CN103261513A - Biomass pretreatment process for a packed bed reactor - Google Patents

Abstract

Biomass pretreatment using anhydrous ammonia was found to be effective in a static vessel when the ammonia can penetrate through the biomass particles or pieces in vapor state. To achieve this condition, total system moisture content is kept below 40 weight % based on total mass in the system. The pretreated biomass product is effectively saccharified to produce fermentable sugars for biocatalyst production of a product.

Description

The biomass pretreatment method that is used for packed bed reactor

Present patent application requires the rights and interests of the U.S. Provisional Application 61/416477 of submission on November 23rd, 2010, and this provisional application is incorporated this paper into way of reference in full.

Technical field

The invention provides and handle living beings to obtain the method for fermentable sugars.Specifically, but provide in packed bed reactor with anhydrous ammonia and handled living beings to produce the method for saccharification material.

Background technology

The raw material of lignocellulose and waste material, for example agriculture residue, timber, forestry waste, provide potential very big renewable raw materials from the sludge of papermaking and municipal administration and industrial solid refuse for chemicals, plastics, fuel oil and feed.The lignocellulosic material and the waste material that comprise carbohydrate polymer (cellulose and hemicellulose) and lignin generally handle to discharge the sugar that is mainly hexose and pentose with the method with enzyme number of chemical, machinery, and these sugar can ferment then becomes useful product.

Preprocess method is used for making carbohydrate polymer or the more accessible cellulase for saccharification of polysaccharide of lignocellulose biomass.The known multiple preprocess method of people comprises the ammonia preliminary treatment of living beings.When handling living beings in the saccharification set-up procedure, ammonia uses with aqueous solution state usually.

For example, the US7 that owns together, 932,063 disclose the method for preprocessing biomass under the condition of highly filled and low ammonia concn.Used ammonia concentration be the pH that is enough to keep living beings-ammonia water mixture at least for the concentration of alkalescence and with respect to the living beings dry weight basis at the most less than about 12 weight %.The initial concentration of living beings dry weight be living beings-ammonia water mixture weight at least about 15% at most about 80%.

US4,064,276 discloses with anhydrous ammonia and has handled straw and other plant material with the purposes of the nutritive value that improves this material.The dry matter content straw of under room temperature and atmospheric pressure, using the anhydrous ammonia of dried straw 15-40kg per ton to handle to have at least 60% weight % at least 10 days.

US7,915,017 discloses with the anhydrous ammonia of liquid state or steam attitude and/or the dense ammonia of liquid state or steam attitude: aqueous mixtures carries out preliminary treatment to living beings, with the ratio that obtains ammonia and dry biomass between the ratio of about 0.2: 1 to 1.2: 1 and water and dry biomass between about 0.2: 1.0 to 1.5: 1 mixture.Used the method that is used for improving the total ammonia umber that is in liquid phase.Temperature is held between about 50 ℃ and 140 ℃ and the living beings that quick release of pressure is handled with formation by discharge ammonia in container.

Packed bed percolation thing reactor uses people such as (, (1995) Applied Biochemistry and Biotechnology51/52:5-19) Yoon being used for the pretreated ammonia recirculation of living beings diafiltration method.In this method, the bedding by living beings carries out recirculation to ammoniacal liquor continuously.

But for use ammonia effectively preprocessing biomass exist demand with the method that produces the saccharification material, it has reduced reactor cost.

Summary of the invention

But the invention provides with anhydrous ammonia in static container preprocessing biomass to produce the method for saccharification material.

Therefore, but the invention provides the method for processing saccharification living beings, described method comprises:

A) provide the living beings that have at least about 60% dry matter content;

B) living beings with described (a) charge into static pretreating containers;

C) total moisture content in described container keeps below under the condition by the gross mass 40 weight % in the described container, makes described living beings and contacts at least about 4% anhydrous ammonia by the living beings dry weight basis in the described container; Thereby the major part of described living beings is by the ammonia vapor permeates; And

D) to described container exhaust to remove steam;

Living beings in the wherein said container are pretreated.

The specific embodiment

When using the ammonia treatment living beings, described ammonia and described living beings contact completely for effectively for the preparation of most important for the living beings of saccharification.Usually use mechanical agitation to mix ammonia and living beings, thereby make the contact maximization.Under condition as herein described, preliminary treatment can be implemented in batch reactor and not carry out mechanical agitation, thereby has eliminated the energy and the maintenance cost that is associated with the mixing reactor that with ammonia is reactant.

Following definition and abbreviation are used for explaining claim and specification.

As used herein, term " comprises ", " comprising ", " having " or " containing ", and perhaps its any other modification is intended to comprise non-exclusive comprising.For example, the composition, mixture, technology, method, goods or the equipment that comprise series of elements needn't only limit to those elements, and can comprise the element that other is not clearly listed, or the intrinsic element of this based composition, mixture, technology, method, goods or equipment.In addition, unless opposite offering some clarification on arranged, " or " refer to inclusive " or ", rather than refer to exclusive " or ".For example, below any one all represent to satisfy condition A or B:A for true (or existence) and B be false (or not existing), A for false (or not existing) and B be true (or existence), and A and B are true (or existence).

Equally, relate to element or component illustration (namely occur) number of times to be positioned at that indefinite article " " before element of the present invention or the component or " a kind of " be intended to be nonrestrictive.Therefore, " one " or " a kind of " should be interpreted as to comprise one or at least one, and the word singulative of element or component also comprises plural form, unless obviously expression odd number of numeral is arranged.

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

As used herein, the term " about " that the amount of composition of the present invention or reactant of modifying is used refers to the variation of the umerical amount that can take place by for example following mode: in real world for the preparation of concentrate or use general measure and the liquid handling operation of solution; By unintentional error in these operations; By for the preparation of the difference in manufacturing, source or the purity of the composition of composition or manner of execution; Deng.Term " about " also contains because with respect to the different amount by the different equilibrium conditions of the composition of specific starting mixt gained.No matter whether modify the equal parts of the claim amount of comprising by term " about ".In one embodiment, term " about " refers in report numerical value 10% scope, preferably in report numerical value 5% scope.

Term " fermentable sugars " refers to during the fermentation can be by compound sugar and the monose of microorganism as carbon source.

Term " lignocellulose " refers to comprise lignin and cellulosic composition.Ligno-cellulosic materials also can comprise hemicellulose.

The composition that term " cellulose " refers to comprise cellulose and comprises the annexing ingredient of hemicellulose.

Term " saccharification " refers to produce fermentable sugars by polysaccharide.

Term " pretreated living beings " means and passed through pretreated living beings before saccharification.

Term " butanols " refers to isobutanol, 1-butanols, 2-butanols or their combination.

Term " lignocellulose biomass " refers to any ligno-cellulosic materials and comprises following material that described material comprises cellulose, hemicellulose, lignin, starch, compound sugar and/or monose.Living beings also comprise annexing ingredient, as protein and/or lipid.Living beings can derive from single source, and perhaps living beings can comprise derived from the mixture more than a kind of source.For example, living beings can comprise the mixture of corn ear and maize straw, or the mixture of grass and leaf.Lignocellulose biomass includes but not limited to bioenergy crop, agriculture residue, municipal solid refuse, industrial solid rubbish, the sludge from paper-making industry, garden rubbish, timber and forestry rubbish.The example of living beings includes but not limited to corn ear, crop residues such as shuck, maize straw, grass, wheat stalk, barley stalk, hay, rice straw, switchgrass, waste paper, bagasse, Chinese sorghum vegetable material, bean plant material, the component that obtains, tree, branch, root, leaf, wood chip, sawdust, shrub and shrubbery, vegetables, fruit and flower from the grinding of cereal.

Term " dry matter content " refers to remove the amount of the weight meter of the main substance that exists after the moisture of material.

Term " biomass by hydrolyzation product " refers to derive from the product of living beings saccharification.Living beings also can be carried out preliminary treatment or preprocessing before saccharification.

Term " biomass by hydrolyzation product fermentation broth " refers to comprise the fluid nutrient medium of product, and described product gets the biocatalyst growth in the culture medium of self-contained biomass by hydrolyzation product and produces.Described fluid nutrient medium comprises not the product that the component of the biomass by hydrolyzation product that is consumed by biocatalyst and biocatalyst itself and biocatalyst produce.

Term " slurries " refers to undissolved material and mixtures of liquids.Slurries also can comprise high-caliber dissolved solid.The example of slurries comprises saccharification liquid, zymotic fluid and stillage.

Term " target product " refers to any product that is produced by the micro-organisms host cell of fermentation.Target product can be the product of the enzymatic pathway of genetic engineering in the host cell or can be produced by endogenous approach.Typical target product includes but not limited to: acid, alcohol, alkane, alkene, aromatic hydrocarbons, aldehyde, ketone, biopolymer, protein, peptide, amino acid, vitamin, antibiotic and medicine.

Term " static container " refers to not comprise the container that charges into the device of material for mixing.Packed bed reactor has used static container.

Term " static pretreating containers " refers to can be used for pretreated static container.Described container is through making up to bear the material that charges into that uses in the inventive method, such as ammonia.

Term " exhaust " refers to allow the gas sealed to leave and does not have explosive effect.

Term " gas " refers to gas and steam, comprises being in the following condensable low-density phase of its critical-temperature, for example, steam.

Term " gross mass in the container " refers to add the combination quality of the whole components in the container.

Term " major part " refers to surpass 50%.Major part can refer to surpass any integer of 50%.

Use the preprocess method of ammonia steam

Method of the present invention relates to uses anhydrous ammonia to handle living beings in the containment of relatively dry in container.When described system relatively dry, ammonia can thoroughly diffuse in the described living beings with the steam attitude, thereby need not mechanical mixture.Thereby by the moisture that keeps moisture in the reactor lowly to make being present in this reactor in conjunction with living beings and do not charge into void space and keep and make biological particles that vapor ammonia passes through or the open void space between the sheet piece.Therefore, the moisture in the described container is also non-constant, and this is because moisture higher at the interface at biological particles or sheet piece, and lower in the void space between biological particles or sheet piece.

Whole total moisture content in the described container keeps below by the gross mass 40 weight % in this container, and described gross mass comprises any other component of living beings, anhydrous ammonia and this container of adding, such as steam.Show according to this paper, in the method for the invention, just but the ammonia steam can penetrate described living beings without mechanical mixture carries out effective preliminary treatment to produce the biomass product of saccharification, and it is measured according to the sugared yield that produces as subsequent saccharification.Charge into the sample of selecting the collection of different distance place can produce high-load between saccharificatinn period sugar at relative ammonia.The sugar that produces in this paper example is in the scope of 57-68% of monomer wood sugar theoretical yield and in the scope of the 80-90% of monomer glucose theoretical yield.

In being used for the container of preprocess method of the present invention, need not mixed organization; It is static container.Described container can be Any shape, such as cylindrical, but and level or vertically towards.Described container has one or more interfaces and charges into being used for, such as, charge into living beings, ammonia and steam.In addition, described container has interface being used for, such as, application of vacuum, add air with destroy vacuum, to the steam exhaust with disengage living beings.Interface can have multi-functional.Described container can move under the pressure between 0.1 absolute atmosphere and 20 the atmospheric pressure scalars (atmospheres gauge) through making up being used for.Described container can have the device of direct its temperature of rising, such as heating jacket.Described container is to be designed for the material that bears the ammonia steam, and can be the reactor of packed bed type.

Living beings are charged into this container.In the method for the invention, living beings (being generally lignocellulose biomass) have the dry matter content at least about 60%.The percentage dry weight of described living beings can be about 60%, 65%, 70%, 75%, 80%, 85%, 90% or higher.For example, the maize straw of fresh collection is generally about 70% dry weight.In addition, described living beings can be dried to higher percentage dry, for example by the air drying.In addition, living beings can reduce size before charging into container.Before living beings are charged into container, with mechanical means it is carried out size usually and reduce, such as by cutting, cut or grinding.Living beings directly can be charged into container or charge into container (such as the basket box in the container), for example by one or more open interfaces.Alternatively, can insert by the container that will be loaded with living beings and charge into living beings in the container.Spendable container can be to railcar from the basket box.

Air can be ejected the generation partial vacuum in container.To produce partial vacuum be preferred, and it has improved the infiltration of the living beings of ammonia steam in the container.In addition, remove air and removed not condensable materials.

By in the container that comprises living beings, charging into anhydrous ammonia and making it penetrate described living beings the living beings in the container are contacted with anhydrous ammonia.In the method, the major part of described living beings is contacted with anhydrous ammonia.Living beings above 50% are contacted with anhydrous ammonia.At least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% living beings are contacted with anhydrous ammonia.

Add anhydrous ammonia with the amount (based on the living beings dry weight basis in the described container) at least about 4%.The concentration range of employed anhydrous ammonia is between about 4% and about 20%.Shown in the example 3 of this paper, compare with the effect of 3% anhydrous ammonia, use 6% anhydrous ammonia to improve wood sugar yield from the anhydrous ammonia preprocessing biomass saccharification that produces.The introducing of anhydrous ammonia has improved the temperature of holding the container endogenous substance.

Can before adding anhydrous ammonia, improve the temperature of described reactor and/or living beings.Temperature can improve by any method directly or indirectly, and described method comprises by this container being executed heat (such as using heating jacket or heater coil), or by in this reaction vessel, introducing hot gas or steam, such as steam.Can inject steam with superheated steam or dry saturated steam form and avoid introducing moisture, in order to the total moisture content in the described container is kept 40 weight % of the gross mass that is lower than in this container.Moisture in the described container may mainly come from the gas of living beings and adding.When handling very dry living beings (such as the living beings that have less than about 15% moisture), then drying-free steam is kept 40% low total moisture content.Yet, all according to appointment 35% if living beings have higher moisture, use dry saturated steam.Desired temperature is generally in the scope of about 70 ℃ and 190 ℃.Described temperature is usually between about 90 ℃ and 150 ℃.

In addition, can improve container inner pressure.Can use steam to inject to improve pressure.During pre-processing, pressure is maintained at 0.1 absolute atmosphere extremely less than 20 atmospheric pressure scalars.The pretreated time of staying be about 2 minutes to a couple of days.The described time of staying can be several minutes to a couple of days.The described time of staying is generally about 20 minutes to 24 hours, and can be about three hours.During this time of staying, keep the pressure and temperature in the described container, penetrate the condition of the void space between described biological particles or the sheet piece so that the ammonia that makes the steam attitude to be provided.

When desired time of staying finishes, to described container exhaust so that gas discharge.Not blasting property gas discharges.Living beings discharge and cool off along with gas.But application of vacuum discharges with the raising that realizes gas between exhaust cycle.If pressure raises or discharges, then this pressure returns atmospheric pressure.The preprocessing biomass product that produces is in dry state, and it utilizes gravity to make it leave described container by opening usually, but also can use mechanical means.The recovery of described pretreated living beings disengages can be by a plurality of openings (such as the situation for the horizontal cylindrical container) or by single opening (such as at vertical hydrostatic column).Alternatively, hydrostatic column at one end can be tilted realize by disengaging that the opening at low side carries out that described opening is in the side of this end or contiguous this end.Alternatively, can come in container, to disengage pretreated living beings by removing the container that is filled with living beings that is in this container.For example, the railcar that is loaded with living beings can be left this container.

But the ammonia steam recirculation of this container is left in exhaust.The commercial viability that this ammonia of recirculation strengthens this method is preferred.Can for example use ammonia steam control system recycle of ammonia steam by method known to those skilled in the art, the ammonia steam can carry out condensation and as ammoniacal liquor recirculation.Alternatively, the ammonia steam can be mainly with anhydrous state recirculation.

Lignocellulose biomass

The living beings that are used for the inventive method are lignocelluloses, and it comprises polysaccharide (such as cellulose and hemicellulose) and lignin.The polysaccharide of living beings also can be known as glucan and xylan.Spendable living beings type includes but not limited to biological energy source crops, agricultural residue, municipal solid refuse, industrial solid rubbish, the sludge from papermaking, garden rubbish, timber and forestry waste.The example of living beings includes but not limited to that corn ear, shuck, maize straw, grass, wheat stalk, barley stalk, oat stalk, Kano draw stalk, hay, rice straw, switchgrass, spire awns, rice grass, reed canary grass, waste paper, bagasse, Chinese sorghum slag or stalk, soybean stalk, the component that obtains, tree, branch, root, leaf, wood chip, sawdust, shrub and shrubbery, vegetables, fruit, flower and animal muck from the grinding of cereal.Living beings can comprise other crop residues, forestry waste such as poplar, other hardwood, cork and sawdust; The paper products rubbish of using with the consumer; With the fiber process residue, such as zein fiber, beet pulp, pulp mill's residue and discarded object; And other ligno-cellulosic materials that enough enriches.

The living beings that the present invention is particularly useful comprise having relative Hi CHO content, intensive relatively and/or be easy to the living beings of collecting, transport, storing and/or handling relatively.

Lignocellulose biomass can come from single source, and perhaps living beings can comprise the mixture that derives from more than one sources; For example, living beings can comprise the mixture of corn ear and maize straw or the mixture of bar or stem and blade.

Living beings can directly be used by the form that obtains from the source, perhaps can carry out some preprocessings, for example energy can be applied on the living beings to reduce size or to reduce moisture.The reducing of size can adopt the method (size that obtains is greater than 0.1mm) of producing the thick size material that reduces to carry out.Spendable method comprises mechanical means, such as cutter milling, crushing, fragmentation, cut, mill and rough forge mill.The size of this type is carried out after reducing or to handle before using anhydrous ammonia to handle, but carries out before processing usually.Drying can be by the device of any routine, as using drying oven, rotary dryer, flash dryer or superheat steam drying device.In addition, the air drying can be enough to reach expectation less than about 40% living beings moisture.For can be used for the inventive method, the dry matter content that living beings have at least about 60 weight %, 65 weight %, 70 weight %, 75 weight %, 80 weight %, 85 weight %, 90 weight % or 93 weight % is desirable.

Pretreated biomass product

The pretreated biomass product that produces by the inventive method is used for saccharification to produce sugar, and it is used for fermenting to produce desirable product by biocatalyst.

Saccharification

Enzymatic saccharification utilize at least a carbohydrase usually and utilize enzyme consortium degraded cellulose usually and hemicellulose to produce hydrolysate, it comprises glucose, wood sugar and arabinose.Carbohydrase is seen Lynd, people's such as L.R. summary (Microbiol.Mol.Biol.Rev., 66:506-577,2002).

Described one or more enzymes generally include one or more glycosidases.The ehter bond of glycosidase hydrolysis disaccharides, compound sugar and polysaccharide, and be present in broad sense " hydrolase " enzyme classification EC3.2.1.x (Enzyme Nomenclature1992 (EC3.), Academic Press, San Diego, CA, and augment 1 (1993), augment 2 (1994), augment 3 (1995), augment 4 (1997) and augment 5[respectively at Eur.J.Biochem., 223:1-5,1994; Eur.J.Biochem., 232:1-6,1995; Eur.J.Biochem., 237:1-5,1996; Eur.J.Biochem., 250:1-6,1997; And Eur.J.Biochem., among the 264:610-6501999]).The glycosidase that can be used in the inventive method can be classified according to the biomass components of their hydrolysis.The glycosidase that can be used in the inventive method comprises cellulose hydrolysis glycosidase (for example, cellulase, endoglucanase, exoglucanase, cellobiohydrolase, beta-glucosidase), hydrolysis of hemicellulose glycosidase (for example zytase, interior zytase, outer zytase, β-xylan glycosides enzyme, arabinose sill dextranase, mannase, galactase, pectase, glucuronidase) and amylum hydrolysate of the sugar glycosides enzyme (for example amylase, α-Dian Fenmei, beta amylase, glucoamylase, alpha-glucosidase, isoamylase).In addition, with the release polysaccharide is useful from other component of living beings to help in other active adding carbohydrase consortium (as peptase (EC3.4.x.y), lipase (EC3.1.1.x and 3.1.4.x), ligninase (EC1.11.1.x) and feruloyl esterase (EC3.1.1.73)).The microorganism of well known production polysaccharide hydrolase usually shows certain activity, and as cellulose degradation, this activity is by some kinds of enzymes or one group of enzymatic with different substrate specificities.Therefore, can comprise one group of enzyme from " cellulase " of microorganism, all enzymes can help the activity of cellulose degradation.Depend on the purification scheme of utilizing when obtaining enzyme, commerce or non-commercial enzyme preparation as cellulase, can comprise plurality of enzymes.

The commercially available acquisition of carbohydrase, as

The CP cellulase,

Zytase,

1500 and

DUET (Danisco U.S.Inc., GenencorInternational, Rochester, NY).Carbohydrase can be unpurified and provides with the form of cell extract or intact cell preparation in addition.Can use and prepare described enzyme through through engineering approaches with the recombinant microorganism of expressing a plurality of carbohydrase.

Multiclass glycoside hydrolase (GH) (as GH3, GH39, GH43, GH55, GH10 and GH11 family) outstanding tool in the present invention is worth.GH is one group of enzyme, the glycosidic bond between two or more carbohydrate of its hydrolysis, or the glycosidic bond between carbohydrate and non-carbohydrate group.GH family classifies based on sequence similarity, and (people (2009) Nucleic Acids Res.37 (database monograph) such as Cantarel: D233-238) can be provided in carbohydrate-organized enzyme (CAZy) database.These enzymes can act on many substrates and be effective in saccharifying.The enzyme of glycoside hydrolysis enzyme family 3 (" GH3 ") has many known activity: β-Pu Tangganmei (EC:3.2.1.21); Xylobiase (EC:3.2.1.37); N-acetyl group beta-amino glucosidase (EC:3.2.1.52); Glucan β-1,3-glucosidase (EC:3.2.1.58); Cellodextrin enzyme (EC:3.2.1.74); Circumscribed-1,3-1,4-dextranase (EC:3.2.1); And beta galactosidase (EC3.2.1.23).The enzyme of glycoside hydrolysis enzyme family 39 (" GH39 ") has the activity of α-L-iduronidase (EC:3.2.1.76) or xylobiase (EC:3.2.1.37).The enzyme of glycoside hydrolysis enzyme family 43 (" GH43 ") has following activity: L-α-arabinofuranosidase (EC3.2.1.55); Xylobiase (EC3.2.1.37); Araban restriction endonuclease (EC3.2.1.99); And galactan 1,3-beta galactosidase (EC3.2.1.145).The enzyme of glycoside hydrolysis enzyme family 51 (" GH51 ") has the activity of L-α-arabinofuranosidase (EC3.2.1.55) or endoglucanase (EC3.2.1.4).The enzyme of glycoside hydrolysis enzyme family 10 (" GH10 ") more intactly be described in people such as Schmidt (1999, Biochemistry38:2403-2412) and in people's (2001, FEBS Lett509:303-308) such as Lo Leggio the document.The enzyme of glycoside hydrolase family 11 (" GH11 ") more intactly be described in people such as Hakouvainen (1996, in document Biochemistry35:9617-24).

These enzymes can separate from their natural host organism, or are expressed in the engineering host organisms and produce.For example, mosaic gene, it is included in the objective expression host cell sequence and the termination signal of the glycoside hydrolase that active promoter, coding provide above, is expressed or is incorporated in the objective expression host cell gene group by standard method well known to those skilled in the art by plasmid vector.Used coded sequence can be at the specific host of expressing usefulness codon optimized.Usually used expression host cell comprises: bacterium such as Escherichia (Escherichia), bacillus (Bacillus), lactobacillus (Lactobacillus), pseudomonas (Pseudomonas) and streptomyces (Streptomyces), saccharomycete such as saccharomyces (Saccharomyces), Schizosaccharomyces (Schizosaccharomyces), Candida (Candida), Hansenula (Hansenula), pichia (Pichia), Kluyveromyces (Kluyveromyces) and Fife's saccharomyces (Phaffia), and filamentous fungi such as the mould genus of branch top spore (Acremonium), aspergillus (Aspergillus), Aureobasidium (Aureobasidium), clarinet Pseudomonas (Bjerkandera), intend wax Pseudomonas (Ceriporiopsis), white-rot fungi belongs to (Chrysoporium), Coprinus (Coprinus), Coriolus Qu61 (Coriolus), the rod softgel shell belongs to (Corynascus), Chaetomium (Chaertomium), Cryptococcus (Cryptococcus), line is deceived powder saccharomyces (Filobasidium), Fusarium (Fusarium), gibberella belongs to (Gibberella), detritus Pseudomonas (Humicola), rice blast Pseudomonas (Magnaporthe), mucor (Mucor), myceliophthora (Myceliophthora), mucor (Mucor), cud fungi (Neocallimastix), Neurospora (Neurospora), paecilomyces (Paecilomyces), Penicillium (Penicillium), flat lead fungi belongs to (Phanerochaete), arteries and veins is penetrated Pseudomonas (Phlebia), Piromyces, Pleurotus (Pleurotus), Scytaldium, Schizophyllum (Schizophyllum), Sporothrix (Sporotrichum), Talaromyces (Talaromyces), heater capsule Pseudomonas (Thermoascus), the mould Pseudomonas of grass roots (Thielavia), curved neck enzyme belongs to (Tolypocladium), Trametes (Trametes) and trichoderma (Trichoderma).

Those skilled in the art can understand the condition of how determining the effective enzyme amount in consortium and being adjusted into best enzymatic activity.Those skilled in the art also can understand how to optimize a plurality of classes that enzyme required in consortium is lived, to obtain the best saccharification of a given prefinished products under selected condition.The example of saccharification is described in US7, in 932,063.

Saccharification can be implemented with approximate number minute (for example, 2 minutes) to about 200 hours time, about 24 hours to about 72 hours usually.Reaction time will be depended on enzyme concentration and specific activity, and employed substrate and environmental condition such as temperature and pH.Those skilled in the art can easily determine the optimum condition of temperature, pH and time that specific substrates and carbohydrase consortium use.

Saccharification can single batch, in batches in or carry out with continuation method.Saccharification also can carry out in a step or multistep is carried out.For example, the required different enzymes of saccharification can show different best pH or temperature.Can under certain temperature and pH, carry out first treated with enzyme, use subsequently different enzymes under different temperatures and/or pH, carry out the second time or for the third time (or more times) handle.In addition, can under identical pH and/or temperature, carry out with the processing that different enzymes carry out in consecutive steps, or under different pH and temperature, carry out, for example use stable handling with the higher hemicellulase of activity under higher pH and temperature, be used in activated cellulase processing under low pH and the temperature subsequently.

The saccharification mixture can concentrate by evaporation before fermentation, for example, increases the concentration of fermentable sugars.Randomly, the liquid in the saccharification product can separate in the solid from method in batches or continuously.Randomly, liquid or whole saccharification product can be sterilized before fermentation.According to the pH that uses in the biocatalyst that uses in the fermentation and the saccharifying, described pH can be adjusted to suitable fermentation.

The biomass by hydrolyzation product that comprises fermentable sugars is included in the fermentation medium with the certain percentage of culture medium usually, thereby provides all or part to be used for the carbon source of biocatalyst growth and product production.Hydrolysate in the fermentation medium is generally about 40% to 90% of fermentation medium.At the US7 that incorporates this paper with way of reference into, the example 9 in 932,063 has provided the example as the hydrolysate of 40% or 80% fermentation medium.Concentration according to fermentable sugars in the described hydrolysate can add additional sugar in the culture medium.For example, when the hydrolysate that contains about 80 grams per liter glucose and about 50 grams per liter wood sugars account for fermentation medium 40% the time, can add additional glucose and wood sugar to the expectation sugared concentration at end.As well known to the skilled person, except hydrolysate, fermentation medium can comprise other nutrients, salt and the factor, and these materials are growths and use specific biological Catalyst Production product necessary.Replenishers can comprise for example yeast extract, specific amino acids, phosphate, nitrogenous source, salt and trace element.Also can comprise by the required component of particular organisms Catalyst Production specific products, as being used for keeping antibiotic or the required co-factor of enzymic catalytic reaction of plasmid.

In the replacement scheme as the preparation hydrolysate, hydrolysate is joined fermentation medium, start fermentation then, synchronous saccharification and fermentation (SSF) can be used for producing biomass by hydrolyzation product fermentation broth.In this method, when they are produced the biocatalyst metabolism, preparation sugar from living beings.

Biocatalyst fermentation and target product

Fermentable sugars in the fermentation medium is to carry out metabolism with the productive target product by suitable biocatalyst.Described sugar contacts with biocatalyst during the fermentation, and wherein biocatalyst is grown under the condition by biocatalyst productive target product.According to the useful condition of particular organisms catalyst to using, temperature and/or head space gas phase can be adjusted at fermentation.Fermentation can be aerobic or anaerobism.The condition of these and other, it comprises temperature and pH, adjusts at the particular organisms catalyst that uses.

Example by the target product of biocatalyst production comprises 1, ammediol, butanols (isobutanol, 2-butanols and 1-butanols) and ethanol.At US7, disclosed in 504,250 is to produce 1, the recombinant microorganism body of ammediol.The production of butanol that yeast by genetic modification carries out is disclosed in, and for example, US7 is among 851,188.The coli strain that has used genetic modification is as the biocatalyst that is used for alcohol production people such as (, (2002) Appl.Environ.Microbiol.68:6263-6272) Underwood.Ethanol is produced in lignocellulose biomass hydrolysate fermentation medium (US7,932,063) by the zymomonas through genetic modification.The zymomonas mobilis bacterial strain of spendable increase ethanol production through genetic modification is described in US7, and 223,575 and US7,998,722.

Example

The present invention will further be set forth in the example below.Should be appreciated that, although these examples have illustrated the preferred embodiments of the present invention, only be that the mode with illustration provides.From above discussion and these examples, those skilled in the art can determine characteristic of the present invention, and under the situation that does not break away from its essence and scope, can carry out variations and modifications to adapt to different purposes and condition to the present invention.

The meaning of used abbreviation is as follows: " s " refers to second, " min " refers to minute, " hr " or " h " refers to hour, and " μ L " refers to microlitre, and " mL " refers to milliliter, " L " refers to rise, " m " refers to rice, and " nm " refers to nanometer, and " mm " refers to millimeter, " cm " refers to centimetre, " μ m " refers to micron, and " mM " refers to every liter of mM, and " M " refers to every liter of mole, " mmol " refers to mM, " μ mole " refers to that micromole, " g " refer to gram, and " μ g " refers to microgram, " mg " refers to milligram, " kg " refers to kilogram, and " rpm " refers to revolutions per minute, and " C " refers to degree centigrade, " ppm " refers to each 1,000,000 parts of parts, and " psi " refers to pound per square inch.

Basic skills

Carbohydrase

1500 (A1500) are available from Danisco U.S.Inc., Genencor, International (Rochester, NY).

Packed bed reactor

Used comprise vertically towards the packed bed pretreatment reaction device of stainless steel filter shell pressure vessel (capping that tool is removable).Described pressure vessel is equipped with stainless removable cylindrical basket box, and it has open top, real facing the wall and meditating and the porous pedestal.This basket box has been realized the static state of living beings packed bed is held and the top enters or the bottom enters liquid, steam and/or gas penetration by the voidage between biological particles.

Filter head housings has the interior length of interior diameter and 29 inches (73.7cm) of 8 inches (20.3cm).The described cylindrical degree of depth with diameter and 29 inches (73.7cm) of 6.5 inches (16.5cm), and about 15.8 liters volume.

Flexible copper tubing has twined this filter head housings external surface area of about 30%.The copper pipe that is used for this external container of heating is connected in the steam supply of 164.7psia (1135.6 kPas).

Described packed bed reactor has been installed tubing to be connected to realize:

Discharge air to produce vacuum wholly or in part

Adding is from the saturated vapor of steam generator, and it is in 84.7psia (584 kPas) operation down

Add anhydrous ammonia

Discharge reacted process steam

After reaction, add air and destroy vacuum

Example 1

Living beings are carried out the packed bed preliminary treatment in order to saccharification

With 4.07 kg corn rods short cut and sort out to obtain particle diameter surpass 1/4 " (0.64cm) and less than 3/8 " (0.375cm) cob component.Described cob is placed the basket box, and it is with being placed in the pretreatment reaction device of describing in the basic skills.Described corn ear has 8.6% moisture and the bulk density of 227.5g/L.The packed bed degree of depth before the described reaction is surveyed and is about 29 inches (73.7cm).

From described reactor, discharge air and reach the partial vacuum of 0.1bar (10 kPas).Restrain the anhydrous ammonia (based on the quality meter of utmost point dried biomass) that ammonia add 8 weight % by charged into 297.6 in the clock time at about 60 minutes.The saturated vapor that adds subsequently under the 84.7psia (584 kPas) transfers to about 70 ℃ with temperature of reactor.At this moment, in 40 minutes stand-by period, make the temperature of described reactor be increased to 90 ℃ from 70 ℃.In waiting time, the pressure in the described reactor is about 85psia (584 kPas).After waiting time, release pressurized gas and steam and its outside that is trapped in cooling held in the container.When described reactor reaches atmospheric pressure, use vavuum pump that described reactor is vented to about 0.1bar (10 kPas) immediately and under this pressure, kept five minutes.Subsequently to the reactor exhaust so that vacuum is alleviated to atmospheric pressure.After reaching atmospheric pressure, this process opens the pretreatment reaction device.

Remove pretreated living beings to the bottom with the quartering from the top of this packed bed, that is, and first in the table 1 (top), four components, the two or four component, the three or four component and the 4th (bottom) four components.Analyze these fractions and the results are shown in Table 2 at moisture and residual ammonia.In water, extract one hour and use the titration of carrying out with 0.1N HCl to measure the acid equivalent that reaches pH5.3 subsequently by the pretreated solid with known quantity, thereby measure remaining ammonia.Subsequently described equivalent is carried out normalization to the amount of the dry in the described extraction system.In addition, mix with each fraction of quality such as being about to, analyze to measure mixing cup (mixing-cup) composition of described preprocessing biomass subsequently.

Table 1: gather four different layers in 29 inches (73.7cm) dark reactor bedSample

168-1	The packed bed top
		168-2	The top is about 10 inches (25.4cm) downwards

168-3	The top is about 20 inches (50.8cm) downwards
		168-4	The packed bed bottom
168-C	Mixture: the thorough packed bed that mixes

Table 2: pretreated species analysis

?	?	168-1	168-2	168-3	168-4	168-C
							Moisture	Weight %	21.42	5.82	6.07	28.78	9.54
NH3Concentration (g/100g DM*)	pH＝5.3	0.275	0.122	0.133	0.331	0.163

* DM is dry

The sample of pretreated each living beings fraction is through monose and the compound sugar yield of enzymatic saccharification with mensuration glucan and xylan.

The saccharification operation

Each fraction of 0.56 gram is weighed and entered the scintillation vial of single 20ml.Add an amount of 50mMpH5 acetate buffer and obtain every bottle 18.6% solid.By adding 1N H₂SO₄PH is adjusted to 5.Described acetic acid buffer solution comprises 0.005% sodium azide to suppress the bacterial growth in the raw material between incubation period.

Use

1500 come 7.2mg/g glucan+xylan or 21.7mg/g glucan+xylan is implemented enzymatic saccharification in conjunction with hemicellulose enzymatic mixture (Xyn3, Fv3A, Fv51A and Fv43D).Described enzyme being added each bottle, adds 1 1/2 subsequently " (1.3cm) stainless steel ball provides between incubation period the abundant grinding to raw material.

Cover tightly bottle and allow in rotating oscillator with 180rpm under about 48 ℃ in rotating on the oscillator saccharification 72 hours.

After hatching, before filtering and analyzing by HPLC, make the water dilute sample.Use HPX-87H chromatographic column (BioRad) to use 0.01N H at 60 ℃₂SO₄With the 0.6mL/min flow velocity institute's sample thief being carried out HPLC as flowing analyzes.

By handling, filter and analyze to obtain the total reducing sugar value by HPLC adding acid hydrolysis, high compressed steam that sulfuric acid carries out.For each sample, calculate the oligomer value by from total sugar concentration, deducting monomer.The result provides in table 3.

Table 3: by the sugar that treated living beings quartering fraction is hydrolyzed and produces

Example 2

Short time is carried out the packed bed preliminary treatment in order to saccharification to living beings under higher temperature

Identical with example 1,3.74 kilograms grinding corn ear is inserted in the basket box of the reactor of describing in the basic skills.Described basket box is inserted the pretreatment reaction device.Described corn ear has 8.6% moisture and the bulk density of 227.5g/L.Packed bed depth survey before the described reaction is about 29 inches (73.7cm).

The steam of 150psig (1034.2 kPas) is applied to the copper coil of described preprocessor.When reaching 145 ℃, described preprocessor top cover discharges air until the partial vacuum that reaches 0.6psia.Steam is directly injected described process to reach the pressure of 17psia (4.14 kPas).Come described preprocessor exhaust to discharge any condensate that forms in this system by opening bottom valve subsequently.Steam is injected described preprocessor top leave until observing the bottom valve of dry saturated steam from described opening, thereby guarantee whole condensates are cleaned out this system.Temperature in the described reactor of this point is about 100 ℃.Stop described vapor stream and close subsequently effluxing valve.Because enter the heat absorption of living beings, the temperature of described reactor is down to 80 ℃, this has caused the partial vacuum of 8psia.In this point, 190.3 anhydrous ammonias that restrain are added the ammonia carrying capacity (based on the dry of injecting reactor) of described process to obtain 6 weight %.After adding ammonia, steam is added described process, it is enough to reach 150 ℃ reaction temperature and the pressure of 70psia (482.6 kPas).After 30 minutes, release pressurized gas and steam and its outside that is trapped in cooling held in the container.When described reactor reached atmospheric pressure, the ammonia that uses vavuum pump further to reduce remnants immediately from described living beings was vented to about 0.1bar (10 kPas) with described reactor and kept five minutes under this pressure.Subsequently to the reactor exhaust so that vacuum is alleviated to atmospheric pressure.After reaching atmospheric pressure, this processing opens the pretreatment reaction device.

Remove pretreated living beings (as table 4) to the bottom with the quartering from the top of this packed bed.Analyze these fractions at moisture and residual ammonia, the results are shown in Table 5.In addition, mix with each fraction of quality such as being about to, analyze to measure the mixing cup composition of described preprocessing biomass subsequently.

Table 4: the sample of gathering four different layers in 30 inches dark reactor bed.

170-1	The packed bed top
		170-2	About 10 inches downwards at top
170-3	About 20 inches downwards at top
		170-4	The packed bed bottom
170-C	Mixture: the thorough packed bed that mixes

Table 5: pretreated species analysis

?	?	170-1	170-2	170-3	170-4	170-C
							The % dry of PT sample	％	54.96	83.68	93.68	92.30	71.29
NH3Concentration (g/100g DM*)	pH＝5.3	0.239	0.150	0.121	0.132	0.192

* DM is dry

According to the description in the example 1, the sample of pretreated each living beings fraction is through monose and the compound sugar yield of enzymatic saccharification with mensuration glucan and xylan.

Table 6: by the sugar that treated living beings quartering fraction is hydrolyzed and produces

Claims (15)

1. handle the method for living beings, comprising:

A) provide the living beings that have at least about 60% dry matter content;

B) living beings with described (a) charge into static pretreating containers;

C) total moisture content in described container keeps below under the condition by the gross mass 40 weight % in the described container, and described living beings are contacted with anhydrous ammonia by the living beings dry weight basis 4% in the described container at least; Thereby the major part of described living beings is by the ammonia vapor permeates; And

D) to described container exhaust to remove steam;

Living beings in the wherein said container are pretreated.

2. method according to claim 1, wherein at the described container of step (b) emptying afterwards to partial vacuum.

3. method according to claim 1 wherein reclaims described pretreated living beings afterwards in step (d).

4. method according to claim 1 wherein makes described anhydrous ammonia contact at least about 2 minutes with described living beings in step (c).

5. method according to claim 4 wherein makes described anhydrous ammonia contact less than about 24 hours with described living beings.

6. the temperature of the living beings in the method according to claim 1, wherein said container is at least about 70 ℃.

7. method according to claim 1, wherein the temperature with described container is elevated at least about 70 ℃ before in step (b).

8. method according to claim 7, wherein said temperature in step (b) before between about 70 ℃ and about 190 ℃.

9. method according to claim 7, wherein said temperature directly raises or raises indirectly by apply heat to described container by injecting hot gas.

10. method according to claim 1, wherein ammonia reclaims and is recycled to pretreating containers or to the ammonia vapor processing system in step (d).

11. method according to claim 10, wherein ammonia is with anhydrous state recirculation.

12. method according to claim 1, wherein at step (c), described anhydrous ammonia concentration is less than the living beings dry weight basis of pressing in the described container about 20%.

12. method according to claim 1, wherein in step (b) to (d), the pressure in the described container is between 0.1 absolute atmosphere with between less than 20 atmospheric pressure scalars.

13. method according to claim 1, the living beings of wherein said (a) are the living beings that reduce through mechanical dimension.

14. method according to claim 1, wherein said living beings are cellulose biomass, and described cellulose biomass comprises cellulose, hemicellulose and lignin.

15. method according to claim 14, wherein said living beings are selected from corn ear, shuck, maize straw, grass, wheat stalk, barley stalk, oat stalk, Kano and draw stalk, hay, rice straw, switchgrass, spire awns, rice grass, reed canary grass, waste paper, bagasse, Chinese sorghum slag or stalk, soybean stalk, the component that obtains, tree, branch, root, leaf, wood chip, sawdust, shrub and shrubbery, vegetables, fruit, flower and animal muck from the grindings of cereal.