CN102803498B - Biomass hydrolysis process - Google Patents
Biomass hydrolysis process Download PDFInfo
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- CN102803498B CN102803498B CN201080058472.2A CN201080058472A CN102803498B CN 102803498 B CN102803498 B CN 102803498B CN 201080058472 A CN201080058472 A CN 201080058472A CN 102803498 B CN102803498 B CN 102803498B
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- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 235000019621 digestibility Nutrition 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 238000009837 dry grinding Methods 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000006052 feed supplement Substances 0.000 description 1
- 238000003872 feeding technique Methods 0.000 description 1
- 150000002240 furans Chemical class 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 229950006191 gluconic acid Drugs 0.000 description 1
- 150000008131 glucosides Chemical group 0.000 description 1
- 229960002989 glutamic acid Drugs 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 239000005431 greenhouse gas Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000000887 hydrating effect Effects 0.000 description 1
- IXCSERBJSXMMFS-UHFFFAOYSA-N hydrogen chloride Substances Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 description 1
- 229910000041 hydrogen chloride Inorganic materials 0.000 description 1
- 238000010335 hydrothermal treatment Methods 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 150000007517 lewis acids Chemical class 0.000 description 1
- 239000012978 lignocellulosic material Substances 0.000 description 1
- 239000004571 lime Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 238000001471 micro-filtration Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000003801 milling Methods 0.000 description 1
- 238000006213 oxygenation reaction Methods 0.000 description 1
- 239000010893 paper waste Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 150000002972 pentoses Chemical class 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 238000007781 pre-processing Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 235000019192 riboflavin Nutrition 0.000 description 1
- 239000002151 riboflavin Substances 0.000 description 1
- 229960002477 riboflavin Drugs 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- APSBXTVYXVQYAB-UHFFFAOYSA-M sodium docusate Chemical compound [Na+].CCCCC(CC)COC(=O)CC(S([O-])(=O)=O)C(=O)OCC(CC)CCCC APSBXTVYXVQYAB-UHFFFAOYSA-M 0.000 description 1
- 235000014347 soups Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-L sulfite Chemical compound [O-]S([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-L 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 238000001238 wet grinding Methods 0.000 description 1
- OENHQHLEOONYIE-JLTXGRSLSA-N β-Carotene Chemical compound CC=1CCCC(C)(C)C=1\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C OENHQHLEOONYIE-JLTXGRSLSA-N 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a biomass process comprising split dosage of the cellulose hydrolysing enzyme.
Description
Technical field
The present invention relates to technique, described technique is included in and uses the gradation of cellulolytic enzyme to drop into (split dosing) in the hydrolysis of lignocellulose-containing materials.
Background of invention
Due to the limited deposit of fossil oil and the worry about greenhouse gas emission, exist such as, about use renewable energy source, tunning, the increasing concern of biological example ethanol.The alcohol production coming authigenic material and lignocellulose-containing materials is known in the art, and can comprise pre-treatment, the enzymically hydrolyse of lignocellulose-containing materials and be fermented into ethanol.The cost of the enzyme used in hydrolysis has been considered as the restriction of the rentability of this type of technique.Therefore, need to provide improvement and the substrate of more effective technique for lignocellulose-containing materials enzymically hydrolyse is become to be suitable for ferment.
Present inventor has surprisingly been found that at present by cellulolytic enzyme being divided at least 2 dosage and adding described dosage in different steps, significantly can improve the hydrolysis of high level cadre's solid biomass slurry.
Summary of the invention
The present invention relates to such technique, it comprises the lignocellulose-containing materials of enzymically hydrolyse high level cadre solids concn, and optionally hydrolysate is fermented into tunning, preferred alcohol, and wherein cellulolytic enzyme two or more stages in the process add.
Correspondingly, in first, the present invention relates to the technique for producing hydrolysate from lignocellulose-containing materials, it comprises the steps, (a) pre-treatment lignocellulose-containing materials; B () forms the slurry comprising water, pretreated lignocellulose-containing materials and cellulolytic enzyme, slurry described in (c) and incubation, d () adds more lytic enzymes, (e) slurry described in incubation, to produce hydrolysate, wherein said slurry has the solid bulk concentration of at least 25%.
Detailed Description Of The Invention
Lignocellulose-containing materials
Term " lignocellulose-containing materials " is used in reference to the material primarily of Mierocrystalline cellulose, hemicellulose and xylogen composition in this article.Lignocellulose-containing materials is commonly referred to " biomass ".
The structure of ligno-cellulose is not that enzymically hydrolyse can be directly close.Therefore, ligno-cellulose such as must carry out pre-treatment by acid hydrolysis under the felicity condition of pressure, humidity and temperature, so that the xylogen that breaks seals and destroys cellulosic crystal structure.This causes solubilising and the saccharification of hemicellulose fraction.Mierocrystalline cellulose fraction can be hydrolyzed subsequently, such as, be hydrolyzed by cellulase enzymatic, and so that carbohydrate polymer is changed into monose and oligosaccharides, it can be fermented into required tunning, such as ethanol.Optionally, tunning is reclaimed, such as, is reclaimed by distillation.
Any lignocellulose-containing materials all takes according to the present invention.Lignocellulose-containing materials can be any material containing ligno-cellulose.In a preferred embodiment, lignocellulose-containing materials contain at least 30 % by weight, preferred at least 50 % by weight, more preferably at least 70 % by weight, even more preferably at least 90 % by weight ligno-celluloses.Be to be understood that lignocellulose-containing materials can also comprise other moietys, such as cellulose materials, comprise Mierocrystalline cellulose and hemicellulose, and other moietys can be comprised, such as proteinacious material, starch, sugar such as fermentable sugars and/or not fermentable sugars.
Lignocellulose-containing materials is general such as to be found in the leaf of the stem of plant, leaf, skin, shell (hull, husk) and cob or tree, branch and timber.Lignocellulose-containing materials can be but be not limited to herbaceous material, agricultural residue, forestry residue, municipal solid waste, waste paper and slurry and paper mill residue.Be to be understood that lignocellulose-containing materials can be the form of the Plant cell wall material containing xylogen, Mierocrystalline cellulose and hemicellulose in mixed-matrix in this article.
Lignocellulose-containing materials can comprise maize straw, hardwood such as aspen (poplar) and birch (birch), soft wood is pine such as, switchgrass (switch grass), cereal straw (cereal straw) and/or shell (husk) are such as from the straw of rice, wheat, barley, rye etc., municipal solid waste (MSW), industrial organic waste, office's paper using, wood chip (wood chip), bagasse, paper or slurry process refuse or its mixture.
In a preferred embodiment, lignocellulose-containing materials is maize straw.In another is preferred, lignocellulose-containing materials is zein fiber.
Pre-treatment
Lignocellulose-containing materials can carry out pre-treatment in any way as suitable.Pre-treatment can be carried out before hydrolysis and/or fermentation.Pretreated object is separated and/or release Mierocrystalline cellulose, hemicellulose and/or xylogen, and this mode improves hydrolysis rate.Pretreatment process such as wet oxidation and oxygenation pretreatment target xylogen, and diluted acid and from target hydrolase to hemicellulose.Vapor explosion is the cellulosic pretreated example of target.
According to the present invention, pre-treatment step (a) can be the conventional pre-treatment step using technology well-known in the art.In a preferred embodiment, pre-treatment is carried out in aqueous slurry.Lignocellulose-containing materials can so that between 10-80 % by weight, preferably, between 20-70 % by weight, especially, between 30-60 % by weight, the amount of such as about 50 % by weight exists in preprocessing process.
Pre-treatment was carried out before hydrolysis and/or fermentation.
Term " chemical treatment " refers to promote the separation of Mierocrystalline cellulose, hemicellulose and/or xylogen and/or any Chemical Pretreatment of release.The example of suitable Chemical Pretreatment comprises by such as diluted acid, lime, alkali, organic solvent, ammonia, sulfurous gas, carbon dioxide treatment.Further, the aquathermolysis that wet oxidation and pH control also is the Chemical Pretreatment considered.
In a preferred embodiment, Chemical Pretreatment is acid treatment, more preferably continuously diluted acid and/or weak acid treatment, such as, by sulfuric acid or another kind of organic acid and/or mineral acid treatment, described acid is acetic acid, citric acid, tartrate, succsinic acid, hydrogenchloride or its mixture such as.Other acid can also be used.Weak acid treatment means process pH and is in the scope of 1-5, preferred pH 1-3.In one particular embodiment, acid concentration is in the scope of 0.1-2.0 % by weight acid (preferably sulfuric acid).Acid can contact with lignocellulose-containing materials, and mixture can at 160-220 DEG C, and the temperature hold-in range of such as 165-195 DEG C of scope is in the period of such as 1-60 minute such as 2-30 minute or 3-12 minute.The interpolation of strong acid such as sulfuric acid can be applied to remove hemicellulose.Which enhance cellulosic digestibility.
Also contemplate other technologies.Cellosolve process has shown about 90% cellulose conversion has been become glucose.Also shown when ligno-cellulose is destructurized, enzymically hydrolyse can greatly be strengthened.Alkali H
2o
2, ozone, organic solvent (organosolv) (be used in (Al) in moisture alcohol
2sO
4, FeCl
3, Lewis acid), glycerine, diox, phenol or ethylene glycol belong to known destruction cellulosic structure and the solvent of facilitation of hydrolysis (people Bioresource Technology 96 (2005), the 673-686 page such as Mosier).
With alkali such as NaOH, Na
2cO
3and/or the alkali electroless pre-treatment of ammonia etc. also takes according to the present invention.The pretreatment process of ammonia is used to describe in such as WO2006110891, WO200611899, WO200611900, WO2006110901 (it is hereby incorporated by).
Wet oxidation techniques relates to the use of oxygenant, such as: based on the oxygenant etc. of sulphite.The example of solvent pre-treatment comprises with process such as DMSO (methyl-sulphoxide).Chemical Pretreatment generally carries out 1-60 minute, such as 5-30 minute, but depends on that material to be pre-treated can carry out shorter or longer period.
Other examples of appropriate pretreatment method are by following description: the people such as Schell (2003) Appl.Biochemand Biotechn. 105-108 rolls up, 69-85 page, people Bioresource Technology96 (2005) 673-686 such as Mosier, the people such as Ahring are in WO2006032282 and WO200160752, the people such as Foody are in WO2006034590, with people such as Ballesteros in US publication 20020164730, described reference is all incorporated herein by reference at this.
Term " mechanical pretreatment " refers to any machinery (or physics) process promoting to be separated and/or to discharge Mierocrystalline cellulose, hemicellulose and/or xylogen from lignocellulose-containing materials.Such as, mechanical pretreatment comprises the grinding of multiple type, irradiation, decatize/vapor explosion, wet oxidation and other hydrothermal treatment consists.
Mechanical pretreatment comprises pulverizing (machinery of size reduces).Pulverizing comprises dry grinding, wet-milling and vibratory milling.Mechanical pretreatment can relate to high pressure and/or high temperature (vapor explosion).In one embodiment of the invention, high pressure to mean in 300-600psi scope, the pressure of preferred 400-500psi such as about 450psi.In one embodiment of the invention, high temperature to mean in about 100-300 DEG C scope, the preferred temperature of about 140-235 DEG C.In a preferred embodiment, mechanical pretreatment is batch process, and vapor gun hydrolysis instrument system (steam gun hydrolyzer system), it uses high pressure as defined above and high temperature.Sunds Hydrolyzer (can obtain from Sunds Defibrator AB (Sweden)) may be used for this.
In a preferred embodiment, chemistry and machinery two kinds of pre-treatment are carried out.Such as, pre-treatment step can relate to diluted acid or weak acid treatment and high temperature and/or pressure treatment.When needing, chemistry and mechanical pretreatment can be carried out continuously or simultaneously.
Correspondingly, in a preferred embodiment, chemistry and machinery two kinds of pre-treatment are implemented, to promote separation and/or the release of Mierocrystalline cellulose, hemicellulose and/or xylogen to lignocellulose-containing materials.
In a preferred embodiment, mechanical pretreatment is carried out before vapor explosion pre-treatment.
In a preferred embodiment, pre-treatment is carried out as diluted acid and/or weak acid vapor explosion step.In a further preferred embodiment, pre-treatment is carried out as ammonia Fibre Explosion step (or AFEX pre-treatment step).
Term " biological pretreatment " as used in the present invention refers to the biological pretreatment that any promotion Mierocrystalline cellulose, hemicellulose and/or xylogen are separated from lignocellulose-containing materials and/or discharge.Biological pretreatment techniques can relate to application lignin dissolution microorganism (see such as, Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol:Production and Utilization, Wyman, C.E., editor, Taylor & Francis, Washington, DC, 179-212; Ghosh, P. and Singh, A., 1993, Physicochemical and biological treatments for enzymatic/microbial conversion of lignocellulosic biomass, Adv.Appl.Microbiol.39:295-333; McMillan, J.D., 1994, Pretreating lignocellulosic biomass:a review, in EnzymaticConversion of Biomass for Fuels Production, Himmel, M.E., Baker, J.O. and Overend, R.P., editor, ACS Symposium Series 566, American Chemical Society, Washington, DC, the 15th chapter; Gong, C.S., Cao, N.J., Du, J. and Tsao, G.T., 1999, Ethanol production from renewable resources, in Advances in Biochemical Engineering/Biotechnology, Scheper, T., editor, Springer-Verlag Berlin Heidelberg, Germany, 65:207-241; Olsson, L. and Hahn-Hagerdal, B., 1996, Fermentation of lignocellulosic hydrolysates for ethanol production, Enz.Microb.Tech.18:312-331; And Vallander, L. and Eriksson, K.-E.L., 1990, Production of ethanol from lignocellulosic materials:State of the art, Adv.Biochem.Eng./Biotechnol.42:63-95).
The washing of pretreated lignocellulose-containing materials
When pre-treatment lignocellulose-containing materials, the degraded product to enzyme inhibition can be produced.Wash pretreated lignocellulose-containing materials and can improve enzymically hydrolyse with the inhibitor removing enzyme.
Inhibitor is ligno-cellulose degraded product, comprises lignin degradation products, cellulose degradation product and hemicellulose degradation product.Lignin degradation products can be substantially phenols.Hemicellulose degradation product comprises the furans from sugar (such as hexose and/or pentose), and described sugar comprises seminose, semi-lactosi, rhamnosyl, pectinose and wood sugar, comprises oligosaccharides.Think the mixture compound of enzyme inhibition being included in wood oligose (XOOs) or XOO and the soluble lignin existed in PCS liquid.According to the present invention, by washing the soluble compound removed from pretreated lignocellulose-containing materials enzyme inhibition with washing soln.Washing soln is preferably Aqueous wash solution.Washing soln can be the substantially pure aqueous solution, or has the water of additive such as stain remover and/or organic solvent of significant quantity, to improve extraction to the compound of enzyme inhibition and/or solubility.
Hydrolysis
Before fermentation and/or with fermentation simultaneously, by pre-treatment and the lignocellulose-containing materials enzymically hydrolyse of washing, so that Mierocrystalline cellulose and hemicellulose are decomposed saccharogenesis and/or oligosaccharides.
In a preferred embodiment, hydrolysis can be carried out as batch feeding technique, wherein by pretreated lignocellulose-containing materials (substrate) progressively feed supplement give such as containing the hydrating solution of enzyme.Pretreated lignocellulose-containing materials in one or more different batches, can be supplied to containing enzymic hydrolysis solution as one or more different Continuous Flow or as the combination of one or more different batches and one or more different Continuous Flow.Hydrolysis solid bulk concentration is from start to finish at least 20%, more preferably at least 25%, at least 26%, at least 27%, at least 28%, at least 29% or even at least 30%.
According to the present invention, use at least enzyme endoglucanase (EC 3.2.1.4); Cellobiohydrolase (EC3.2.1.91) and beta-glucosidase enzyme (EC 3.2.1.21) are hydrolyzed pretreated lignocellulose-containing materials.
The other enzyme can applied in hydrolytic process describes in hereafter " enzyme " part, and comprises zytase, arabinofuranosidase, acetyl xylan esterase, feruloyl esterase, glucuronidase, inscribe Galactanase, mannonase inscribe or circumscribed arabinase, inscribe or exogalactanase and the mixture of two or more thereof.
The pretreated lignocellulose-containing materials of washing directly or indirectly can be changed into fermentable sugars by one or more enzymes for being hydrolyzed, and it can be fermented into required tunning, such as ethanol.
According to the present invention, the amount of enzyme is divided at least two dosage, wherein apply when hydrolysing step is initial for first, and one or more doses remaining is applied subsequently in hydrolytic process.
In a preferred embodiment, the amount of enzyme is divided into two dosage of roughly equal size, the first half is applied when hydrolysing step is initial, and before application doses remaining, slurry incubation is about 12-48 hour, preferably about 18-36 hour, more preferably from about 20-30 hour.Hydrolysis continues other 48-72 hour.
Consider several dosage of wherein amount of enzyme being divided into roughly equal and/or different size equally and with the embodiment of roughly regular and/or irregular interval application in hydrolytic process.Enzyme even can as the dilution enzyme prepared product application added continuously in hydrolysis or the first half process be at least hydrolyzed.
The full duration of hydrolysis established technology step (c)+(e) is preferably between 72-120 hour.
Not bound by theory, think from using the positively effect of gradation enzyme dosage to be deactivation because of generally speaking enzyme in hydrolysing step process, described enzyme is cellobiohydrolase I and cellobiohydrolase II and the lower endoglucanase of degree especially.This is the reason observing positively effect under acting on the stronger high level cadre's solids concn of the shearing force of enzyme why wherein.Drop into by using gradation enzyme and postpone one or more dosage and add in incubation, the residual enzyme activity in the subsequent stage of hydrolysis is increased.
Enzymically treat is carried out under the condition that easily can be measured by those skilled in the art in suitable water surrounding.Preferably, to be hydrolyzed between 25 DEG C-70 DEG C, preferably between 40 DEG C-60 DEG C, the temperature of especially about 50 DEG C carries out.The pH of this optimal process in 3-8 scope, preferred pH 4-6, especially about pH 5 carry out.
In one embodiment, can by hydrolysate fermentation to produce tunning.
Consider that hydrolysis and fermentation can (SHHF technique) or continuous (SHF technique) be carried out simultaneously.
Fermentation
According to the present invention, by least one fermenting organism fermentation pretreated (and hydrolysis) lignocellulose-containing materials that fermentable sugars such as glucose, wood sugar, seminose and semi-lactosi directly or indirectly can be fermented into required tunning.
Fermentation is preferably carried out between 8-96 hour, preferred 12-72, more preferably 24-48 hour.In one embodiment, to ferment between 20-40 DEG C, the temperature of preferably 26-34 DEG C, particularly about 32 DEG C carries out.In one embodiment, pH is pH 3-6, preferably about pH 4-5.
What be preferred for ethanol fermentation is the yeast belonging to species yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), preferred pin is to high-level ethanol namely up to such as about 10,12 or 15 volume % ethanol or more, and such as 20 volume % ethanol have the bacterial strain of resistance.
According to the present invention it is considered that be hydrolyzed and ferment (SHF) simultaneously.In one embodiment, there is not the independent reservation phase for being hydrolyzed, meaning one or more lytic enzymes and adding together with fermenting organism.When ferment with hydrolysis carry out simultaneously time, temperature preferably between 26 DEG C-35 DEG C, more preferably between 30 DEG C-34 DEG C, such as about 32 DEG C.The temperature program(me) of at least two reservation phases being included in differing temps can be applied according to the present invention.
In the washing process of pretreated lignocellulose-containing materials, the sugar of dissolving can be accumulated in the Aqueous wash solution of recirculation.The C5 sugar that the sugar dissolved will comprise from hemicellulose degradation, such as wood sugar.These sugar can ferment with suitable fermenting organism, and C5 sugar can be changed into required tunning by described fermenting organism.This C5 fermentation can separately be carried out, or can add in the lignocellulose-containing materials of hydrolysis by the sugar of the dissolving accumulated in the Aqueous wash solution of recirculation, for C6 and the C5 fermentation of combining.This type of fermentation is preferably carried out with can ferment C6 sugar (such as glucose) and both at least one biologies of C5 sugar (such as wood sugar).Alternately, fermentation can be carried out to utilize at least two kinds of C6 or C5 sugar independent biologies by respective optimizing, or in a fermentation step under the condition of permission two kinds of biological fermentations, or as at least two fermentation steps, under each leisure allows the condition of one of described biology fermentation.
Technique of the present invention can as in batches, batch feeding or carry out as continuous processing.Preferably, fermentation step carries out as continuously fermenting.
reclaim
After fermentation, tunning can be separated with fermenting broth.Meat soup can carry out distilling to extract tunning, or tunning can be extracted from fermenting broth by micro-filtration or membrane filtration technique.Alternately, tunning can pass through stripping (stripping) and reclaims.Recovery method is well-known in the art.
tunning
Technique of the present invention may be used for producing any tunning.Especially the tunning considered comprises alcohol (such as ethanol, methyl alcohol, butanols); Organic acid (such as citric acid, acetic acid, methylene-succinic acid, lactic acid, glyconic acid); Ketone (such as acetone); Amino acid (such as L-glutamic acid); Gas (such as H
2and CO
2); Microbiotic (such as penicillin and tsiklomitsin); Enzyme; VITAMIN (such as riboflavin, B12, β-carotene); And hormone.
The product also considered comprises consumable alcohol Industrial products, such as beer and grape wine.In a preferred embodiment, tunning is alcohol, especially ethanol.Fuel alcohol/ethanol can be preferably according to the tunning such as ethanol that the present invention obtains.But in the case of ethanol, it can also be used as drinking alcohol.
fermenting organism
Term " fermenting organism " refers to any biology being suitable for producing required tunning, comprises bacterium and fungal organism.Sugar such as glucose directly or indirectly can be fermented according to the fermenting organism that the present invention is especially suitable, namely change into required tunning.What be also applicable to is the fermenting organism that C5 sugar such as xylose can be become required tunning.The example of fermenting organism comprises fungal organism, especially yeast.Preferred yeast comprises Saccharomyces sp (Saccharomyces spp.) bacterial strain, particularly yeast saccharomyces cerevisiae or saccharomyces uvarum (Saccharomyces uvarum) bacterial strain; Pichia (Pichia) bacterial strain, particularly pichia stipitis (Pichia stipitis), such as pichia stipitis CBS 5773; Mycocandida (Candida) bacterial strain, particularly Candida utilis (Candida utilis), Di Dansi candiyeast (Candida diddensii) or Candida boidinii (Candida boidinii) bacterial strain.Other yeast considered comprise zymomonas (Zymomonas); Hansenula (Hansenula), particularly Hansenula anomala (H.anomala); Genus kluyveromyces (Klyveromyces), particularly Kluyveromyces fragilis (K.fragilis); With Schizosaccharomyces (Schizosaccharomyces), particularly schizosaccharomyces pombe (S.pombe) bacterial strain.
The yeast be obtained commercially comprises such as ETHANOL RED
tMyeast (can obtain from Fermentis/Lesaffre, USA), FALI (can obtain from Fleischmann ' s Yeast, USA), SUPERSTART and THERMOSACC
tMfresh yeast (can from Ethanol Technology, WI, USA obtains), BIOFERM AFT and XR (can from NABC-North American Bioproducts Corporation, GA, USA obtains), GERT STRAND (can obtain from Sweden Gert Strand AB), and FERMIOL (can obtain from DSM Specialties).ANQI YEAST (can from Anqi yeast (CHIFENG) CO., LTD, China obtains).
Enzyme
Although specifically do not mention in the background of technique of the present invention, be to be understood that enzyme (and other compounds) uses with " significant quantity ".
cellulase: term " cellulase " is interpreted as comprising cellobiohydrolase (EC 3.2.1.91) as used herein, such as cellobiohydrolase I and cellobiohydrolase II, and endoglucanase (EC 3.2.1.4) and beta-glucosidase enzyme (EC 3.2.1.21).Cellulase is applied in hydrolysing step.
In order to effectively, the digestion of Mierocrystalline cellulose and hemicellulose needs the enzyme of synergistic several type.The enzyme of at least 3 classifications is required for cellulose conversion being become fermentable sugars: random endoglucanase (EC 3.2.1.4) of cutting cellulose chain; From the cellobiohydrolase (EC 3.2.1.91) of cellulose chain end cutting fibre disaccharide unit, and cellobiose and soluble cello-dextrins are changed into the beta-glucosidase enzyme (EC 3.2.1.21) of glucose.In the enzyme of these 3 classifications relating to cellulose biodegradation, cellobiohydrolase is for the cellulosic key enzyme of native crystal of degrading.Term " cellobiohydrolase I " is defined as Mierocrystalline cellulose 1 in this article, 4-beta fibers disaccharides Glycosylase is (also referred to as exoglucanase, exocellobiohydrolase or 1,4-beta fibers disaccharide-hydrolysing enzymes) active, as defined in the other EC 3.2.1.91 of enzyme, it is by the non-reducing end release cellobiose from chain, Isosorbide-5-Nitrae-β-D-glucoside bond hydrolysis in catalyse cellulose and cellotetrose.The definition of term " cellobiohydrolase II is active " is identical, except cellobiohydrolase II is from the reducing end attack of chain.
In endoglucanase (EC numbering 3.2.1.4) catalyse cellulose, derivatived cellulose (such as carboxymethyl cellulose and Natvosol), moss starch 1,4-β-D-glycosidic link, the β-1 mixed, 3 dextran such as cereal beta-D-glucans or xyloglucan and the interior hydrolysis containing the β-Isosorbide-5-Nitrae key in the other plant material of cellulosic part.Approved name is inscribe-Isosorbide-5-Nitrae-callose 4-dextran (glucano) lytic enzyme, but uses abbreviation term endoglucanase in this manual.
In a preferred embodiment, cellulase activity can derived from originated from fungus, such as Trichoderma (Trichoderma) bacterial strain, preferred Trichodermareesei (Trichoderma reesei) bacterial strain; Humicola (Humicola) bacterial strain, such as Humicola insolens (Humicola insolens) bacterial strain; Or Chrysosporium (Chrysosporium) bacterial strain, preferred Chrysosporium lucknowense bacterial strain.
In a preferred embodiment, Cellulase preparation comprises the polypeptide with cellulolytic enhancing activity (GH61A), is preferably disclosed in the polypeptide in WO2005074656.Cellulase preparation can comprise beta-glucosidase enzyme further, such as, be disclosed in US 60/832, the fusion rotein in 511.In one embodiment, Cellulase preparation also comprises CBH II, preferred autochthonal shuttle spore shell (Thielavia terrestris) cellobiohydrolase II CEL6A.In one embodiment, Cellulase preparation also comprises the cellulase derived from Trichodermareesei.In a preferred embodiment, Cellulase preparation is the Cellulase preparation using in embodiment 1 and be disclosed in WO 2008/151079, described Cellulase preparation comprises the cellulolytic enzyme derived from Trichodermareesei, be disclosed in WO2005074656 the polypeptide with cellulolytic enhancing activity (GH61A), and be disclosed in Aspergillus fumigatus (Aspergillus fumigatus) beta-glucosidase enzyme in WO 2008/151079.
Cellulase can be the product be obtained commercially, such as
1.5L or CELLUZYME
tMor Cellic Ctec
tM(all from Novozymes A/S, Denmark) or ACCELLERASE
tM1000 or ACCELLERASE
tM1500 (all from Genencor Int.).
Cellulase can drop into the scope of 0.1-100FPU/ gram of solid body (DS), preferred 0.5-50FPU/ gram of DS, especially 1-20FPU/ gram of DS.
Cellulase can drop into the scope of 0.1-10000mg zymoprotein (EP)/solid body of kg (DS), preferably 0.5-5000mgEP/kg DS, especially 1-2500mg EP/kg DS.
hemicellulase: hemicellulose can be decomposed by hemicellulase and/or acid hydrolysis, with discharge its five and hexose component.
Any hemicellulase being suitable for using in hydrolyzed hemicellulose can be used.Preferred hemicellulase comprises zytase, arabinofuranosidase, acetyl xylan esterase, feruloyl esterase, glucuronidase, inscribe Galactanase, mannonase inscribe or circumscribed arabinase, inscribe or exogalactanase and two or more mixture in them.Preferably, hemicellulase for using in the present invention is the hemicellulase of circumscribed effect, and more preferably, hemicellulase is the hemicellulase of so circumscribed effect, it has in the ability lower than pH 7, preferably the hydrolyzed under acidic conditions hemicellulose of pH 3-7.The example being suitable for the hemicellulose enzyme composition used in the present invention comprises VISCOZYME
tMand ULTRAFLO
tM(can from NovozymesA/S, Denmark obtains).
For what use in the present invention
zytase (EC 3.2.1.8)preferably inscribe-Isosorbide-5-Nitrae-beta-xylanase, and preferably belong to glycoside hydrolase Families 10 or 11 (GH10 or GH11).GH10 or GH11 the people such as Cantarel (2008) in Nucl.Acids Res.2009 37:D233-D238 and
www.cazy.orgupper definition.
Zytase can be any source, comprises Mammals, plant or animal-origin; But preferred zytase is microbe-derived.Especially, zytase can be can that derived from filamentous fungus or yeast.Preferably, zytase belongs to bacterial classification, Rhizomucor bacterial classification (Rhizomucor sp.) or Trichoderma derived from filamentous fungus such as aspergillus bacterium (Aspergillus sp.), Bacillus sp (Bacillus sp.), Humicola bacterial classification (Humicola sp.), myceliophthora bacterial classification (Myceliophotora sp.), Poitrasia.Zytase is preferably GH10 zytase.
Most preferably be disclosed in the zytase in WO 1994/021785 as xylanase I I derived from microorganism Aspergillus aculeatus (Aspergillus aculeatus).
The zytase that the zytase for the treatment of used washing soln applied in technique of the present invention is preferably fixing.If use revocable zytase, so it is with the amount of 0.001-1.0g/kg DS substrate, preferably with the amount of 0.005-0.5g/kg DS substrate and most preferably 0.05-0.10g/kg DS substrate adds.
Zytase can also with the amount of 0.001-1.0g/kg DS substrate, preferably with the amount of 0.005-0.5g/kg DS substrate and most preferably 0.05-0.10g/kg DS substrate adds in hydrolysing step of the present invention.
feruloyl esterase(EC 3.1.1.73) catalysis 4-hydroxy-3-methoxy cinnyl (asafoetide acyl) base, from the hydrolysis of the sugar of esterification, it typically is the pectinose in araboxylan.Suitable feruloyl esterase can derive from filamentous fungus (such as, Trichoderma, sub-Grifola frondosa Pseudomonas (Meripilus), Humicola, Aspergillus, fusarium (Fusarium)) or bacterium (such as bacillus) bacterial strain, such as derive from aspergillus niger (Aspergillus niger), such as by the people such as Faulds 1994, Microbiology, 140, the FAEIII feruloyl esterase that 779-787 page describes.
arabinofuranosidasethe hydrolysis of the non-reduced α of the end-L-arabinofuranosidase glucosides residue in (EC 3.2.1.55) catalysis α-L-cytosine arabinoside.
galactanase(EC 3.2.1.89), arabinogalactan endo-Isosorbide-5-Nitrae-beta-galactosidase enzymes, the interior hydrolysis of the Isosorbide-5-Nitrae-D-semi-lactosi glycosidic bond in catalysis arabogalactan.
polygalacturonasethe hydrolysis of Isosorbide-5-Nitrae-α-D-galacturonic acid (galactosiduronic) key in (EC 3.2.1.15) catalysis pectate and other polygalacturonic acids.
xyloglucanasethe hydrolysis of catalysis xyloglucan.
Hemicellulase can add with the amount of effective hydrolyzed hemicellulose, such as, with the amount of the solid body of about 0.001-0.5 % by weight (DS), and more preferably from about 0.05-0.5 % by weight DS.
Materials and methods
enzyme:
Cellulase preparation comprises the cellulolytic enzyme derived from Trichodermareesei, is disclosed in WO2005074656 the polypeptide with cellulolytic enhancing activity (GH61A), and is disclosed in the Aspergillus fumigatus beta-glucosidase enzyme in WO2008/151079.Described Cellulase preparation is disclosed in WO2008/151079.
Embodiment 1
Maize straw is used vapor explosion pre-treatment 5 minutes at 205 DEG C.Obtained PCS (pretreated maize straw) is used tap water, to remove soluble inhibitors.The PCS of batch feeding hydrolysis through washing carries out with 15%, 20% and 30%TS (total solids) respectively.All batch feedings through the PCS of washing are initial with 12.6%TS, and need 1,3 or 4 of PCS extra carrying capacity with the final TS of the appointment reaching 15%, 20% or 30%.Cellulase preparation added with the cellulosic enzyme dosage of 6mg EP/g at 0 hour, or was divided into equal two halves, and half added at 0 hour, and second half added at 24 hours.Be hydrolyzed and carry out 96 hours at 50 DEG C and pH 5.0.
Every sample extracting whole slurry for 24 hours, and measure sugared concentration by HPLC.The results are shown in table 1.Glucose equivalent conversion calculations is the per-cent of cellobiose and the glucose produced in the total glucose potentiality (potential) in PCS.
Embodiment 2
Repeat above-mentioned test, to obtain the data of about 25%TS content, except reactor arrange slightly different except.In embodiment 1, vertical reactor is used to arrange, and in the present embodiment, usage level reactor.Generally speaking, this will cause lower sugared yield.
Claims (20)
1., for producing a technique for hydrolysate from lignocellulose-containing materials, it comprises the steps,
(a) pre-treatment lignocellulose-containing materials;
B () forms the slurry comprising water, pretreated lignocellulose-containing materials and cellulolytic enzyme,
The described slurry formed in (c) incubation step (b),
D () adds more lytic enzymes to the slurry of the described incubation of step (c) and does not add fermenting organism, and
The described slurry formed in (e) incubation step (d), to produce hydrolysate,
Wherein said slurry has the solid bulk concentration of at least 25%.
2. the technique of claim 1, wherein front in step (d), by the slurry incubation 12-48 hour of described step (c).
3. the technique of claim 2, wherein by the slurry incubation 18-36 hour of described step (c).
4. the technique of claim 3, wherein by the slurry incubation 20-30 hour of described step (c).
5. the technique any one of claim 1-4, the time length of wherein said step (c)+(e) is 72-120 hour.
6. the technique any one of claim 1-4, wherein said cellulolytic enzyme comprise in cellobiohydrolase I, cellobiohydrolase II and endoglucanase one or more.
7. the technique any one of claim 1-4, wherein ferments the hydrolysate of described step (e) to produce tunning.
8. the technique any one of claim 1-4, it comprises step (f) further and is hydrolyzed simultaneously and ferments, and this step (f) is after step (e).
9. the technique any one of claim 1-4, it comprises the step comprising and reclaim described tunning further.
10. the technique any one of claim 1-4, wherein said tunning is ethanol.
Technique any one of 11. claim 1-4, wherein said lignocellulose-containing materials comes from and is selected from following material: maize straw, hardwood, soft wood, cereal straw, switchgrass, rice husk, municipal solid waste, industrial organic waste, office's paper using or its mixture.
The technique of 12. claims 11, wherein said lignocellulose-containing materials comes from and is selected from following material: aspen and birch.
The technique of 13. claims 11, wherein said lignocellulose-containing materials comes from wheat straw.
Technique any one of 14. claim 1-4, wherein said lignocellulose-containing materials comes from zein fiber.
Technique any one of 15. claim 1-4, the pre-treatment in wherein said step (a) uses organic acid and/or mineral acid to carry out low-kappa number.
The technique of 16. claims 15, the pre-treatment in wherein said step (a) uses sulfuric acid, acetic acid, citric acid, tartrate, succsinic acid and/or its mixture to carry out low-kappa number.
Technique any one of 17. claim 1-4, wherein carries out low-kappa number by the acid of the lignocellulose-containing materials 0.1-2.0 % by weight in described step (a).
The technique of 18. claims 17, wherein said acid is sulfuric acid.
Technique any one of 19. claim 1-4, wherein carries out mechanical pretreatment at high temperatures and/or high pressures by the lignocellulose-containing materials in described step (a).
The technique of any one of 1-4 in 20. claims, wherein carries out pre-treatment by the lignocellulose-containing materials in described step (a) by vapor explosion, dilute acid steam explosion and/or wet oxidation.
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| 木质纤维素原料酶水解产乙醇工艺的研究进展;王铎等;《生物加工过程》;20100731;第8卷(第4期);全文 * |
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