fermentor tank is at different time points.See Figure 11 A as an example, fermentor tank 300 is in time point comparatively early compared to fermentor tank 400, and the latter is in time point comparatively early relative to fermentor tank 500.The fermented liquid comprising product alcohol 302 from fermentor tank 300 can contact with extraction agent 307 in extractor 305, and product alcohol can be transferred to extraction agent, generates the extraction agent 309 of by-pass product alcohol.Extraction agent 309 from the by-pass product alcohol of extractor 305 can be imported into extractor 405.The fermented liquid comprising product alcohol 402 from fermentor tank 400 can be imported into extractor 405, produces the extraction agent 409 of by-pass product alcohol.The extraction agent 409 of by-pass product alcohol can be imported into extractor 505.The fermented liquid comprising product alcohol 502 from fermentor tank 500 can be imported into extractor 505.Extraction agent 509 from the by-pass product alcohol of extractor 505 can be processed, to reclaim product alcohol.The fermented liquid 304,404,504 of poor product alcohol can be returned to fermentor tank 300,400 and 500 respectively.The quantity of fermentor tank and extractor can change according to exercisable facility.The benefit of this method is, such as, and the minimizing of total extraction agent process and the size of extractor.

In another embodiment of above-mentioned example, other fermentor tank F ' and other extractor E ' (Figure 11 B) can be there is.In this embodiment, when fermentor tank 500 (it is in more late time point compared to fermentor tank 300 and 400) has completed fermentation, fermentor tank 500 off-line can be made, and fermentor tank 500 in certain embodiments, can be made to experience health and/or the sterilizing process of such as situ cleaning (CIP) and disinfect in situ (SIP) process.When making fermentor tank 500 off-line, fermentor tank F ' can be made to reach the standard grade.In this embodiment, fermentor tank F ' is in time point comparatively early compared to fermentor tank 300, and the latter is in time point comparatively early relative to fermentor tank 400.Be similar to the description to Figure 11 A, the fermented liquid comprising product alcohol F '-02 from fermentor tank F ' can be made to contact with extraction agent in extractor E ', and product alcohol can be transferred to extraction agent, generate the extraction agent E '-09 of by-pass product alcohol.Extraction agent E '-09 from the by-pass product alcohol of extractor E ' can be imported into extractor 305.The fermented liquid comprising product alcohol 302 from fermentor tank 300 can be imported into extractor 305, produces the extraction agent 309 of by-pass product alcohol.The extraction agent 309 of by-pass product alcohol can be imported into extractor 405.The fermented liquid comprising product alcohol 402 from fermentor tank 400 can be imported into extractor 405.Extraction agent 409 from the by-pass product alcohol of extractor 405 can be processed, to reclaim product alcohol.The fermented liquid (F '-04,304,404) of poor product alcohol can be returned to fermentor tank F ', 300 and 400 respectively.In certain embodiments, said process can be repeated multiple circulation, such as, at least one, at least two, at least three, at least four, at least five, at least ten, at least ten five, at least two ten, or more circulation.In certain embodiments, fermentor tank off-line and the process that other fermentor tank is reached the standard grade is made can be manual or automatic.The benefit of this method is reduced to the extractor flow that product reclaims (such as, distilling).

In certain embodiments, extraction agent can reduce the flash-point (that is, inflammableness) of product alcohol.Flash-point refers to the minimum temperature that the propagation of flame of crossing over fluid surface occurs.Flash-point can be measured, such as, uses ASTM D93-02 method (" standard method by Pensky-Martens closed tester test flash-point ").The reduction of the flash-point of product alcohol can improve the safety case of alcohol production facility, such as, minimizes by making the fire hazard of possible inflammable product alcohol.By improving safety case, the risk of the risk damaged and property damage and revenue losses is minimized.In some embodiments needing inactivating microbial, extraction agent can improve the deactivation of microorganism.

In certain embodiments, technique as herein described can be have employed in the concentration of such as fermented liquid and extraction agent and the line of other physical property, in line, line comprehensive extraction zymotechnique that is other and/or that measure in real time.These are measured and can be used for such as feedback loop with the condition of regulation and control fermentation condition and/or extractor.In certain embodiments, the concentration of the product alcohol in fermented liquid and/or other metabolite and substrate can use that any suitable measuring apparatus carries out on line, in line, line is other and/or measure in real time.In certain embodiments, measuring apparatus can be following in one or more: Fourier transform infrared spectroscopy (FTIR), near infrared spectrum (NIR), Raman spectrum, high pressure liquid chromatography (HPLC), viscometer, specific gravity hydrometer, tonometer, drop size analyser, pH meter, dissolved oxygen (DO) probe etc.In certain embodiments, the exhaust gas emission of fermentor tank such as can be analyzed by on-line mass spectroscopy instrument.The exhaust gas emission of measuring fermentor tank can be used as identifying the method for the material be present in fermentation reaction.Use the techniques described herein and equipment, also can measure the concentration being dissolved in product alcohol in extraction agent and other metabolite and substrate.

In certain embodiments, the input of measuring can be sent to controller and/or Controlling System, and the condition (temperature in fermentor tank, pH, nutritive substance, enzyme and/or concentration of substrate) can be changed, to maintain concentration, concentration distribution and/or the condition (fermented liquid flow, fermented liquid are to extraction agent flow, stir speed (S.S.), drop size, temperature, dissolved oxygen (DO) content) in extractor.Similarly, the condition in extractor can be changed, to maintain concentration in fermentor tank and/or concentration distribution.By utilizing this Controlling System, can by this way keep-process parameter to improve integral production ability and the economic goal of factory.In certain embodiments, by change the component (such as, sugar, enzyme, nutritive substance etc.) in fermentor tank concentration, change condition in extractor or both, the real-time control to fermenting can be realized.

As the example of isobutanol fermentation technique, the efficiency that in tower, isopropylcarbinol extracts continues change along with the change of starch, sugar and isobutanol concentration in fermented liquid.In order to make the maximizing efficiency of extractor, maybe advantageously change the speed removing isopropylcarbinol from fermented liquid, to mate the productive frontiers of isobutanol fermentation.Isobutanol concentration in extraction agent can be maximized, and causes more energy-conservation distillation procedure.

As a part for technology controlling and process strategy, can with the real-time measuring coupling to the isopropylcarbinol in extraction agent and poor fermented liquid to the real-time measurement of isopropylcarbinol in fermented liquid (such as, the charging of tower).These measurements can be used to be adjusted to the fermented liquid of extractor to extraction agent ratio (flow).The handiness that the speed that the speed that isopropylcarbinol is extracted and isopropylcarbinol generate matches can make the whole process that extractor is extracting effectively run.In addition, by maintaining the high density of isopropylcarbinol in extraction agent, can make to minimize to the volumetric flow rate of distillation tower, causing the energy saving of distillation procedure.Be separated also can use and measure monitoring in real time, such as, by monitoring the composition of the speed, extraction agent drop size and/or the fermented liquid that are separated.In certain embodiments, be separated by conductivity measurement, dielectric medium measurement, viscoelasticity measurement and or ultrasonic measurement monitored.In certain embodiments, the detection system that is separated of automatization can be used to monitoring and is separated.Above-mentioned automation system can be used to regulate and to flow to or from the fermented liquid of extractor and the flow rate of extraction agent and/or the drop size regulating extraction agent, such as, after mixed fermentation liquid and extraction agent.By using these real-time monitoring systems, being clearly separated containing aqueous phase and organic phase can be realized.

As another example that technology controlling and process is measured, sreen analysis can be utilized to measure drop size, such as course grain analyser (JM Canty, Inc., Buffalo, NY), Focused Beam Reflectance measuring technology or particulate is observed and is measured technology (Mettler-Toledo, LLC, Columbus OH).In certain embodiments, these measurements can be that real-time in-situ microparticulate systems characterizes.By Real-Time Monitoring drop size, the change of droplet profile and size can be detected, and adjustable processing step is to change the speed of drop size and raising mass transfer.Such as, drop size can be used to the amount of the extraction agent of monitoring in fermented liquid.After phase separation, some extraction agents may reside in fermented liquid, and are recirculated in some embodiments of fermentor tank at fermented liquid, and monitoring drop size makes the minimized method of the amount of the extraction agent be back in the fermented liquid of fermentor tank by providing.If the amount of the extraction agent in fermented liquid is too high, then can improves and be separated, such as, by regulating the drop size of the extraction agent in extractor and/or being adjusted to the fermented liquid of extractor and the flow rate of extraction agent.These adjustment in processing step can make the amount of extraction agent in fermented liquid minimize, and the amount making rare tower heat up in a steamer extraction agent in thing and DDGS minimizes.

In an embodiment of above-mentioned control strategy, the concentration that the isopropylcarbinol in fermented liquid can not exceed isopropylcarbinol becomes concentration to harmful microorganism or set(ting)value.Along with the fermentation based on fermentation path is carried out, isobutanol fermentation liquid set(ting)value can be adjusted to higher or lower.Such as, in fermented liquid the concentration of isopropylcarbinol and the set(ting)value of isobutanol concentration compare the flow rate that can be used to change the ratio of fermented liquid to extraction agent or the fermented liquid to extractor and extraction agent continuously.In order to monitor the isobutanol concentration in fermented liquid, the in site measurement that Fourier transform infrared spectroscopy (FTIR), near infrared spectrum (NIR) and/or Raman spectrum carry out fermented liquid can be used.In addition, the measurement that FTIR, Raman spectrum and/or mass spectrum carry out fermentor tank headspace can be used.

In certain embodiments, effective extraction agent runs and can carry out in the degree close to extractor overflow.The use utilizing the Real-time process from the concentration data of entrance and exit stream to control, can make extractor reliably to run in the degree close to overflow.In certain embodiments, extract real-time agent monitoring can be used to detection from the distribution of the by product of fermented liquid or the pollutent entering extraction agent.The by product of such as alcohol, lipid, oil and other fermentation component can reduce the extraction efficiency of extraction agent.Kinds of processes Monitoring techniques can be applied to above-mentioned measurement, includes but not limited to Fourier transform infrared spectroscopy (FTIR), near infrared spectrum (NIR), high performance liquid chromatography (HPLC) and nucleus magnetic resonance (NMR).Be selected for the analytical technology of the existence monitoring extraction agent for by product or pollutent, can be and the different technology being used to the real time measure alcohol.Real time data can be used to trigger to pollute extraction agent reparation or to pollute extraction agent from the removing technique.These technology and gas-chromatography (GC) and supercritical fluid chromatography (SFC) also can be used to the heat collapse of monitoring extraction agent.

See Figure 12, technique of the present invention and system can comprise in line, line, line other and/or the device (circle represents measuring apparatus and dotted line represents feedback loop) of measurement in real time.Figure 12 is similar to Fig. 9, unlike with the addition of in line, line, line measuring apparatus that is other and/or that measure in real time, therefore will no longer describe in detail.

Such as, to the line of aqueous stream 22 being measured the concentration of oxygen that can be used to monitor fermentable carbon source (such as, polysaccharide), oil and/or dissolve.Such as, FTIR can be used to monitor the dispersion of oil in aqueous stream 22, and process imaging can be used to monitor concentration and size that the fluid in aqueous stream 22 drips.In certain embodiments, ferment 30 line on measure can be used to monitor product alcohol remove speed.What the measurement of the oxygen of fermentable carbon source, dissolving, product alcohol and by product can be used to regulate product alcohol removes speed, to maintain the product alcohol concentration in fermentation 30 that microorganism can tolerate.By maintaining the set(ting)value of product alcohol concentration, the retarding effect of product and toxicity can be minimized.

The line of convection current 105 and stream 122 is measured and can be used to operating procedure control feedback loop.Such as, the concentration flowing product alcohol in 105 can be used to control the flow rate that this flow to extractor 120; And the concentration flowing product alcohol in 122 can be used to control this flow rate flowing to separation 130 and arrange the ratio of fermented liquid to extraction agent.In addition, the line of convection current 105 and stream 122 is measured and also can be used to set up real-time product alcohol mass balance.The quality be separated of monitoring extraction agent and fermented liquid can be used to for extractor 120 and the Process control feedback loop being separated 130.Such as, on line, measuring apparatus can be used to the balance be separated of Detection and Extraction agent and fermented liquid, and correspondingly can regulate the feeding rate of extraction agent and fermented liquid, is separated to improve.On the line of such as Optical devices, device can be used to detect heterogeneous interlayer (such as, the mixture of oil, the aqueous solution and solid) existence in such as extractor 120, and adjustable fermented liquid minimizes to make the formation of heterogeneous interlayer the ratio of extraction agent.To come self-separation 130 stream 135 line on measure and can be used to monitoring fermented liquid existence in this stream, and the existence of fermented liquid in stream 135 can indicate not good enough being separated.If the concentration of fermented liquid exceedes a certain set(ting)value in stream 135, then can the change of implementing process, the adjustment of such as flow rate or to the adjustment of fermented liquid to the ratio of extraction agent, is separated to improve.In addition, the concentration flowing product alcohol in 135 can be used as Process control feedback loop, to guarantee effective operation of separation 130.

And for example, in convection current 127 product alcohol concentration line on measure the product alcohol concentration that can be used to monitor extraction efficiency and maintain in the microorganism fermentation 30 that can tolerate.In addition, monitoring stream 127 can be there is, as making the minimized method of the amount of the extraction agent returning fermentation 30 for extraction agent.Such as, spectroscopy and process imaging technique can be used to the existence of extraction agent in monitoring stream 127.In addition, certain density extraction agent in stream 127 can be maintained, to improve extraction efficiency and to be separated.

In another embodiment, the stream 135 carrying out self-separation 130 can be imported into purifying 150 for further processing, comprises and reclaims product alcohol and extraction agent 152.Extraction agent 152 can be imported into extractor 120.Line is measured and can be used to for pollutent and degraded product monitoring stream 152.By monitoring stream 152, the possibility of the pollution of extractor 120 and fermentation 30 is minimized.If there is the rising of pollutent in stream 152, this stream can be further processed, such as, to remove these pollutents, by absorption or chemical reaction.

In leaching process, heterogeneous interlayer can be formed in the interface containing aqueous phase and organic phase, and the heterogeneous interlayer be made up of solid and extraction agent (such as, the drop of extraction agent) can gather and may hinder and is separated.In order to reduce the formation of heterogeneous interlayer, can adopt the stirring containing aqueous phase and organic phase.Such as, impeller can be used to the heterogeneous interlayer disperseing moisture-organic interface place.In addition, fluid flowing such as recirculation loop or vibrations/vibration can be used to the formation destroying heterogeneous interlayer.Figure 13 A and 13B shows the illustrative processes of the formation for reducing heterogeneous interlayer.Figure 13 A exemplified with static mixer in conjunction with the agitating unit in settling vessel or decantor downstream for the treatment of the use of heterogeneous interlayer, and Figure 13 B exemplified with static mixer in conjunction with the agitating unit of settling vessel or decantor upstream for the treatment of the use of heterogeneous interlayer.In certain embodiments, other device of such as coalescer or sonic agitation can be used to disperse heterogeneous interlayer.In certain embodiments, these devices can be integrated into settling vessel or decantor.

Technique as herein described and system can use in batches, batch feeding or operation of continuously fermenting.Batch fermentation is closed system, and wherein the composition of fermented liquid is determined when fermenting and starting, and during the fermentation without undergoing manual change.In some embodiments of batch fermentation, extraction agent can be added into fermentor tank.In certain embodiments, the volume of extraction agent can be about 20% of fermentor tank working volume to about 60%.

Fed-batch fermentation is the modification of batch fermentation, and wherein bottom (fermentable sugars) is added with incremental mode during the fermentation.When catabolite repression can suppress the metabolism of microorganism and have limited amount substrate in expectation substratum, batch feeding system is useful.In certain embodiments, the concentration of substrate and/or nutritive substance can be monitored during the fermentation.In certain embodiments, the oxygen of such as pH, dissolving and gas (such as, CO ₂) parameter can be monitored during the fermentation.Speed or the amount of the interpolation of substrate and/or nutritive substance can be determined from these measurements.In certain embodiments, along with the level of fermented liquid or amount reduce during the fermentation, other wine with dregs can be added into fermentor tank, to maintain level and the amount of fermented liquid, such as, maintains the level of fermented liquid or amount at the initial level of fermenting process.In some embodiments of fed-batch fermentation, extraction agent can be added into fermentor tank.

Continuously fermenting is open system, and wherein fermented liquid is added into fermentor tank continuously, and a certain amount of fermented liquid is removed, for processing further (such as, reclaiming product alcohol).In certain embodiments, fermented liquid interpolation and to remove can be simultaneously.In certain embodiments, can add from fermentor tank and remove the fermented liquid of equivalent.In some embodiments of continuously fermenting, extraction agent can be added into fermentor tank.In certain embodiments, the volume of extraction agent can be about 3% of fermentor tank working volume to about 50%.In certain embodiments, the volume of extraction agent can be about 3% of fermentor tank working volume to about 20%.In certain embodiments, the volume of extraction agent can be about 3% of fermentor tank working volume to about 10%.

In some embodiments of technique as herein described and system, air lift can be used to remove product alcohol from fermented liquid.By providing one or more gases of such as air, nitrogen or carbonic acid gas to carry out air lift to fermented liquid, thus form the gas phase comprising product alcohol.Such as, air lift is carried out by jet flow stream by fermented liquid.In certain embodiments, gas can be provided by fermentation reaction.Such as, carbonic acid gas can be used as fermentable carbon source and is provided by the by product of microbial metabolism.In certain embodiments, air lift can be used, to remove product alcohol from fermented liquid with extraction agent simultaneously.Use methods known in the art, such as, use cold water trap make product alcohol condensation or use solvent wash gas phase, product alcohol can be reclaimed from the gas phase comprising product alcohol.

recombinant microorganism and biosynthetic pathway

Not bound by theory, it is believed that technique described herein can with any microorganism that can produce tunning, comprise produce alcohol microorganism, be especially combined to produce the recombinant microorganism of alcohol higher than the titre of its durability level.

The microorganism producing alcohol is known in the art.Such as, methyl alcohol is produced by methane-oxidizing bacteria (such as Methylosimus trichosporium (Methylosinus trichosporium)) fermentation oxidizing of methylene, and yeast strain CEN.PK113-7D (CBS 8340, the Centraal Buro voor Schimmelculture; The people such as van Dijken, Enzyme Microb.Techno.26:706-714,2000) produce ethanol.The recombinant microorganism producing alcohol is also (such as, the people such as Ohta, Appl.Environ.Microbiol.57:893-900,1991 known in the art; The people such as Underwood, Appl.Environ.Microbiol.68:1071-1081,2002; Shen and Liao, Metab.Eng.10:312-320,2008; The people such as Hahnai, Appl.Environ.Microbiol.73:7814-7818,2007; United States Patent (USP) 5,514,583; United States Patent (USP) 5,712,133; PCT public announcement of a patent application WO 1995/028476; The people such as Feldmann, Appl.Microbiol.Biotechnol.38:354-361,1992; The people such as Zhang, Science 267:240-243,1995; U.S. Patent Application Publication 2007/0031918 A1; United States Patent (USP) 7,223,575; United States Patent (USP) 7,741,119; United States Patent (USP) 7,851,188; U.S. Patent Application Publication 2009/0203099 A1; U.S. Patent Application Publication 2009/0246846 A1; With PCT public announcement of a patent application WO 2010/075241, they are all incorporated herein by reference).

In addition, microorganism can use recombinant technology to carry out modifying to generate recombinant microorganism, and it can produce product alcohol such as ethanol and butanols.Can reorganized modification to be comprised the member of following genus by the microorganism of biosynthetic pathway production product alcohol: fusobacterium (Clostridium), zymomonas (Zymomonas), Escherichia (Escherichia), salmonella (Salmonella), serratia (Serratia), erwinia (Erwinia), Klebsiella (Klebsiella), Shigella (Shigella), Rhod (Rhodococcus), Rhodopseudomonas (Pseudomonas), bacillus (Bacillus), lactobacillus (Lactobacillus), enterococcus spp (Enterococcus), Alkaligenes (Alcaligenes), Klebsiella (Klebsiella), series bacillus belongs to (Paenibacillus), genus arthrobacter (Arthrobacter), corynebacterium (Corynebacterium), brevibacterium sp (Brevibacterium), Schizosaccharomyces (Schizosaccharomyces), kluyveromyces spp (Kluyveromyces), Ye Shi yeast belong (Yarrowia), Pichia (Pichia), mycocandida (Candida), Hansenula (Hansenula), Issatchenkia (Issatchenkia) or yeast belong (Saccharomyces).In certain embodiments, recombinant microorganism is optional from intestinal bacteria (Escherichia coli), plant lactobacillus (Lactobacillus plantarum), Kluyveromyces lactis (Kluyveromyces lactis), kluyveromyces marxianus (Kluyveromyces marxianus) and yeast saccharomyces cerevisiae (Saccharomyces cerevisiae).In certain embodiments, recombinant microorganism is yeast.In certain embodiments, recombinant microorganism is the Crabtree positive (crabtree-positive) yeast, some bacterial classifications that it is selected from yeast belong (Saccharomyces), zygosaccharomyces belongs to (Zygosaccharomyces), Schizosaccharomyces (Schizosaccharomyces), moral gram yeast belong (Dekkera), torulopsis (7orulopsis), Brettanomyces belong to (Brettanomyces) and mycocandida (Candida).The bacterial classification of Crabtree positive yeast includes but not limited to yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), kluyveromyces (Saccharomyces kluyveri), schizosaccharomyces pombe (Schizosaccharomyces pombe), saccharomyces bayanus (Saccharomyces bayanus), budding yeast (Saccharomyces mikitae), Saccharomyces paradoxus (Saccharomyces paradoxus), Lu Shi zygosaccharomyces (Zygosaccharomyces rouxii) and Candida glabrata (Candida glabrata).

Yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) is for known in the art and can derive from multiple source, include but not limited to American type culture collection (Rockville, MD), Centraalbureau voor Schimmelcultures (CBS) fungal organism center of diversity, LeSaffre, Gert Strand AB, Ferm Solutions, North American Bioproducts, Martrex and Lallemand.Yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) includes but not limited to BY4741, CEN.PK 113-7D, Ethanol yeast, Ferm Pro ^tMyeast, Bio- xR yeast, Gert Strand Prestige Batch Turbo alcohol yeast, Gert Strand Pot Distillers yeast, Gert Strand Distillers Turbo yeast, FerMax ^tMgreen yeast, FerMax ^tMgold yeast, yeast, BG-1, PE-2, CAT-1, CBS7959, CBS7960 and CBS7961.

In certain embodiments, microorganism can be fixed or encapsulate.Such as, microorganism can use alginate esters, Protanal TXF 200 or polyacrylamide gel to fix or encapsulate, or by inducing biofilm formation to fix on multiple high surface area carrier matrix such as diatomite (diatomite), diatomite (celite), diatomite (diatomaceous earth), silica gel, plastics or resin or encapsulating.In certain embodiments, ISPR can use with microbial association that is fixing or that encapsulate.This combination can improve throughput such as specific volume throughput, metabolic rate, product alcohol yield and product alcohol tolerance.In addition, fixing and encapsulation can make processing condition such as shear to minimize the impact of microorganism.

The microorganism of fermentative production butanols and production butanols is utilized to be disclosed in such as United States Patent (USP) 7,851,188 and U.S. Patent Application Publication 2007/0092957; 2007/0259410; 2007/0292927; 2008/0182308; 2008/0274525; 2009/0155870; 2009/0305363; In 2009/0305370, their each full text is incorporated herein by reference.In certain embodiments, microorganism can be engineered and comprise biosynthetic pathway.In certain embodiments, biosynthetic pathway is the butanol biosynthetic pathway of through engineering approaches.In certain embodiments, conversion of pyruvate is become tunning by biosynthetic pathway.In certain embodiments, biosynthetic pathway is by pyruvic acid and amino acid converting one-tenth tunning.In certain embodiments, the heterologous polynucleotides in microorganism encode at least one, at least two, at least three or at least four polypeptide, in described polypeptide catalyzes approach, substrate is to the conversion of product.In certain embodiments, the heterologous polynucleotides in microorganism is encoded all polypeptide, and in described polypeptide catalyzes approach, substrate is to the conversion of product.In certain embodiments, catalytic substrate is to produce acetylactis to 2, and the polypeptide of the conversion of 3-dihydroxyisovalerate and/or catalytic substrate can utilize nicotinamide adenine dinucleotide reduced (NADH) as cofactor to produce isobutyric aldehyde to the polypeptide of the conversion of isopropylcarbinol.

biosynthetic pathway

Can be comprised by the biosynthetic pathway for the production of isopropylcarbinol used and be described in United States Patent (USP) 7,851, those in 188, this patent is incorporated herein by reference.In one embodiment, isobutanol biosynthetic pathway comprises the conversion of following substrate to product:

A) pyruvic acid is to acetylactis, and it can by such as acetolactate synthase catalysis;

B) acetylactis to 2,3-dihydroxyisovalerate, it can by such as Acetohydroxy acid isomeroreductase catalysis;

C) 2,3-dihydroxyisovalerate are to alpha-ketoisocaproic acid, and it can by such as acetohydroxy acid dehydratase catalysis;

D) alpha-ketoisocaproic acid is to isobutyric aldehyde, and it can by such as branched-chain α-keto acid decarboxylase catalysis; And

E) isobutyric aldehyde is to isopropylcarbinol, and it can be dehydrogenase catalyzed by such as branched-chain alcoho.

In another embodiment, isobutanol biosynthetic pathway comprises the conversion of following substrate to product:

A) pyruvic acid is to acetylactis, and it can by such as acetolactate synthase catalysis;

B) acetylactis to 2,3-dihydroxyisovalerate, it can by such as Ketol-acid Reductoisomerase catalysis;

C) 2,3-dihydroxyisovalerate are to a-ketoisovaleric acid, and it can by such as dihydroxyacid dehydratase catalysis;

D) alpha-ketoisocaproic acid is to α-amino-isovaleric acid, and it can by such as transaminase or valine dehydrogenase catalysis;

E) α-amino-isovaleric acid is to isobutylamine, and it can by such as valine decarboxylase catalysis;

F) isobutylamine is to isobutyric aldehyde, and it can be transaminase-catalyzed by such as ω; And

G) isobutyric aldehyde is to isopropylcarbinol, and it can be dehydrogenase catalyzed by such as branched-chain alcoho.

In another embodiment, isobutanol biosynthetic pathway comprises the conversion of following substrate to product:

A) pyruvic acid is to acetylactis, and it can by such as acetolactate synthase catalysis;

B) acetylactis to 2,3-dihydroxyisovalerate, it can by such as Acetohydroxy acid isomeroreductase catalysis;

C) 2,3-dihydroxyisovalerate are to alpha-ketoisocaproic acid, and it can by such as acetohydroxy acid dehydratase catalysis;

D) alpha-ketoisocaproic acid is to isobutyryl-CoA, and it can be dehydrogenase catalyzed by such as branched-chain keto acids;

E) isobutyryl-CoA is to the conversion of isobutyric aldehyde, and it can by such as acidylate acetaldehyde-dehydrogenase enzyme catalysis; And

F) isobutyric aldehyde is to isopropylcarbinol, and it can be dehydrogenase catalyzed by such as branched-chain alcoho.

Biosynthetic pathway for the production of spendable n-butyl alcohol is included in those that describe in U.S. Patent Application Publication 2008/0182308, and the document is incorporated to herein by reference.In one embodiment, n-butyl alcohol biosynthetic pathway comprises the conversion of following substrate to product:

A) acetyl-CoA is to acetoacetyl-CoA, and it can by such as acetyl-CoA acetyltransferase catalyzes;

B) acetoacetyl-CoA to 3-hydroxybutyric acid-CoA, it can be dehydrogenase catalyzed by such as 3-hydroxybutyric acid-CoA;

C) 3-hydroxybutyric acid-CoA is to crotonoyl-CoA, and it can by such as enoyl-CoA hydratase catalysis;

D) crotonoyl-CoA is to butyryl-CoA, and it can by such as butyryl-CoA is dehydrogenase catalyzed;

E) butyryl-CoA is to butyraldehyde, and it can be dehydrogenase catalyzed by such as butyraldehyde; And

F) butyraldehyde is to n-butyl alcohol, and it can be dehydrogenase catalyzed by such as butanols.

Biosynthetic pathway for the production of spendable 2-butanols is included in those that describe in U.S. Patent Application Publication 2007/0259410 and U.S. Patent Application Publication 2009/0155870, and they are incorporated to herein by reference.In one embodiment, 2-butanol biosynthetic pathway comprises the conversion of following substrate to product:

A) pyruvic acid is to α-acetylactis, and it can by such as acetolactate synthase catalysis;

B) α-acetylactis is to acetoin, and it can by such as acetolactate decarboxylase catalysis;

C) acetoin is to 3-amino-2-butanols, and it can by such as acetoin aminase catalysis;

D) 3-amino-2-butanols is to phosphoric acid-3-amino-2-butanols, and it can by such as aminobutanol kinase catalysis;

E) phosphoric acid-3-amino-2-butanols is to 2-butanone, and it can by such as phosphorylated amino butanols Starch phosphorylase catalysis; And

F) 2-butanone is to 2-butanols, and it can be dehydrogenase catalyzed by such as butanols.

In another embodiment, 2-butanol biosynthetic pathway comprises the conversion of following substrate to product:

A) pyruvic acid is to α-acetylactis, and it can by such as acetolactate synthase catalysis;

B) α-acetylactis is to acetoin, and it can by such as acetolactate decarboxylase catalysis;

C) acetoin is to 2,3-butanediol, and it can by such as butanediol dehydrogenation enzyme catalysis;

D) 2,3-butanediol is to 2-butanone, and it can by such as diol dehydratase enzyme catalysis; And

E) 2-butanone is to 2-butanols, and it can be dehydrogenase catalyzed by such as butanols.

Biosynthetic pathway for the production of spendable 2-butanone is included in those that describe in U.S. Patent Application Publication 2007/0259410 and U.S. Patent Application Publication 2009/0155870, and they are incorporated to herein by reference.In one embodiment, 2-butanone biosynthetic pathway comprises the conversion of following substrate to product:

A) pyruvic acid is to α-acetylactis, and it can by such as acetolactate synthase catalysis;

B) α-acetylactis is to acetoin, and it can by such as acetolactate decarboxylase catalysis;

C) acetoin is to 3-amino-2-butanols, and it can by such as acetoin aminase catalysis;

D) 3-amino-2-butanols is to phosphoric acid-3-amino-2-butanols, and it can by such as aminobutanol kinase catalysis; And

E) phosphoric acid-3-amino-2-butanols is to 2-butanone, and it can by such as phosphorylated amino butanols Starch phosphorylase catalysis.

In another embodiment, 2-butanone biosynthetic pathway comprises the conversion of following substrate to product:

A) pyruvic acid is to α-acetylactis, and it can by such as acetolactate synthase catalysis;

B) α-acetylactis is to acetoin, and it can by such as acetolactate decarboxylase catalysis;

C) acetoin is to 2,3-butanediol, and it can by such as butanediol dehydrogenation enzyme catalysis; And

D) 2,3-butanediol is to 2-butanone, and it can by such as diol dehydratase enzyme catalysis.

Term " acetohydroxy acid synthase ", " acetolactate synthase " and " acetolactate synthestase " (being abbreviated as " ALS ") are used interchangeably herein, are used in reference to and have catalysis pyruvic acid one or more polypeptide to the enzymic activity of the conversion of acetylactis and CO2.The example of known acetolactate synthase is EC numbering 2.2.1.6 (Enzyme Nomenclature 1992, Academic Press, San Diego).These not modified enzymes can derive from multiple source, include but not limited to subtilis (Bacillus subtilis) (GenBank No:CAB15618 (SEQ ID NO:1), Z99122 (SEQ ID NO:2), NCBI (National Center for Biotechnology Information) aminoacid sequence, NCBI nucleotide sequence), Klebsiella pneumonia (Klebsiella pneumoniae) (GenBank No:AAA25079 (SEQ ID NO:3), M73842 (SEQ ID NO:4)) and Lactococcus lactis (Lactococcus lactis) (GenBank No:AAA25161 (SEQ ID NO:5), L16975 (SEQ ID NO:6)).

Term " Ketol-acid Reductoisomerase " (" KARI "), " acetohydroxy acid isomeroreductase " and " Acetohydroxy acid isomeroreductase " are used interchangeably herein, be used in reference to and there is catalysis (S)-acetylactis to 2, one or more polypeptide of the enzymic activity of the reaction of 3-dihydroxyisovalerate.The example of KARI enzyme can classify as EC numbering EC 1.1.1.86 (Enzyme Nomenclature 1992, Academic Press, San Diego), and purchased from multiple-microorganism, include but not limited to intestinal bacteria (Escherichia coli) (GenBank No:NP_418222 (SEQ ID NO:7), NC_000913 (SEQ ID NO:8)), yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) (GenBank No:NP_013459 (SEQ ID NO:9), NC_001144 (SEQ ID NO:10)), sea natural pond methanosarcina (Methanococcus maripaludis) (GenBank No:CAF30210 (SEQ ID NO:11), BX957220 (SEQ ID NO:12)) and subtilis (Bacillus subtilis) (GenBank No:CAB14789 (SEQ ID NO:13), Z99118 (SEQ ID NO:14)).KARI comprises excrement anaerobism corynebacterium (Anaerostipes caccae) KARI variant " K9G9 " and " K9D3 " (being respectively SEQ ID NO:15 and 16).Described by Ketol-acid Reductoisomerase (KARI) has in U.S. Patent Application Publication 2008/0261230,2009/0163376 and 2010/0197519 and PCT public announcement of a patent application WO/2011/041415, they are incorporated to herein by reference.The example of KARI disclosed herein be from Lactococcus lactis (Lactococcus lactis), vibrio cholerae (Vibrio cholera), Pseudomonas aeruginosa (Pseudomonas aeruginosa) PAO1 and Pseudomonas fluorescens (Pseudomonas fluorescens) PF5 mutant those.In certain embodiments, KARI utilizes NADH.In certain embodiments, KARI utilizes NADPH (NADPH).

Term " acetohydroxy acid dehydratase " and " dihydroxyacid dehydratase " (" DHAD ") refer to have catalysis 2,3-dihydroxyisovalerate one or more polypeptide to the enzymic activity of the conversion of alpha-ketoisocaproic acid.The example of acetohydroxy acid dehydratase is known as EC numbering EC 4.2.1.9.This fermentoid can derive from multiple-microorganism, include but not limited to intestinal bacteria (Escherichia coli) (GenBank No:YP_026248 (SEQ ID NO:17), NC000913 (SEQ ID NO:18)), yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) (GenBank No:NP_012550 (SEQ ID NO:19), NC 001142 (SEQ ID NO:20), sea natural pond methanosarcina (M.maripaludis) (GenBank No:CAF29874 (SEQ ID NO:21), BX957219 (SEQ ID NO:22)), subtilis (B.subtilis) (GenBank No:cAB14105 (SEQ ID NO:23), Z99115 (SEQ ID NO:24)), Lactococcus lactis (L.lactis) and Neurospora crassa (N.crassa).U.S. Patent Application Publication 2010/0081154 and United States Patent (USP) 7,851,188 (they are incorporated to herein by reference) describe dihydroxyacid dehydratase (DHAD), comprise the DHAD from Streptococcus mutans (Streptococcus mutans).

Term " branched-chain α-keto acid decarboxylase ", " alpha-ketoacid decarboxylase ", " alpha-ketoisocaproic acid decarboxylase " or " 2-ketoisovaleric acid decarboxylase " (" KIVD ") refer to have the polypeptide (or polypeptide) of catalysis alpha-ketoisocaproic acid to the enzymic activity of the conversion of isobutyric aldehyde and CO2.The example of known branched-chain α-keto acid decarboxylase is EC numbering 4.1.1.72, and derive from many sources, include but not limited to Lactococcus lactis (Lactococcus lactis) (Genbank No:AAS49166 (SEQ ID NO:25), AY548760 (SEQ ID NO:26), CAG34226 (SEQ ID NO:27), AJ746364 (SEQ ID NO:28), Salmonella typhimurium (Salmonella typhimurium) (GenBank No:NP_461346 (SEQ ID NO:29), NC_003197 (SEQ ID NO:30)), clostridium acetobutylicum (Clostridium acetobutylicum) (GenBank No:NP_149189 (SEQ ID NO:31), NC_001988 (SEQ ID NO:32)), separate junket micrococci (M.caseolyticus) (SEQ ID NO:33) and listeria grayi (L.grayi) (SEQ ID NO:34).

Term " branched-chain alcoho desaturase " (" ADH ") refers to have the polypeptide (or multiple polypeptides) of catalyzing iso-butane aldehyde to the enzymic activity of the conversion of isopropylcarbinol.The example of branched-chain alcoho desaturase is known as EC numbering 1.1.1.265, but also can be classified as other alcoholdehydrogenase (specifically, EC 1.1.1.1 or 1.1.1.2).Alcoholdehydrogenase can be NADPH dependent form or NADH dependent form.These enzymes can derive from multiple source, include but not limited to yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) (GenBank No:NP_010656 (SEQ ID NO:35), NC_001136 (SEQ ID NO:36), NP_014051 (SEQ ID NO:37), NC_001145 (SEQ ID NO:38)), intestinal bacteria (Escherichia coli) (GenBank No:NP_417484 (SEQ ID NO:39), NC_000913 (SEQ ID NO:40)), clostridium acetobutylicum (C.acetobutylicum) (GenBank No:NP_349892 (SEQ ID NO:41), NC_003030 (SEQ ID NO:42), NP_349891 (SEQ ID NO:43), NC_003030 (SEQ ID NO:44)).U.S. Patent Application Publication 2009/0269823 describes alcoholdehydrogenase (ADH) SadB from Achromobacter xylosoxidans (Achromobacter xylosoxidans).Alcoholdehydrogenase also comprises horse liver ADH and India Bai Yelin kirschner bacterium (Beijerinkia indica) ADH (as described in U.S. Patent Application Publication 2011/0269199, the document is incorporated herein by reference).

Term " butanols desaturase " refers to polypeptide (or multiple polypeptides), and it has catalyzing iso-butane aldehyde to the enzymic activity that isopropylcarbinol transforms or catalysis 2-butanone transforms to 2-butanols.Butanols desaturase is a subset in alcoholdehydrogenase extended familys.Butanols desaturase can be NAD-or NADP-dependent form.NAD-dependent form enzyme is known as EC 1.1.1.1 and can derives from such as Rhodococcus ruber (Rhodococcus ruber) (GenBank No:CAD36475, AJ491307).NADP dependent form enzyme is known as EC 1.1.1.2 and can derives from such as fierce hot-bulb bacterium (Pyrococcus furiosus) (GenBank No:AAC25556, AF013169).In addition, butanols desaturase derives from intestinal bacteria (Escherichia coli) (GenBank No:NP 417484, NC_000913), and cyclohexanol dehydrogenation enzyme can derive from acinetobacter (Acinetobacter sp.) (GenBank No:AAG10026, AF282240).Term " butanols desaturase " also refer to that catalysis butyraldehyde changes into the enzyme of n-butyl alcohol, it uses NADH or NADPH as cofactor.Butanols desaturase derives from such as clostridium acetobutylicum (C.acetobutylicum) (GenBank No:NP_149325, NC_001988; This enzyme has aldehyde and alcohol dehydrogenase activity simultaneously); NP_349891, NC_003030; With NP_349892, NC_003030) and intestinal bacteria (Escherichia coli) (GenBank No:NP_417-484, NC_000913).

Term " branched-chain keto acids desaturase " refers to one or more polypeptide with the enzymic activity that catalysis alpha-ketoisocaproic acid transforms to isobutyryl-CoA (isobutyryl-coenzyme A), usually uses NAD+ (Reduced nicotinamide-adenine dinucleotide) as electron acceptor(EA).The example of branched-chain keto acids desaturase is known as EC numbering 1.2.4.4.This type of branched-chain keto acids desaturase is made up of four subunits, and can multiple-microorganism be derived from from the sequence of all subunits, include but not limited to subtilis (Bacillus subtilis) (GenBank No:CAB14336 (SEQ ID NO:45), Z99116 (SEQ ID NO:46); CAB14335 (SEQ ID NO:47), Z99116 (SEQ ID NO:48); CAB14334 (SEQ ID NO:49), Z99116 (SEQ ID NO:50); With CAB14337 (SEQ ID NO:51), Z99116 (SEQ ID NO:52)) and pseudomonas putida (Pseudomonas putida) (GenBank No:AAA65614 (SEQ ID NO:53), M57613 (SEQ ID NO:54); AAA65615 (SEQ ID NO:55), M57613 (SEQ ID NO:56); AAA65617 (SEQ ID NO:57), M57613 (SEQ ID NO:58); With AAA65618 (SEQ ID NO:59), M57613 (SEQ ID NO:60)).

Term " acylating aldehyde dehydrogenase " refers to have the polypeptide (or multiple polypeptides) of catalyzing iso-butane acyl-CoA to the enzymic activity of the conversion of isobutyric aldehyde, usually uses NADH or NADPH as electron donor.The example of acidylate acetaldehyde dehydrogenase is known as EC numbering 1.2.1.10 and 1.2.1.57.This fermentoid derives from multiple source, includes but not limited to Bai Shi clostridium (Clostridium beijerinckii) (GenBank No:AAD31841 (SEQ ID NO:61), AF157306 (SEQ ID NO:62)), clostridium acetobutylicum (Clostridium acetobutylicum) (GenBank No:NP_149325 (SEQ ID NO:63), NC_001988 (SEQ ID NO:64); NP_149199 (SEQ ID NO:65), NC_001988 (SEQ ID NO:66)) and pseudomonas putida (Pseudomonas putida) (GenBank No:AAA89106 (SEQ ID NO:67), U13232 (SEQ ID NO:68)) and thermus thermophilus (Thermus thermophilus) (GenBank No:YP_145486 (SEQ ID NO:69), NC_006461 (SEQ ID NO:70)).

Term " transaminase " refers to have catalysis alpha-ketoisocaproic acid one or more polypeptide to the enzymic activity of the conversion of Valine, uses L-Ala or L-glutamic acid as amine donor.The example of transaminase is known as EC numbering 2.6.1.42 and 2.6.1.66.These enzymes can derive from multiple source.The example relying on the source of the enzyme of L-Ala includes but not limited to intestinal bacteria (Escherichia coli) (GenBank No:YP_026231 (SEQ ID NO:71), NC_000913 (SEQ ID NO:72)) and Bacillus licheniformis (Bacillus licheniformis) (GenBank No:YP_093743 (SEQ ID NO:73), NC_006322 (SEQ ID NO:74)).The example relying on the source of the enzyme of L-glutamic acid includes but not limited to intestinal bacteria (Escherichia coli) (GenBank No:YP_026247 (SEQ ID NO:75), NC_000913 (SEQ ID NO:76)), yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) (GenBank No:NP_012682 (SEQ ID NO:77), NC_001142 (SEQ ID NO:78)) and addicted to hot autotrophic methane bacteria (Methanobacterium thermoautotrophicum) (GenBank No:NP_276546 (SEQ ID NO:79), NC_000916 (SEQ ID NO:80)).

Term " valine dehydrogenase " refers to have catalysis alpha-ketoisocaproic acid one or more polypeptide to the enzymic activity of the conversion of Valine, usually uses NAD (P) H to be electron donor and uses ammonia as amine donor.The example of valine dehydrogenase is known as EC numbering 1.4.1.8 and 1.4.1.9 and this fermentoid can derive from multiple source, includes but not limited to streptomyces coelicolor (Streptomyces coelicolor) (GenBank No:NP_628270 (SEQ ID NO:81), NC_003888 (SEQ ID NO:82)) and subtilis (Bacillus subtilis) (GenBank No:CAB14339 (SEQ ID NO:83), Z99116 (SEQ ID NO:84)).

Term " valine decarboxylase " refers to have catalysis Valine one or more polypeptide to the enzymic activity of the conversion of isobutylamine and CO2.The example of valine decarboxylase is known as EC numbering 4.1.1.14.This fermentoid is present in streptomyces (Streptomyces), such as raw green streptomycete (Streptomyces viridifaciens) (GenBank No:AAN10242 (SEQ ID NO:85), AY116644 (SEQ ID NO:86)).

Term " ω transaminase " refers to have the polypeptide (or multiple polypeptides) of catalyzing iso-butane amine to the enzymic activity of the conversion of isobutyric aldehyde, uses suitable amino acid as amine donor.The example of known ω transaminase is EC numbering 2.6.1.18, and derive from many sources, include but not limited to alcaligenes dentrificans (Alcaligenes denitrificans) (AAP92672, (SEQ ID NO:87), AY330220 (SEQ ID NO:88)), Ralstonia eutropha (Ralstonia eutropha) (GenBank No:YP_294474 (SEQ ID NO:89), NC_007347 (SEQ ID NO:90)), Oneida lake Shewanella (Shewanella oneidensis) (GenBank No:NP_719046 (SEQ ID NO:91), NC_004347 (SEQ ID NO:92)) and pseudomonas putida (Pseudomonas putida) (GenBank No:AAN66223 (SEQ ID NO:93), AE016776 (SEQ ID NO:94)).

Term " acetyl-CoA Transacetylase " refers to one or more polypeptide of the enzymic activity of the conversion of acetyl-CoA to acetoacetyl-CoA and the coenzyme A (CoA) with catalysis two molecules.The example of acetyl-CoA Transacetylase is the acetyl-CoA Transacetylase of the substrate preference (reacting forward) had short chain acyl-CoA and acetyl-CoA, and this enzyme classifies as E.C.2.3.1.9 [Enzyme Nomenclature 1992, Academic Press, San Diego]; But the enzyme (E.C.2.3.1.16) with more extensive substrate spectrum also will have function.Acetyl-CoA Transacetylase derives from multiple source, such as, and intestinal bacteria (Escherichia coli) (GenBank No:NP_416728, NC_000913; NCBI aminoacid sequence, NCBI nucleotide sequence), clostridium acetobutylicum (Clostridium acetobutylicum) (GenBank No:NP_349476.1, NC_003030; NP_149242, NC_001988, subtilis (Bacillus subtilis) (GenBank No:NP_390297, NC_000964) and yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) (GenBank No:NP_015297, NC_001148).

Term " 3-hydroxyl butyryl-CoA desaturase " refers to one or more polypeptide of the enzymic activity of the conversion with catalysis acetoacetyl-CoA to 3-hydroxyl butyryl-CoA.The example of 3-hydroxyl butyryl-CoA desaturase can be NADH dependent form, have substrate Preference to (S)-3-hydroxyl butyryl-CoA or (R)-3-hydroxyl butyryl-CoA.Example can classify as E.C. 1.1.1.35 and E.C. 1.1.1.30 respectively.In addition, 3-hydroxyl butyryl-CoA desaturase can be NADPH dependent form, there is the substrate Preference to (S)-3-hydroxyl butyryl-CoA or (R)-3-hydroxyl butyryl-CoA, and classified as E.C. 1.1.1.157 and E.C. 1.1.1.36 respectively.3-hydroxyl butyryl-CoA desaturase derives from multiple source, such as clostridium acetobutylicum (Clostridium acetobutylicum) (GenBank No:NP_349314, NC_003030), subtilis (Bacillus subtilis) (GenBank No:AAB09614, U29084), Ralstonia eutropha (Ralstonia eutropha) (GenBank No:YP_294481, NC_007347) and alcaligenes eutrophus (Alcaligenes eutrophus) (GenBank No:AAA21973, J04987).

Term " enoyl-CoA hydratase " refers to one or more polypeptide of the enzymic activity of the conversion with catalysis 3-hydroxyl butyryl-CoA to crotonoyl-CoA and H2O.The example of enoyl-CoA hydratase can have the substrate Preference to (S)-3-hydroxyl butyryl-CoA or (R)-3-hydroxyl butyryl-CoA, and can be classified as E.C. 4.2.1.17 and E.C. 4.2.1.55 respectively.Enoyl-CoA hydratase derives from multiple source, such as intestinal bacteria (Escherichia coli) (GenBank No:NP_415911, NC_000913), clostridium acetobutylicum (Clostridium acetobutylicum) (GenBank No:NP_349318, NC_003030), subtilis (Bacillus subtilis) (GenBank No:CAB13705, Z99113) and Aeromonas caviae (Aeromonas caviae) (GenBank No:BAA21816, D88825).

Term " butyryl-CoA desaturase " refers to have catalyzing butene acyl-CoA one or more polypeptide to the enzymic activity of the conversion of butyryl-CoA.The example of butyryl-CoA desaturase can be NADH-dependent form, NADPH-dependent form or flavine-dependent form, and E.C. 1.3.1.44, E.C. 1.3.1.38 and E.C. 1.3.99.2 can be classified as respectively.Butyryl-CoA desaturase derives from multiple source, such as clostridium acetobutylicum (Clostridium acetobutylicum) (GenBank No:NP_347102, NC_003030), euglena gracilis (Euglena gracilis) (GenBank No:Q5EU90, AY741582), massif streptomycete (Streptomyces collinus) (GenBank No:AAA92890, U37135) and streptomyces coelicolor (Streptomyces coelicolor) (GenBank No:CAA22721, AL939127).

Term " butyraldehyde desaturase " refers to have the polypeptide (or multiple polypeptides) of catalysis butyryl-CoA to the enzymic activity of the conversion of butyraldehyde, uses NADH or NADPH as cofactor.Butyraldehyde desaturase has the Preference to NADH, and this enzyme is known as E.C. 1.2.1.57 and derives from such as Bai Shi clostridium (Clostridium beijerinckii) (GenBank No:AAD31841, AF157306) and clostridium acetobutylicum (Clostridium acetobutylicum) (GenBank No:NP.sub.--149325, NC.sub.--001988).

Term " isobutyryl-CoA mutase " refers to have catalysis butyryl-CoA one or more polypeptide to the enzymic activity of the conversion of isobutyryl-CoA.This enzyme uses actimide as cofactor.The example of isobutyryl-CoA mutase is known as EC numbering 5.4.99.13.These enzymes are present in multiple streptomyces (Streptomyces), include but not limited to Chinese cassia tree ground streptomycete (Streptomyces cinnamonensis) (GenBank No:AAC08713 (SEQ ID NO:95), U67612 (SEQ ID NO:96); CAB59633 (SEQ ID NO:97), AJ246005 (SEQ ID NO:98)), streptomyces coelicolor (Streptomyces coelicolor) (GenBank No:CAB70645 (SEQ ID NO:99), AL939123 (SEQ ID NO:100); CAB92663 (SEQ ID NO:101), AL939121 (SEQ ID NO:102)) and Avid kyowamycin (Streptomyces avermitilis) (GenBank No:NP_824008 (SEQ ID NO:103), NC_003155 (SEQ ID NO:104); NP_824637 (SEQ ID NO:105), NC_003155 (SEQ ID NO:106)).

Term " acetolactate decarboxylase " refers to have one or more polypeptide that catalysis α-conversion of acetolactate becomes the enzymic activity of acetoin.The example of acetolactate decarboxylase is known as EC 4.1.1.5 and can be from, such as, subtilis (Bacillus subtilis) (GenBank No:AAA22223, L04470), Klebsiella terrigena (Klebsiella terrigena) (GenBank No:AAA25054, L04507) and Klebsiella pneumonia (Klebsiella pneumoniae) (GenBank No:AAU43774, AY722056).

Term " acetoin aminase " or " acetoin transaminase " refer to have catalysis acetoin one or more polypeptide to the enzymic activity of the conversion of 3-amino-2-butanols.Acetoin aminase can utilize cofactor 5 '-pyridoxal phosphate or NADH or NADPH.Products therefrom can have (R) or (S) three-dimensional chemical configuration in 3-site.Pyridoxal phosphate dependent form enzyme can use amino acid such as L-Ala or L-glutamic acid as amino group donor.NADH-and NADPH-dependent form enzyme can use ammonia as the second substrate.The suitable example of NADH dependent form acetoin aminase is also known as amino alcohol desaturase, is described by the people such as Ito (United States Patent (USP) 6,432,688).The example of pyridoxal dependent form acetoin aminase is amine: pyruvate aminotransferase (also referred to as amine: Pyruvic Transaminase), as described in Shin and Kim (J.Org.Chem.67:2848-2853,2002).

Term " acetoin kinases " refers to have catalysis acetoin one or more polypeptide to the enzymic activity of the conversion of phosphoric acid acetoin.Acetoin kinases can utilize ATP (Triphosaden) or phosphoenolpyruvic acid as the phosphodonor in reaction.The enzyme of the similar reaction of similar substrate otan catalysis is comprised to the enzyme (people such as Garcia-Alles, Biochemistry 43:13037-13046,2004) being such as known as EC 2.7.1.29.

Term " phosphoric acid acetoin aminase " refers to have catalytic phosphatase acetoin one or more polypeptide to the enzymic activity of the conversion of adjacent phosphoric acid-3-amino-2-butanols.Phosphoric acid acetoin aminase can utilize cofactor 5 '-pyridoxal phosphate, NADH or NADPH.Products therefrom can have (R) or (S) three-dimensional chemical configuration in 3-site.Pyridoxal phosphate dependent form enzyme can use amino acid such as L-Ala or L-glutamic acid.The enzyme relying on NADH-and NADPH-can use ammonia as the second substrate.Although not to the report of the enzyme of this phosphoric acid acetoin reaction of catalysis, but there is a kind of pyridoxal phosphate dependent form enzyme, propose it and similar reaction (people such as Yasuta, Appl.Environ.Microbial.67:4999-5009,2001) is carried out to similar substrate phosphoric acid serinol.

Term " phosphorylated amino butanols Starch phosphorylase ", also referred to as " adjacent phosphorylated amino alcohol lyase ", refers to have catalysis adjacent phosphoric acid-3-amino-2-butanols one or more polypeptide to the enzymic activity of the conversion of 2-butanone.Phosphorylated amino butanols phosphoroclastic cleavage enzyme can utilize cofactor 5 '-pyridoxal phosphate.There is the report (people such as Jones, Biochem.J.134:167-182,1973) of the enzyme of the similar reaction to catalysis similar substrates phosphoric acid-1-amino-2-propyl alcohol.U.S. Patent Application Publication 2007/0259410 describes the phosphorylated amino butanols phosphoroclastic cleavage enzyme from biological carrot soft rot Erwinia (Erwinia carotovora).

Term " aminobutanol kinase " refers to have one or more polypeptide that the amino 2-butanols of catalysis 3-changes into the enzymic activity of adjacent phosphoric acid 3-amino-2-butanols.Aminobutanol kinase can utilize ATP as phosphodonor.Although not to the report of the enzyme of this reaction of 3-amino-2-butanols catalysis, report catalysis to the enzyme (people such as Jones, see above) of similar substrates cholamine with the similar reaction of 1-amino-2-propyl alcohol.U.S. Patent Application Publication 2009/0155870 describes the amino alcohol kinases of the black shin subspecies of carrot soft rot Erwinia (Erwinia carotovora subsp.Atroseptica) in example 14.

Term " butanediol dehydrogenase ", also referred to as " acetoin reductase enzyme ", refers to have catalysis acetoin one or more polypeptide to the enzymic activity of the conversion of 2,3-butanediol.Suceinic aldehyde desaturase is a subset in alcoholdehydrogenase extended familys.Butanediol dehydrogenase can have specificity for the generation of (R)-in alcohol product or (S)-three-dimensional chemical configuration.(S)-specificity butanediol dehydrogenase is known as EC 1.1.1.76 and can be from, such as, and Klebsiella pneumonia (Klebsiella pneumoniae) (GenBank No:BBA13085, D86412).(R)-specificity butanediol dehydrogenase is known as EC 1.1.1.4 and can be from, such as, and bacillus cereus (Bacillus cereus) (GenBank No:NP 830481, NC_004722; AAP07682, AE017000) and Lactococcus lactis (Lactococcus lactis) (GenBank No.AAK04995, AE006323).

Term " Butanediol enzyme ", also referred to as " dioldehydrase " or " propanediol dehydratase ", refers to have catalysis 2,3-butanediol one or more polypeptide to the enzymic activity of the conversion of 2-butanone.Butanediol enzyme can utilize cofactor adenosylcobalamin (also referred to as coenzyme Bw or vitamin B12; But vitamin B12 also can refer to it is not the cobalami of actimide, other form).Adenosylcobalamin-dependent form enzyme is known as EC 4.2.1.28 and can derives from such as Klebsiella oxytoca (Klebsiella oxytoca) (GenBank NO:AA08099 (α subunit), D45071; BAA08100 (β subunit), D45071; With BBA08101 (γ subunit), D45071 (all three subunits are needed for activity) and Klebsiella pneumonia (Klebsiella pneumonia) (GenBank No:AAC98384 (α subunit), AF102064; GenBank No:AAC98385 (β subunit), AF102064, GenBank No:AAC98386 (γ subunit), AF102064).Other suitable dioldehydrase includes but not limited to B12 dependent form dioldehydrase, and it derives from Salmonella typhimurium (Salmonella typhimurium) (GenBank No:AAB84102 (large subunit), AF026270; GenBank No:AAB84103 (middle subunit), AF026270; GenBank No:AAB84104 (small subunit), AF026270); With Lactobacillus collinoides (Lactobacillus collinoides) (GenBank No:CAC82541 (large subunit), AJ297723; GenBank No:CAC82542 (middle subunit); AJ297723; GenBank No:CAD01091 (small subunit), AJ297723); With from short lactobacillus (Lactobacillus brevis) (especially bacterial strain CNRZ 734 and CNRZ 735, the people such as Speranza, J.Agric.Food Chem.45:3476-3480,1997) enzyme, and the nucleotide sequence of corresponding enzyme of encoding.Dioldehydrase gene isolation method is this area well known (such as United States Patent (USP) 5,686,276).

Term " pyruvic carboxylase " refers to have catalysis pyruvate decarboxylation one or more polypeptide to the enzymic activity of acetaldehyde and carbonic acid gas.Pyruvic oxidase is known as EC numbering 4.1.1.1.These enzymes are present in multiple yeast, comprise yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) (GenBank No:CAA97575 (SEQ ID NO:107), CAA97705 (SEQ ID NO:109), CAA97091 (SEQ ID NO:111)).

Should be appreciated that the microorganism comprising isobutanol biosynthetic pathway as herein provided also can comprise one or more additional modification.U.S. Patent Application Publication 2009/0305363 (being incorporated to way of reference) discloses and improves pyruvic acid to acetolactic conversion with expression and localization in the acetolactate synthase of cytosol and basic removing Pyruvate decarboxylase activity by engineered yeast.In certain embodiments, the modification that microorganism can comprise as described in U.S. Patent Application Publication 2009/0305363 (being incorporated to by reference herein) has the gene of the polypeptide of Pyruvate decarboxylase activity to reduce glycerol-3-phosphate activity and/or to destroy at least one coding or destroys the gene of the controlling element that at least one coding-control Pyruvate Decarboxylase Gene is expressed, and/or provides the modification by the raising Carbon flux of En Tena-Dao Pierre-Marie Deloof approach (Entner-Doudoroff Pathway) or the active balance of reduction as described in U.S. Patent Application Publication 2010/0120105 (being incorporated to by reference herein).Other is modified and comprises the integration that coding catalysis utilizes at least one polynucleotide of the polypeptide of the step in the biosynthetic pathway of pyruvic acid.Other is modified, and comprising encodes has at least one disappearance, the sudden change in the endogenous polynucleotide of the polypeptide of acetylactis reductase activity and/or replaces.In certain embodiments, the polypeptide described in acetylactis reductase activity is the YMR226C of yeast saccharomyces cerevisiae (Saccharomyces cerevisae) (SEQ ID NO:127,128) or its homologue.Additional modification comprises the disappearance had in the endogenous polynucleotide of the polypeptide of aldehyde dehydrogenase and/or Aldehyde oxidase activity of encoding, sudden change and/or replacement.In certain embodiments, the polypeptide with aldehyde dehydrogenase activity is ALD6 from yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) or its homologue.Wherein yeast production host cell is pdc-, has and reduce described by the genetic modification of resistance to glucose has in U.S. Patent Application Publication 2011/0124060, and the document is incorporated to herein by reference.In certain embodiments, the pyruvic carboxylase of disappearance or down-regulation is selected from: PDC1, PDC5, PDC6 and their combination.In certain embodiments, pyruvic carboxylase is selected from those enzymes in table 1.In certain embodiments, microorganism can containing coding catalysis glyceraldehyde 3-phosphate to 1, the disappearance of the polynucleotide of the polypeptide of the conversion of 3-diphosphoglyceric acid or downward.In certain embodiments, the enzyme of this reaction of catalysis is glyceraldehyde 3-phosphate dehydro-genase.

the sequence number of table 1.PDC target gene coding region and protein

In certain embodiments, any one specific nucleic acid molecule or polypeptide can be at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical with nucleotide sequence as herein described or peptide sequence.As known in the art, term " percentage identities " is the relation between two or more peptide sequences or between two or more polynucleotide sequences, and this relation is determined by comparing sequence.In this area, " identity " also means the serial correlation degree between polypeptide or polynucleotide sequence, depends on the circumstances, as measured by the coupling of this class sequence of characters string.Identity and similarity can easily be calculated by currently known methods, described method to include but not limited in following documents disclosed those: Computational Molecular Biology (Lesk, A.M. edit) Oxford University:NY (1988); Biocomputing:Informatics and Genome Projects (Smith, D.W. edit) Academic:NY (1993); ComputerAnalysis of Sequence Data, Part I (Griffin, A.M. and Griffin, H.G. edits) Humania:NJ (1994); Sequence Analysis in Molecular Biology (von Heinje, G. edits) Academic (1987); With Sequence Analysis Primer (Gribskov, M. and Devereux, J. edits) Stockton:NY (1991).

Setting determines that the method for identity is for providing the optimum matching between sequence to be tested.Determine that the method for identity and similarity has been compiled to code in the computer program that can openly obtain.Sequence alignment and percentage identities calculate the MegAlign that can use in LASERGENE information biology computation software package (LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, WI)) ^tMprogram is carried out.The multiple ratio of sequence is carried out using Clustal comparison method, the method contains several different algorithms, the Clustal V comparison method comprised corresponding to the comparison method being called as Clustal V (is disclosed in Higgins and Sharp, CABIOS.5:151-153,1989; The people such as Higgins, Comput.Appl.Biosci.8:189-191 (1992)) in, and can MegAlign in LASERGENE information biology computation software package (DNASTAR Inc.) ^tMthe comparison method found in program.For multiple ratio pair, default value corresponds to GAP PENALTY=10 and GAP LENGTH PENALTY=10.The default parameters carrying out the percentage identities calculating of paired comparison and protein sequence by Clustal method is KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.For nucleic acid, these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4.After carrying out sequence alignment by Clustal V program, likely obtain percentage identities by the sequence distance table observed in same program.Described " Clustal W comparison method " is available and corresponding to the comparison method (Higgins and Sharp, CABIOS.5:151-153,1989 that are labeled as Clustal W in addition; The people such as Higgins, Comput.Appl.Biosci.8:189-191, (1992)), and can MegAlign in LASERGENE information biology computation software package (DNASTAR Inc.) ^tMthe comparison method found in v6.1 program.For the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=0.2, Delay Divergen Seqs (%)=30, DNA Transition Weight=0.5, Protein Weight Matrix=Gonnet series, DNA Weight Matrix=IUB) that multiple ratio is right.After use Clustal W program is compared to sequence, likely by checking that the sequence distance table in same program obtains percentage identities.

Standard recombinant dna and molecule clone technology are known in the art and have in such as Publication about Document and more fully describe: the people such as Sambrook (Sambrook, J., Fritsch, E.F. and Maniatis, T. (Molecular Cloning:A Laboratory Manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989, be called Maniatis herein) and the people (people such as Ausubel such as Ausubel, Current Protocols in Molecular Biology, published by Greene Publishing Assoc.and Wiley-Interscience, 1987).The example comprising the structure method of microorganism of butanol biosynthetic pathway is disclosed in such as United States Patent (USP) 7,851,188 and U.S. Patent Application Publication 2007/0092957; 2007/0259410; 2007/0292927; 2008/0182308; 2008/0274525; 2009/0155870; 2009/0305363; In 2009/0305370, their each full text is incorporated herein by reference.

In addition, although there have been described multiple embodiment of the present invention, be to be understood that they only provide by way of illustration, and nonrestrictive.Those skilled in the relevant art be it is evident that and can not depart from the multiple modification can carrying out form and details under the spirit and scope of the invention to it.Therefore, range of the present invention and scope by the restriction of any above-mentioned exemplary embodiment, but should only should not limit according to following claim and equivalents thereof.

The all announcements mentioned in this manual, patent and patent application all indicate the state of the art of those skilled in the art in the invention, and be incorporated herein by reference to as each independent announcement, patent or patent application by specifically and be indicated as being the same degree be incorporated to way of reference individually.

example

Following limiting examples will illustrate the present invention further.Although should be appreciated that following instance relates to corn as raw material, such as sugarcane can be used as raw material and does not deviate from the present invention other biomass sources.In addition, although following instance relates to ethanol and butanols, other alcohol or tunning can be prepared and do not deviate from the present invention.

The implication of abbreviation is as follows: " atm " refers to air, " ccm " refers to cubic centimetres per minute, " g/L " refers to gram often liter, " g " refers to gram, " gpl " refers to gram often liter, " gpm " refers to gallons per minute, and " h " or " hr " refers to hour, and " HPLC " refers to high performance liquid chromatography, " kg " refers to kilogram, " L " refers to liter, and " min " refers to minute, and " mL " refers to milliliter, " ppm " refers to every 1,000,000 parts of number, " psig " refers to pound per square inch, standard size, and " wt% " refers to weight percent.

example 1

for the production of the method with the butanols reclaimed by fermentation generation

Methods described herein can use computation model such as Aspen model show (see, such as, United States Patent (USP) 7,666,282).Such as, business prototype software (Aspen Technology, Inc., Burlington, MA) can such as can purchased from American Institute of Chemical Engineers with physical characteristic data storehouse, Inc., the DIPPR of (New York, NY) uses together, develops the Aspen model of the butylic fermentation for integrating, purifying and water management process.This process model can carry out many basic engineering calculation, such as quality and energy balance, and gas/liquid balance and speed of reaction calculate.In order to generate Aspen model, information input can comprise such as experimental data, water-content and raw material composition, wine with dregs boils and the temperature of flash distillation, saccharification condition (such as, enzyme charging, Starch Conversion, temperature, pressure), fermentation condition (such as, microorganism charging, conversion of glucose, temperature, pressure), degassing conditions, solvent tower, preflash tower, condenser, vaporizer, whizzer etc.

Establish Aspen model with strict material and energy balance, wherein the corn of 53400kg/h is also fermented to produce isopropylcarbinol by wine with dregsization, and wherein most isopropylcarbinol is extracted during the fermentation and distills.This model comprises as processed continuously approximate sequence batch (fermentation.An example of above-mentioned fermentation, extraction and distil process as shown in figure 14.

Be pumped across heat exchanger and watercooler by the corn mash 601 clarified to the liquefaction comprising 1.5 % by weight suspended solidss by with 170.7 tons/hour and 85 DEG C, and be fed to fermentation 600 at 32 DEG C.Under atmospheric pressure from fermentation 600, discharge steam stream 602 to scrubbers with 17.2 tons/hour, described steam stream has following on average continuous mole of composition: 95.8% carbonic acid gas, 3.4% water and 0.8% isopropylcarbinol.The average beer stream 603 comprising 12.6gpl isopropylcarbinol is discharged from fermentation 600 continuously, and before being distilled to reclaim isopropylcarbinol by interchanger by wine with dregs 601 preheating.

The stream 604 with the average discharge that 3875 tons/hour merge is removed from fermentation 600 by with the medial temperature of the average isobutanol concentration of 11.1gpl and 32 DEG C, and is recycled by extractor 610 partly to remove isopropylcarbinol.The moisture fermented liquid 605 left comprising 7.9gpl isopropylcarbinol before reentering fermentation 600 by being cooled to 30 DEG C with the heat exchange of cooling tower water (CTW).The solvent comprising diisopropylbenzene(DIPB) enters extractor 610 and leaves as the stream 606 comprising 30.1gpl isopropylcarbinol.Extractor 610 effectively provides for making five of fermented liquid and solvent contacts theoretical liquid-liquid equilibrium column plates.Stream 606 passes through interchanger with 340 tons/hour, and enters the centre of 12 theoretical trays of distillation tower 620.Reboiler uses 150psig steam to run with 0.6atm and 183 DEG C, diisopropylbenzene(DIPB) is comprised and the solvent streams 607 substantially not containing isopropylcarbinol to produce, it is by interchanger and solvent streams 606 exchanging heat, and cooled water CTW cools further before again entering extractor 610.The steam that the tower top of distillation tower 620 distillates is cooled by CTW, and condensation 630 is to form the backflow of 23.1 tons/hour, the vapor exhaust 608 of remnants of 0.2 ton/hour and the product distillate 609 comprising 99.2% isopropylcarbinol, 0.6% water and 0.2% diisopropylbenzene(DIPB) of 13.2 tons/hour.

example 2

extraction column is used to reclaim the method for ethanol

1 " diameter extraction column (Koch Modular Process Systems, Paramus, NJ) is used to process the fermented liquid produced in alcohol fermentation processes.Described tower comprises a series of plate run along the length of tower downwards, and they are connected on central shaft.Axle is connected on driving mechanism, and it can move up and down perforation plate (1/4 " diametric hole) with reciprocating manner.The frequency of motion is variable at test period, but the spacing of the stroke (0.75 ") of vibration and tower tray (2 ") be all fixing.Described tower has the plate stacks as high of 3000mm.

Top to tower provides the aqueous feed be made up of fermented liquid, and the bottom simultaneously to tower provides corn oil fatty acid (COFA) charging as extraction agent.Two kinds of chargings each other upstream stream wear tower, and to be collected as product at the relative two ends of tower.

Fermented liquid uses the fermentation code for ethanol fermentation to obtain from the corn mash of also saccharification of liquefying, and in some cases, eliminates some solids by centrifugal from described wine with dregs.In some cases, extract test and be through that the process of some days completes, the test making a part is at CO ₂exhaust gas emission reaches or close to complete while reaching its maximum value, and another part is completed when exhaust gas emission effectively stops.The COFA used in this operation is the distillation level from Emery Oleochemicals (Cincinnati, OH).

Some experiments are what to carry out using COFA as the external phase in tower, and other the continuous print that uses carries out containing aqueous phase.Test while also there is or do not have internals in position.Test the internals of two types: stainless steel and tetrafluoroethylene (PTFE).Measure a series of flow rate, there is no the flow state of overflow to determine tower can run with it.

from the impact of the dynamic charging of fermentation

In test process, determine in some cases, the performance of tower changes along with the carrying out of fermentation.Early stage in fermentation, the fermented liquid sugar degree of composition charging is high, in the intermediate time, and the CO of sizable amount ₂(it can affect fluid flowing) separates out from fermented liquid, and in the later stage, the alcohol concn in fermented liquid is high.This time variations in charging is reflected in the change of extraction output of column.

Under the condition using PTFE plate and continuous print COFA phase (without stirring), when be used in fermentation close to gas evolution peak period (" middle fermented liquid ") and collect close to (" fermented liquid in latter stage ") during fermentation ends fermented liquid time, observe the difference of performance.Use fermented liquid in latter stage, achieve 14gpm/ft ²the liquid throughput rate of (sample 3E) and do not have the overflow of tower.With regard to middle fermented liquid, the maximum throughput that can reach before there is overflow is for being less than 9gpm/ft ²(sample 4D), and there is the size of water-containing drop and the significant difference of outward appearance.Compared with middle fermented liquid, latter stage contains aqueous phase drop size in fermented liquid is larger (having the formation of the globule).

external phase

Maximum tower throughput capacity is also by the characteristics influence of external phase.With regard to fermentation ends condition, run containing aqueous phase and stainless steel (S.Steel) internals with continuous print, achieve close to 14gpm/ft ²total liquid production ability (sample 2B).With regard to continuous print organic phase and PTFE internals, described speed is for being less than 9gpm/ft ²(sample 4D).Result is shown in Table 2.Abbreviation AQ refers to containing aqueous phase, and the ORG that abridges refers to organic phase.See table 2, " phase " is continuous print, " sample " refers to operational conditions, " internals " refers to the material of internals, " nominal AQ " refers to that nominal contains aqueous phase flow rate, " nominal ORG " refers to nominal organic phase flow rate, and " total flux (ccm) " refers to the total flux of moisture and organic charging, and " total flux (gpm/ft ²) " refer to the total flux of per unit cross-sectional area.

table 2

Use the charging be made up of the fermented liquid close to fermentation ends, when tower does not run with having internals, the selection of external phase affects output of column.Contain with regard to aqueous phase, likely with about 25gpm/ft with regard to continuous print ²run (sample 2G and 2H).But adopting continuous print COFA phase, the problem of overflow is at 18gpm/ft ²occur (sample 2I).Result is shown in Table 3.

table 3

example 3

fermentation condition is to the effect of extraction column throughput

The character of fermented liquid is not static, but changes along with the carrying out of fermenting process.In fermentation, along with carbohydrate is by microbial metabolism, the concentration of carbohydrate reduces.This composition change in fermented liquid will change the viscosity of such as fermented liquid and capillary physical parameter, and they have impact to leaching process.Except the change of concentration, in the intermediate time, generate the CO of sizable amount ₂; And these CO ₂moisture for impact and organic liquid are passed the flowing of tower.

Be equipped with 1 " diameter glass of PTFE internals extraction column (Koch Modular Process Systems, Paramus, NJ) is used to process the fermented liquid from ethanol fermentation.Process some time point during the fermentation carries out.Organic extraction agent (COFA) is the external phase in tower, and fermented liquid passes through tower as drop.Before fermented liquid is introduced tower, make fermented liquid have passed a threeway in pipeline, be present in the CO in charging ₂bubble is removed by ventilating pit herein.

When with static internal components (without stirring), when fermented liquid (" the middle fermented liquid ") in gas evolution peak period is taken from use, with take from close to compared with the fermented liquid (" fermented liquid in latter stage ") during fermentation ends, observe the difference of performance.Use middle fermented liquid, achieve 14gpm/ft ²liquid throughput rate.With regard to latter stage fermented liquid, maximum throughput (before tower overflow) is for being less than 9gpm/ft ².Significant difference is there is in the size and outward appearance of water-containing drop.Compared to middle fermented liquid, with regard to latter stage fermented liquid, the drop size containing aqueous phase is obviously larger.

example 4

isobutanol concentration is to the effect of extraction column efficiency

In typical fermenting process, the level of product changes in time.This dynamic change in concentration can affect the mass transfer of leaching process.

In order to show the effect of isobutanol concentration, be equipped with 1 " diameter glass of stainless steel internals extraction column (Koch Modular Process Systems, Paramus, NJ) is used to the fermented liquid from fermentation of pack processing containing about 3g/L isopropylcarbinol.Fermented liquid forms external phase in extractor, and organic extraction agent (COFA) passes through tower as drop simultaneously.Although CO ₂generation substantially stop, before charging enters extraction column, make fermented liquid have passed a threeway in pipeline, be present in any CO in charging ₂bubble is removed herein.

By the sample of stream that liquid chromatography (LC) or gas-chromatography (GC) analyze charging for isopropylcarbinol and leave.Result is shown in Table 4.Carry out mass balance, and use Kremser formulae discovery to balance transfer plate height (HETS).With regard to two data points of above-mentioned fermented liquid, HETS value is 10 and 13 feet.

Then isopropylcarbinol is added into fermented liquid, so that concentration is adjusted to 20g/L.Carry out extracting test, and found that HETS is 18 feet from these data.The value that above-mentioned value ratio blank run liquid obtains high about 50%, and with the data consistent (see Figure 15) using the rare tower being doped with about 20g/L isopropylcarbinol to heat up in a steamer thing to obtain.

table 4

example 5

use the ISPR of outside extraction column

Fermented liquid from isobutanol fermentation (10 liters of scales) is circulated to 5/8 " diameter platform-type tower.Extraction solvent (COFA) is circulated to by from extraction agent reservoir tower.Carried out control fermentation, wherein the COFA of certain volume is added into fermentor tank, with continuously from broth extraction isopropylcarbinol.

tower has run twice during the fermentation.First time run be fermentation the time point of 4 to 7 hours, and second time run be fermentation the time point of 22 to 33 hours.Such as pO has all been monitored to two kinds of fermentations ₂with the parameter of pH.With do not use the contrast of tower runs to be compared, and is employing pO measured in the operation of tower ₂lower.Just tower and in contrast, absolute pH value is similar, but is differences with regard to two kinds of pH curves running speech.Relative to the peak of just single in contrast gradual change, the operating pH of tower comparatively early reaches peak value, flattens and then reaches peak value.

From tower analyzes two aliquots containigs (each 1.8 liters) of Extraction solvent.Take out sample from each aliquots containig, and analyze isopropylcarbinol content.Use the amount of the isopropylcarbinol that tower is produced in fermentation is much the same with what produce in control fermentation.Use the fermentation of tower creates total 82.4 grams of isopropylcarbinols, about 34 grams be in the organic phase of 3.6 liters and 48 grams be containing in aqueous phase.Contrast (30% organic phase is added into fermented liquid by volume) creates 90g/L, 60 grams in the organic phase of 3 liters and 30 grams containing in aqueous phase.Due to relative to the initial extraction of non-zero during tower runs, lights the existence of COFA from time zero in control fermentation tank, isobutanol concentration in contrast containing being lower in aqueous phase.Just tower is constantly little 22, and isopropylcarbinol is produced rapider from the extraction of fermentor tank than it.From contrast and tower generates glucose curve.Above-mentioned curve is similar, shows that the metabolism and growth of cell is much the same.Result is shown in Figure 16 A and 16B.Bracket represents the time point (4 to 7 hours and 22 to 33 hours) that tower runs.

example 6

use the ISPR of mixer-settler

The active-fermented broth that external mix settling vessel system is used to from comprising the microorganism (that is, isopropylcarbinol bacterium) producing isopropylcarbinol removes isopropylcarbinol continuously.This research employs about 100 liters and is vaccinated with the fermented liquid of microorganism (that is, isopropylcarbinol bacterium) producing isopropylcarbinol.The content of fermentor tank by from fermentor tank recirculated through mixer-settler extraction system.Comprise and do not used with single-pass manner containing the extraction agent of the COFA of isopropylcarbinol.

Test two kinds of static mixers.Great majority test uses static stainless-steel mixer (diameter 1/2 ", with 36 hybrid elements).Between the run the 12nd and 24 hours, employ plastic mixer (StaMixCo HT-11-12.6-24, StaMixCo LLC, Brooklyn, NY).Fermented liquid and COFA are fed to the relative both sides of threeway, and mixture flows through static mixer from it.The material leaving static mixer is fed to settling vessel.Settling vessel is made up of the glass pot of five liters.Leaching material pipe by the top of settling vessel, periphery, and extends downward the only about half of place of settling vessel.Take out organic phase by a mouth at settling vessel top place, shift out fermented liquid from the bottom of settling vessel simultaneously.Settling vessel is equipped with as moisture-organic interface provides the gentle agitator mixed, and to help the separation of these two kinds of liquid phases, thus solid is minimized gathering of interface.In table 5, and Figure 17 shows the isopropylcarbinol realized during the fermentation to the data presentation of collecting in above-mentioned operational process removes speed.As seen from above-mentioned data, the isopropylcarbinol level in moisture fermented liquid keeps relative constancy, shows that isopropylcarbinol is to be removed by from fermented liquid by the approximately identical speed of producing with it.See table 5, " elapsed time " is the time from fermentation, and " AQ flow " is aqueous feed flow, and " ORG flow " is organic feed rate, " iB in AQ charging " is the isopropylcarbinol in aqueous feed, and " iB in ORG product " is the isopropylcarbinol in rich organic product.

table 5:

* A:1/2 " stainless steel mixing tank, 32 elements

B:StaMixCo HT-11-12.6-24, plastic mixer

example 7

on line, line is other and measure in real time

The wine with dregs stream prepared from maize raw material is imported into three-phase centrifuge, generates three kinds of streams: wine with dregs, Semen Maydis oil and wet cake.The measurement of on line or line side process is used to, and such as, improves the recovery of starch/sugar and the quality of Semen Maydis oil, and the amount of the starch/sugar extracted from wet cake is maximized.Real-time measurement is used to, and such as, controls the interpolation to serum pot of adverse current, process water or water, to maintain starch/sugared concentration set point.Use the water of minimum interpolation, and reduce the hydraulic load on three-phase centrifuge, the amount of the starch/sugar extracted from wet cake is maximized.

The Fourier transform infrared spectroscopy (FTIR) (it allows the measurement to the existence of solid) of popping one's head in diamond attenuated total reflectance attenuated total refraction (ATR) is used to analyze corn mash sample.By collecting the spectrum of the standard model that wherein total starch/sugar determination has used HPLC to complete, calibrate FTIR.HPLC data are used to build multivariate offset minimum binary (PLS) model for FTIR.Have collected FTIR spectrum and obtain total starch concentration.Figure 18 shows the starch concentration scope for calibrating FTIR.

The corn mash with the average initial concentrations of 250g/L is fed to three-phase centrifuge.Wet cake is subsequently repulped, and measures the concentration of starch on two kinds of samples: 80g/L and 70g/L.Then use three-phase centrifuge to be separated above-mentioned slurries, and make wet cake repulped.The starch concentration of these slurries is measured as 28.9g/L.Result is shown in Figure 19.These measure the proper amount of water being used to determine to make wet cake repulped at every one-phase.The optimization of the interpolation of water makes starch concentration maximize, and the hydraulic load of separating step is minimized.Near infrared spectrum (NIR) is used to measure the water content of wet cake.

Real-Time Monitoring Semen Maydis oil quality, and these data are used to the variable (such as, feeding rate, g power, entrance flow rate, rolling speed) controlling three-phase centrifuge.By being brought into the concentration of the water of Semen Maydis oil in monitoring sepn process, measure the quality of the Semen Maydis oil generated by three-phase centrifuge.The FTIR with diamond ATR probe is used to the spectrum collecting Semen Maydis oil when Semen Maydis oil have left three-phase centrifuge.The detectability of diamond ART probe method with regard to water is used to be about 500ppm.By using the flow cell with longer active path length, achieve lower detectability.

Figure 20 comprises a series of infrared spectras of Semen Maydis oil, and described Semen Maydis oil comprises from exceeding percentage ratio level to being low to moderate in the water concentration of the ppm scope of 100.Water concentration uses the-OH stretching region between 3700cm-1 and 3050cm-1 to measure.Data show, process FTIR can be used to generate real-time W/O concentration data.Real-time Water concentration data can be used to the process variable (such as, feeding rate, g power, entrance flow rate, rolling speed) controlling three-phase centrifuge.The operation of three-phase centrifuge can be controlled, and to produce first-chop Semen Maydis oil or to make throughput-maximized, is no more than the set(ting)value of water simultaneously.

Extract real-time agent monitoring is used to the heat collapse detecting and monitor extraction agent.Be used to trigger to the reparation of extraction agent or contaminated extraction agent from the removing technique to the real-time detection of these heat collapse products.

Figure 21 is the example of the real-time measurement of COFA to rich isopropylcarbinol.Use the Metter-Toledo ReactIR of the diamond ATR sampling probe adopted in flow cell ^tM247 have collected data.From 1 inch diameter the outlet of tower have collected COFA stream, and uses peristaltic pump to deliver to FTIR.Be doped with the COFA standard substance of isopropylcarbinol by generation and generate multivariate PLS model, having calibrated FTIR.

example 8

drop size is analyzed

This example describes the analysis to liquid extraction agent drop after the process flow that will comprise fermented liquid and extraction agent (COFA) imports static mixer.After process flow leaves static mixer about 24 hours, will probe (Mettler-Toledo, LLC, Columbus OH) insertion process stream. probe is used to collect an image in every two minutes in fermentation operational process.Image shows size at the COFA drop of 50 to 80 μm of diameter ranges and the size CO in the scope of 200 to 400 μm of diameters ₂the existence of bubble.Be used to guarantee that drop keeps below specific mean diameter to the monitoring comprising drop size in the process flow of fermented liquid and COFA after static mixer, to guarantee the good mass transfer of isopropylcarbinol to COFA drop.

also be used to before stream is back to fermentor tank the COFA drop imaging in poor fermentation broth stream.The instruction of the amount of the COFA being back to fermentor tank to the detection of COFA drop in above-mentioned stream. probe is used to the image collecting once this stream during the fermentation for every two minutes.Unlike the stream leaving static fermentation tank, poor fermentation broth stream has less and less drop (10-40 μm).These measure the feasibility illustrating use procedure imaging to monitor the amount of the COFA being back to fermentor tank.

Real-time average droplet size data from two sampling spots are all used to monitor being separated of fermented liquid and COFA.The little concentration of COFA drop detected in poor fermented liquid recirculation flow (after the extraction of isopropylcarbinol) or the increase of quantity can be that be separated deterioration and the too much COFA of fermented liquid and COFA is leaving the instruction of fermentor tank.In order to improve the quality that is separated and reduce the quantity or the concentration that are back to the COFA drop of fermentor tank in poor fermentation broth stream, after static mixer, increase average COFA drop size.

The other process variable that can affect average COFA drop size comprises the concentration of polysaccharide in fermentation liquid, the ratio of fermented liquid to COFA and the total flow rate by static mixer.Along with fermentation is carried out, flow rate and/or fermented liquid can be changed the ratio of COFA, to maintain constant average COFA drop size.

example 9

extractor designs

This example describes the method designing extensive extractor unit.The data extracted from pilot scale are used to the size estimating extensive extractor unit.The impact that the stream of presence or absence on the extractor unit from pilot scale determining flow rate, stir speed (S.S.) and internals is separated.In fixing temperature, with the process change total flux of fermentation and fermented liquid flow to the ratio of extraction agent flow, and observe the condition being separated and stopping.What have recorded the extractor unit flow meter area of every square feet realizes flow to the maximum of extractor.Following formula is used to the flow determining per unit area:

$U=\frac{F}{A}$ (formula 1)

The flow (gallon per minute/square feet) of U=per unit area

F=fermented liquid and extraction agent are to the total flux (gallon per minute) of extractor unit

The cross-sectional area (square feet) of A=flow direction

For extraction column, this is calculated as

D=tower diameter (foot).

Use following formula, be have estimated the diameter of large scale fermentation tank unit by fermented liquid and extraction agent to the desired flow of extractor unit:

(formula 2)

F _{on a large scale}=fermented liquid and extractor are to the total flux (gallon per minute) of large scale fermentation tank.

The height of pilot scale extractor unit be the different flow states comprising different flow rates, have and do not have internals, different stir speed (S.S.)s and different product alcohol concentration under measure.Use this data, use Kremser formula to estimate theoretical plate number (Seader and Henley, the Separation Process Principles realized with the height of extractor unit, 2nd edition, John Wiley & Sons, 2006,358-359 page):

$n=\frac{\ln [\left(\frac{x_f-\frac{y_s}{m}}{x_n-\frac{y_s}{m}}\right)(1-\frac{1}{E})+\frac{1}{E}]}{\ln \left(E\right)}$ (formula 3)

E=extraction factor

F _{fermented liquid}=fermented liquid is to the flow (gallon per minute) of extractor unit

F _{extraction agent}=extraction agent is to the flow (gallon per minute) of extractor unit

M=product alcohol fermented liquid and extraction agent mutually in partition ratio (the every g/L of g/L)

The concentration of Xf=product alcohol in fermented liquid charging (g/L)

The concentration of Xn=product alcohol in the fermented liquid leaving extractor unit (g/L)

The concentration of Ys=product alcohol in the extraction agent entering extractor unit (g/L)

The theoretical plate number that n=realizes with the height of extractor unit

Formula 3 is only effective when E ≠ 1.

By will pilot scale extract in the height of extraction column that uses divided by the theoretical plate number realized in given experiment, give the theoretical plate height with regard to this extractor unit.The operational conditions using to expect on a large scale in formula 4 has estimated the theoretical plate number realized needed for large-scale separation:

$n=\frac{\ln [\left(\frac{x_f^{\′}-\frac{t_s^{\′}}{m}}{x_n^{\′}-\frac{y_s^{\′}}{m}}\right)(1-\frac{1}{E^{\′}})+\frac{1}{E^{\′}}]}{\ln \left(E^{\′}\right)}$ (formula 4)

Wherein ' represent the condition of extensive extractor unit.

The theoretical plate number measured for similar flow condition and the product of theoretical plate height provide the estimation of the total height to extensive extractor unit.The flow using dynamic fermentation model assessment to expect with extensive extractor unit and concentration (such as, the people such as Daugulis, Biotech.Bioeng.27:1345-1356,1985).

Although multiple embodiment of the present invention is as described herein, be to be understood that they only exist by way of example, and unrestricted.Those skilled in the relevant art be it is evident that and can not depart from the multiple modification can carrying out form and details under the spirit and scope of the invention to it.Therefore, range of the present invention and scope by the restriction of any above-mentioned exemplary embodiment, but should only should not limit according to following claim and equivalents thereof.

The all announcements mentioned in this manual, patent and patent application all indicate the state of the art of those skilled in the art in the invention, and be incorporated herein by reference to as each independent announcement, patent or patent application by specifically and be indicated as being the same degree be incorporated to way of reference individually.

Claims (39)

1., for reclaiming a method for product alcohol from fermented liquid, described method comprises:

Providing package contains the fermented liquid of microorganism, the raw product alcohol of wherein said production by biological;

Described fermented liquid is contacted with at least one extraction agent; And

Reclaim described product alcohol.

2. method according to claim 1, the contact of wherein said fermented liquid and at least one extraction agent occur in fermentor tank, external unit or both in.

3. method according to claim 2, wherein said external unit is extractor.

4. method according to claim 3, wherein said extractor is selected from siphon pipe, decantor, whizzer, gravitational settler, phase splitter, mixer-settler, column extractor, centrifugal extractor, stirs extractor, hydrocyclone, spray tower or their combination.

5. method according to claim 1, wherein said at least one extraction agent is selected from C ₇-C ₂₂fatty alcohol, C ₇-C ₂₂lipid acid, C ₇-C ₂₂the ester of lipid acid, C ₇-C ₂₂alkanoic, C ₇-C ₂₂fatty amide and their mixture.

6. method according to claim 1, wherein said at least one extraction agent is selected from oleyl alcohol, behenyl alcohol, hexadecanol, lauryl alcohol, tetradecyl alcohol, stearyl alcohol, oleic acid, lauric acid, linolic acid, linolenic acid, tetradecanoic acid, stearic acid, sad, capric acid, undeeanoic acid, Myristicin acid methylester, Witconol 2301, 1 nonyl alcohol, 1-decanol, 2-undecyl alcohol, 1-aldehyde C-9, 1-undecyl alcohol, the undecyl aldehyde, lauryl aldehyde, the 2-methyl undecyl aldehyde, oleylamide, sub-oleylamide, palmitic amide, stearylamide, 2-ethyl-1-hexanol, 2-hexyl-1-decanol, 2-octyl group-1-lauryl alcohol, and their mixture.

7. method according to claim 6, is wherein added into described fermented liquid by hydrophilic solutes.

8. method according to claim 7, wherein said hydrophilic solutes is selected from polyol, polycarboxylic acid, polyol compound, ion salt or their mixture.

9. method according to claim 1, the contact of wherein said fermented liquid and at least one extraction agent occurs in two or more external units.

10. method according to claim 1, the contact of wherein said fermented liquid and at least one extraction agent occurs in two or more fermentor tanks.

11. methods according to claim 10, wherein said fermentor tank comprises for improving the internals or device that are separated.

12. methods according to claim 11, wherein said internals or device are selected from coalescer, baffle plate, perforation plate, hole, tilted plate separator, cone-shaped body or their combination.

13. methods according to claim 1, wherein use the extraction of measuring in real time and monitoring product alcohol.

14. methods according to claim 13, wherein monitor the extraction of product alcohol by measuring to be separated in real time.

15. methods according to claim 14, the composition wherein by measuring the speed, extraction agent drop size and/or the fermented liquid that are separated is monitored and is separated.

16. methods according to claim 15, are wherein monitored by conductivity measurement, dielectric medium measurement, viscoelasticity measurement or ultrasonic measurement and are separated.

17. methods according to claim 1, wherein providing package occurs in two or more fermentor tanks containing the fermented liquid of microorganism.

18. methods according to claim 1, wherein said product alcohol is selected from ethanol, propyl alcohol, butanols, amylalcohol, hexanol and fusel.

19. methods according to claim 1, wherein said microorganism comprises butanol biosynthetic pathway.

20. methods according to claim 19, wherein said butanol biosynthetic pathway is n-butyl alcohol biosynthetic pathway, 2-butanol biosynthetic pathway or isobutanol biosynthetic pathway.

21. methods according to claim 19, wherein said microorganism is recombinant microorganism.

22. methods according to claim 1, described method is further comprising the steps of:

Providing package is containing the raw slurry of fermentable carbon source, undissolved solid, oil and water;

Be separated by described raw slurry, form the aqueous solution that (i) comprises fermentable carbon source thus, (ii) comprises the wet cake of solid, and (iii) oil; And

The described aqueous solution is added into described fermented liquid.

23. methods according to claim 22, wherein by described profit solution to form lipid acid.

24. methods according to claim 23, wherein make described fermented liquid contact with described lipid acid.

25. methods according to claim 23, wherein said oil is by enzymic hydrolysis.

26. methods according to claim 25, wherein said enzyme is one or more lipase or Phospholipid hydrolase.

27. methods according to claim 22, wherein generate described raw slurry by hydrolysis material.

28. methods according to claim 27, its Raw is selected from rye, wheat, corn, sugarcane, barley, Mierocrystalline cellulose or ligno-cellulosic materials or their combination.

29. methods according to claim 22, are wherein separated described raw slurry by following: decanter type bowl formula is centrifugal, three phase centrifugation, dish stacked are centrifugal, filter centrifugation, decanter type are centrifugal, filtration, vacuum filtration, belt filter, press filtration, the filtration of use screen cloth, screening, grid filter, the filter of porous grid, flotation, hydrocyclone, pressure filter, helical pressure device, gravitational settler, vortex separator or their combination.

30. methods according to claim 22, being wherein separated described raw material is single step process.

31. methods according to claim 22, wherein by described wet cake and described aqueous solution.

32. methods according to claim 22, described method also comprises makes the described aqueous solution and the catalyst exposure oil in the described aqueous solution being changed into lipid acid.

33. methods according to claim 32, are wherein added into described fermented liquid by the described aqueous solution and lipid acid.

34. methods according to claim 32, wherein by described catalyst deactivation.

35. 1 kinds of systems, described system comprises:

One or more fermentor tank, described fermentor tank comprises:

For receiving the entrance of raw slurry; With

For discharging the outlet of the fermented liquid comprising product alcohol; With

One or more extractor, described extractor comprises:

For receiving the first entrance of described fermented liquid;

For receiving the second entrance of extraction agent;

For discharging the first outlet of poor fermented liquid; With

For discharging the second outlet of rich extraction agent.

36. systems according to claim 35, described system also comprises:

One or more liquefaction unit;

One or more tripping device; With

Optionally, one or more washing system.

37. systems according to claim 36, wherein said tripping device is selected from that decanter type is centrifugal, three phase centrifugation, dish stacked are centrifugal, filter centrifugation, decanter type are centrifugal, filtration, vacuum filtration, belt filter, press filtration, membrane filtration, micro-filtration, the filtration of use screen cloth, screening, grid filter, the filter of porous grid, flotation, hydrocyclone, pressure filter, helical pressure device, gravitational settler, vortex separator and their combination.

38. systems according to claim 35, wherein said system also comprises measuring apparatus on line.

39. according to system according to claim 38, and on wherein said line, measuring apparatus is selected from particle-size analyzer, Fourier transform infrared spectroscopy, near infrared spectrum, Raman spectrum, high pressure liquid chromatography, viscometer, specific gravity hydrometer, tonometer, drop size analyser, pH meter, dissolved oxygen probe or their combination.