CN101509024B - Method for preparing monosaccharide by raw materials containing cellulose - Google Patents
Method for preparing monosaccharide by raw materials containing cellulose Download PDFInfo
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- CN101509024B CN101509024B CN200810057730XA CN200810057730A CN101509024B CN 101509024 B CN101509024 B CN 101509024B CN 200810057730X A CN200810057730X A CN 200810057730XA CN 200810057730 A CN200810057730 A CN 200810057730A CN 101509024 B CN101509024 B CN 101509024B
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Abstract
A method which uses cellulose-containing raw materials for preparing monosaccharide comprises the steps as follows: the raw materials which contain cellulose is carried out steam blasting; a product which is obtained by steam blasting is mixed with enzyme and then experiences enzymolysis, wherein, the method which leads the steam blasting product to be mixed with the enzyme comprises: firstly, a first batch of steam blasting products are mixed with the enzyme for obtaining a mixture; and then the remaining steam blasting products are continuously added to be mixed with the mixture; the speed for adding the remaining steam blasting products causes the concentration of the steam blasting products in a reaction system not to be higher than 150 grams/liter; the weight of the first batch of steam blasting products which are mixed with the enzyme is10-30% of the total steam blasting products; the water content of the raw materials which contain cellulose is 30-60%. The method can effectively improve the sugar conversion rate of raw materials, namely the yield of monosaccharide; in addition, the method of continuously supplementing materials can also significantly shorten the enzymolysis time and improve the enzymolysis efficiency.
Description
Technical field
The invention relates to the preparation method of sugar, more specifically is the method about the feedstock production monose that adopts cellulose.
Background technology
Prior art can be used the feedstock production monose of cellulose; Gained monose mainly is glucose; Glucose can be used for food, medicine industry, in the printing and dyeing tanning industry, can be used as reductive agent, and glucose commonly used is made reductive agent in industry of system mirror and the glass liner of a thermos flask silver plating process; Gained monose also comprises wood sugar, can make Xylitol through hydrogenation, as the non-caloric sweetener of food.Said monose also can pass through mikrobe (for example yeast) alcohol prepared by fermenting.
The raw material of cellulose is generally root, stem and the leaf of plant, comprises stalk.Said stalk is root, stem and the leaf that stays behind farm crop (being generally herbage) the results fruit, is mainly stem and leaf, like stalk, the wheat straw of corn straw, broomcorn straw, cereal grass.The staple of stalk is a lignocellulose.Said lignocellulose is the mixture of Mierocrystalline cellulose, semicellulose and xylogen.The wood fibre that Mierocrystalline cellulose, semicellulose and xylogen are combined closely and formed through covalent linkage or non covalent bond in the stalk accounts for the 70-90% of stalk gross weight.The content of Mierocrystalline cellulose, semicellulose and xylogen can the difference to some extent with the different piece of the farm crop of different sources or farm crop in the stalk.
Mierocrystalline cellulose is commonly referred to enzyme digestion reaction by the reaction of cellulase hydrolysis, can be called saccharification react again, and the primary product of enzymolysis is a monose.Mierocrystalline cellulose can change into glucose under the effect of cellulase; Semicellulose can change into monose such as wood sugar under the effect of hemicellulase; Glucose and wood sugar may be used to fermentative production of ethanol; Can prepare ethanol by glucose fermentation such as yeast saccharomyces cerevisiae, pichia stipitis can prepare ethanol by xylose-fermenting.Cellulosic non-crystalline structure is easy to broken, and it can be degraded into glucose fully, becomes ethanol by glucose fermentation then.But cellulosic crystalline texture is difficult to be destroyed.In addition, xylogen is a kind of non-polysaccharide material that is polymerized by phenyl-propane, by the verivate of aromatic hydrocarbon with-C-C-key ,-the O-key is crosslinked together in length and breadth; Its side chain again with semicellulose with covalent bonds, form a very fine and close network structure, Mierocrystalline cellulose tightly is wrapped in the inside; Be unfavorable for that cellulase is to cellulosic attack; Make cellulase can't contact substrate, therefore, need earlier stalk to be carried out pre-treatment with hemicellulase; Contact with cellulase and/or hemicellulase again after steam explosion or the s.t. as stalk is carried out, Mierocrystalline cellulose is hydrolyzed.Though acid hydrolysis can destroy xylogen, also can destroy Mierocrystalline cellulose, reduce the productive rate of glucose then, and also need add after the acid hydrolysis in a large amount of alkali and could carry out enzymolysis and fermentation.Therefore more tendency is only accomplished preprocessing process through steam explosion, reaches the embedding effect of xylogen to Mierocrystalline cellulose and semicellulose that destroy.
Document " to the introduction of straw biomass to alcohol conversion process " for example, Zhang Xuefeng, orchid are kindly helped secure the success of, wine brewing, the 29th the 4th phase of volume; The 37-38 page or leaf discloses a kind of method of utilizing straw to produce alcohol in July, 2002, and this method comprises to be removed corn straw after the stone deironing cleans; Cut into 1.5 centimetres segment, water logging 40 minutes feeds 2.5 MPa steam, is incubated 8 minutes; Pressure release, the steam explosion products of washing 1/10th, and this wooden enzyme produces cellulase in said 1/10th steam explosion products, cultivating, and the gained culture is mixed with other 9/10ths steam explosion products; Be incubated 50 ℃ of hydrolysis 24 hours, through filtering 6% liquid glucose, mineral membrane is condensed into the liquid glucose more than 20%, adds Xiu Hata yeast (0.8-1.2 hundred million/milliliter); Fermented 24 hours, and produced wine part 10%, distill 99.5% alcohol.
CN1117835C discloses a kind of method of dissolve with ethanol steam burst straw lignin prepare liquid fuel, and this method is a raw material with the steam puffed stalk, in autoclave, with the xylogen in the alcohol extraction steam puffed stalk; Obtain filtrating with vacuum filtration again, and steam puffed stalk is extracted slag carry out the synchronous saccharification solid state fermentation of ethanol through, alcohol extraction liquid and vacuum filtration gained filtrating are the liquid fuel of this method preparation.
The preparation method of said steam puffed stalk raw material comprises plant materials such as wheat straw, straw or the cornstalk steam-explosion jar of packing into of chopping, under 1.2-1.7 MPa pressure, keeps pressure 5-10 minute, and afterwards, instant decompression discharges.
The method of the xylogen in the described alcohol extraction steam puffed stalk is in autoclave, adds steam puffed stalk and ethanol, and steam puffed stalk and alcoholic acid volume ratio are 1: 10-1: 50, in the pressure range of 1.5-2.0 MPa, kept 1-2 hour.
Describedly steam puffed stalk is extracted slag carry out the synchronous saccharification solid state fermentation of ethanol through and be meant: in 33-35 ℃ of scope (fermentation), kept 14-16 hour, in 38-40 ℃ of scope (enzymolysis), keep 68 hours, and fermentation 2-3 days that hockets.
CN1193099C discloses a kind of use heat resistant type yeast and from the lignin fiber cellulosic biomass, has produced the alcoholic acid method, and this method may further comprise the steps:
It is the 15-30 millimeter that the lignin fiber cellulosic biomass is ground into particle size; Lignin fiber cellulosic biomass to pulverizing carries out the vapor explosion pre-treatment, keeps the pressure of 1-3 MPa and 190-230 ℃ temperature 1-10 minute time according to the type of material therefor, causes explosive decompression afterwards; Collect pretreated material and pass through filtering separation liquid and solid part, solid part is introduced in the fermentation storage; Adding concentration to the fermentation storage is that 15FPU/ restrains the cellulosic beta-glucosidase of cellulosic cellulase and 12.6 ius/restrain; Give fermentation storage inoculation Marx Crewe Vickers yeast CECT the suspension-s of 10875 heat resistant type yeast cultures; Mixture was shaken under 42 ℃ 72 hours.
Adopt aforesaid method all can realize purpose, but the productive rate of monose is lower, is generally about 35% by the feedstock production monose of cellulose.
Summary of the invention
The objective of the invention is to overcome the defective that the monose productive rate is lower, enzymolysis efficiency is lower that the method for the feedstock production monose of existing employing cellulose prepares, the method for the feedstock production monose of the employing cellulose that a kind of monose productive rate is higher, enzymolysis efficiency is higher is provided.
Contriver of the present invention finds; In the method for the feedstock production monose of the employing cellulose of prior art; The raw material of cellulose normally exists with the form of liquid slurry like stalk, when the whole stalks with the slurries shape directly mix with enzyme; Enzyme concn is reduced, enzyme activity is descended.Moreover; To pass through pretreated stalk and the method for enzyme mixed enzymolysis is generally in the mixed solution that stalk particle with fragmentation once all joins enzyme and water and mixes with enzyme liquid; Carry out the enzymatic saccharification reaction, perhaps, earlier with used reaction mass---mix with water in retort through pretreated stalk particle; Then under hydrolysis temperature, enzyme directly joined in the retort react.Although above-mentioned mixing is carried out under agitation condition, stalk particle still is not easy to mix with enzyme.Because; Diminish through pretreated stalk granularity, particle and clastic stalk particle are easy to mutual adhesion and reunion, therefore-inferior whole stalks are mixed with enzyme fully; Only there is the part stalk fully to contact with enzyme; Because the adhesion between the stalk also has the part stalk with reunion and can not fully contact with enzyme, even under agitation, can not fully stalk be broken up.Therefore, behind the enzymolysis cellulose conversion be sugar transformation efficiency low.In addition, owing to once whole stalks are mixed with enzyme fully, stalk can not fully contact with enzyme, and therefore, the time of enzymolysis is also longer relatively, and enzymolysis efficiency is lower.
In addition, during the raw material of prior art steam blasting cellulose-containing, the raw material that it has been generally acknowledged that said cellulose is corn straw for example; Fully water logging can better reach the effect of steam explosion, because fully water logging can make the raw material of said cellulose for example contain a large amount of water in corn straw inside; When steam explosion, the raw material of the said cellulose for example inner water of corn straw can be gasificated into water vapor, is for example gone out corn straw inside by the raw material of said cellulose during pressure release; With the raw material that fully the destroys said cellulose from inside to outside xylogen sheath of corn straw embedding Mierocrystalline cellulose and semicellulose for example; Such as (Zhang Xuefeng, orchid are kindly helped secure the success of, wine brewing, the 29th the 4th phase of volume in above-mentioned " to the introduction of straw biomass to alcohol conversion process "; The 37-38 page or leaf; In July, 2002) disclosed method in need just be carried out steam explosion with the corn straw water logging after 40 minutes.But contriver of the present invention is surprised to find that, with corn straw (sapphire rice stalk and the dry corn straw) water logging of various water cut after 40 minutes; The water cut of corn straw is all up to more than the 80 weight %; And the water cut of the raw material of the cellulose of steam explosion is low, and it is no doubt bad for example to be lower than 30 weight %, if but the water cut of the raw material of cellulose; As be higher than 60 weight %, also can reduce the productive rate of monose.
The invention provides a kind of method that adopts the feedstock production monose of cellulose, this method comprises the raw material of steam blasting cellulose-containing; With the steam explosion products that obtains mix with enzyme, enzymolysis; Wherein, Said steam explosion products and enzyme method of mixing are comprised earlier first steam explosion products being mixed with enzyme obtain mixture; Then remaining steam explosion products is added continuously and mix with said mixture, the adding speed of said remaining steam explosion products makes the concentration of steam explosion products in reaction system not be higher than 150 grams per liters; The weight of said first and enzyme blended steam explosion products is the 10-30 weight % of whole steam explosion products weight; The water cut of the raw material of said cellulose is 30-60 weight %.
Method of the present invention obtains mixture for earlier a collection of steam explosion products being mixed with enzyme; Then remaining steam explosion products is added continuously and mix with said mixture; It is the method for continuous feeding; This method can make steam explosion products evenly, contact, mix and react with enzyme fast, the reaction that makes steam explosion products and enzyme more evenly, more abundant, reaction system is more stable; And the concentration of enzyme can remain on higher level always, thereby the sugared transformation efficiency that has improved raw material effectively is the productive rate of monose.And the further strict water cut of controlling the raw material of the cellulose that is used for steam explosion is 30-60 weight %; Can guarantee under the steam explosion condition, to make the inner aqueous vapor of raw material of cellulose to change into water vapor; Water vapour is gone out by the raw material inside of cellulose when pressure release; Fully destroy the xylogen sheath of embedding Mierocrystalline cellulose and semicellulose in the raw material of cellulose,, thereby can further improve the productive rate of monose with the cellulose ratios that can contact with enzyme in the raw material that has increased cellulose.In addition, the method for said continuous feeding can also shorten enzymolysis time greatly, improves enzymolysis efficiency.For example, under the raw materials pretreatment condition situation all identical, in the method for embodiment 1 with other enzymatic hydrolysis condition; Per hour the cellulosic amount of enzymolysis is 7.0 grams, and the monose productive rate reaches 46.6%, and in the method for Comparative Examples 1; Per hour the cellulosic amount of enzymolysis is 4.75 grams, and the monose productive rate is merely 36.9%, in the method for Comparative Examples 2; Per hour the cellulosic amount of enzymolysis is 4.67 grams; The monose productive rate is merely 36.3%, and the increase rate of enzymolysis efficiency reaches 47.4-49.9%, and the increase rate of monose productive rate is up to 26.3-28.4%.
Description of drawings
Fig. 1 is the electron scanning micrograph of natural corn stalk;
Fig. 2 is the electron scanning micrograph according to stalk behind the method steam explosion of Comparative Examples 1;
Fig. 3 is the electron scanning micrograph according to stalk behind the method steam explosion of Comparative Examples 2;
Fig. 4 is the electron scanning micrograph according to stalk behind the method steam explosion of the embodiment of the invention 1.
Embodiment
According to the present invention, this method comprises the raw material of steam blasting cellulose-containing; With the steam explosion products that obtains mix with enzyme, enzymolysis; Wherein, Said steam explosion products and enzyme method of mixing are comprised earlier first steam explosion products being mixed with enzyme obtain mixture; Then remaining steam explosion products is added continuously and mix with said mixture, the adding speed of said remaining steam explosion products makes the concentration of steam explosion products in reaction system that obtains not be higher than 150 grams per liters; The weight of said first and enzyme blended steam explosion products is the 10-30 weight % of the steam explosion products weight that all obtains; The water cut of the raw material of said cellulose is 30-60 weight %, is preferably 35-50 weight %.
Under the preferable case; In order to enhance productivity and can to guarantee simultaneously thorough mixing, the reaction of steam explosion products and enzyme; Obtain the enzyme speed of action of higher steam explosion products concentration and higher level, the adding speed of said remaining steam explosion products makes the concentration of steam explosion products in reaction system be preferably the 100-150 grams per liter.
In order to guarantee that enzyme has best reactive behavior; With first steam explosion products with before enzyme mixes, the pH value of steam regulation explosion product is greater than 3 less than 7, is preferably 4.0-5.5; So that steam explosion products is with after enzyme mixes, enzyme has best reactive behavior.Because the fluctuation of pH value is little in the enzymolysis process, therefore the pH value of said enzymolysis can be regulated before adding enzyme according to this area method commonly used, and the method for said conditioned reaction material pH value can adopt and well known to a person skilled in the art the whole bag of tricks.For example earlier steam explosion products and water or substratum (enzyme-addedly generally mixing the general substratum with this mikrobe of adding microbes producing cellulase with water) are mixed, according to the pH value of gained mixture, adding acidic substance or alkaline matter in this mixture.For example, said acidic substance can be one or more in sulfuric acid, hydrochloric acid and the phosphoric acid; Said alkaline matter can be sodium hydroxide and/or Pottasium Hydroxide.Under the preferable case; With first steam explosion products with after enzyme mixes; Before the continuous adding of remaining steam explosion products, also comprise the timed interval, fully contact and fully hydrolysis in order to make first steam explosion products with enzyme; Improve the transformation efficiency of sugar, the said timed interval is preferably 10-30 minute.
Said enzymolysis time begins when enzyme is mixed with steam explosion products to calculate; Promptly; Used time when said enzymolysis time refers to steam explosion products mixed beginning to the content that steam explosion products is converted into sugar back sugar and no longer increase with enzyme; Said with the continuous 15-35% that accounts for enzymolysis time with the said mixture blended time that adds of remaining steam explosion products, be preferably 20-30%.In addition, according to the present invention, said with first steam explosion products with after enzyme mixes, the timed interval before remaining steam explosion products added continuously is also included within the said enzymolysis time.
In order to make enzyme and reaction raw materials blended more even, the enzymolysis of said steam explosion products preferably carries out under the water existence condition, more preferably in the suspension-s of the raw material that contains water and cellulose, carries out.That is to say, the present invention is preferred earlier first steam explosion products and water are mixed with suspension-s after, the more required whole enzymes of enzymolysis are mixed with suspension-s, and then add remaining steam explosion products according to method continuation successive of the present invention.Do not limit the amount of the water that added is special, under the preferable case, the add-on of said water is 1-4 with whole weight ratios of steam explosion products: 1.More preferably under the situation; Mixture to obtain after first steam explosion products, water and the enzyme mixing is a benchmark, and with the dry weight basis of steam explosion products, the content of first steam explosion products is at least 1 weight %; Be preferably 1-20 weight %, more preferably 1.2-10 weight %.Prepared by steam explosion products in the alcoholic acid method existing, when enzymolysis, because the concentration of steam explosion products is too rare, cause the production cycle long, production efficiency is low.And contriver of the present invention finds; The concentration of steam explosion products in the mixture of when enzymolysis, controlling first steam explosion products, water and obtaining after enzyme mixes, with first steam explosion products, water and the mixture that obtains after enzyme mixes be benchmark, with the dry weight basis of steam explosion products; Make the content of steam explosion products be at least 1 weight %; Be preferably 1-20 weight %, more preferably 1.2-10 weight % can either guarantee that the cycle of enzymolysis is short; Further guarantee to have high production efficiency simultaneously, promptly the productive rate of monose is high.
According to the present invention, the concentration of said steam explosion products in reaction system is meant, ratio * 100% of solid-liquid gross weight in the weight of the steam explosion products of be not liquefied (not with enzyme reaction) and the reaction system.
The raw material water content difference of the cellulose of different sources is very big; Water content difference such as the corn straw raw material of different times purchase is just very big; The fresh straw water cut that harvesting is no more than a week reaches more than the 80 weight %, but possibly have only 10% through the air-dry storage stalk water cut of airing.Existing method all adopts the similarity condition water logging to handle for the raw material of the cellulose of different moisture content without exception; Cause the water cut of raw material of cellulose excessive; Under the steam explosion condition; The inner moisture of the raw material of cellulose is gone out with liquid form during pressure release, does not reach the effect of the xylogen sheath of embedding Mierocrystalline cellulose and semicellulose in the raw material that destroys cellulose from inside to outside.
According to method provided by the invention, can obtain the raw material that water cut is the cellulose of 30-60 weight % through variety of way.The raw material of the different batches cellulose that there are differences for water cut can carry out different processing, such as for the high stalk of water cut, can 45-90 ℃ of oven dry part moisture, and make it water cut and be reduced to 30-60 weight %; For the low stalk of water cut, can adopt control water logging time method, the water cut that increases stalk is to 30-60 weight %; Just in time at stalk, can directly carry out steam explosion for water cut to 30-60 weight %.The water cut of the raw material of the preferred said cellulose that is used for steam explosion is 35-50 weight %.Only if specified otherwise; The water cut of the raw material of cellulose according to the invention all is meant the initial weight W1 of cellulose containing raw material and under 70-100 ℃, dries poor to the dried meter weight W 2 of the cellulose containing raw material of constant weight; With the weight percent of the initial weight W1 of cellulose containing raw material, i.e. water cut (weight %)=(W1-W2)/W1 * 100%.
Because the present invention only relates to the qualification to the water cut of the raw material of the improvement of steam explosion products feed way and said cellulose, therefore other step with the method for the feedstock production monose of cellulose is had no particular limits.
Method according to the feedstock production monose with cellulose according to the invention; Can adopt the conventional steam explosion condition of prior art to accomplish steam explosion to the raw material of cellulose; For example; The temperature of said steam explosion can be 180-200 ℃, and the pressure of said steam explosion can be the 1.4-2.0 MPa, and holding time of said steam explosion pressure can be 3-7 minute.More preferably the temperature of said steam explosion is 185-195 ℃, and the pressure of said steam explosion is the 1.4-1.7 MPa, and holding time of said steam explosion pressure is 4-5 minute.Under above-mentioned steam explosion condition, can both fully destroy the xylogen sheath of stalk embedding Mierocrystalline cellulose and semicellulose, realize the object of the invention.
The enzyme that said enzymolysis uses comprises cellulase.Said cellulase can obtain through variety of way, for example is commercially available, perhaps through using the microbes producing cellulase secretion to obtain.
Because the enzyme that uses the microbes producing cellulase secretion to obtain can contain various by products, the therefore preferred enzyme that directly adds.The consumption of said enzyme is The more the better, and from cost consideration, preferably with the dry weight basis of every gram steam explosion products, the consumption of said cellulase is the 8-20 enzyme activity unit, more preferably the 10-15 enzyme activity unit.The enzyme activity of cellulase according to the invention is according to American National renewable energy source laboratory (NationalRenewable Energy Laboratory; NREL) standard method that provides---cellulase activity is measured NREL LAP-006 and is measured; The enzyme activity unit of said cellulase is under the condition determination of this standard method regulation, restrains the micrograms that Whatman No.1 filter paper is converted into the required enzyme of glucose with 1 in 1 minute.
The temperature of said enzymolysis can be any optimum temperature of cellulase, is generally 45-55 ℃, more preferably 48-52 ℃.The longer the better on the time theory of said enzymolysis; But, because the method for the present invention's employing continuous feeding after feeding in raw material has for the first time guaranteed the uniform mixing of raw material and enzyme, therefore; Can effectively shorten enzymolysis time; Can guarantee sugared transformation efficiency again, therefore, the time of said enzymolysis is preferably 30-40 hour.
According to of the present invention one preferred embodiment; Said method with the steam explosion products enzymolysis comprises earlier mixes first steam explosion products afterwards in the adding enzymatic vessel with water, the pH value of steam regulation explosion product is greater than 3 to less than 7, and is warming up to 48-52 ℃ to reach the optimum response reactive conditions of enzyme; Then; Under agitation, in enzymatic vessel, add enzyme, stir after 10-30 minute; Add continuously remaining steam explosion products again, the adding speed of said remaining steam explosion products makes and contains the concentration of steam explosion products in reaction system and be not higher than 150 grams per liters.The amount of said water is 1-4 with the weight ratio of whole steam explosion products: 1; The amount of said first and enzyme blended steam explosion products is the 10-30% of whole steam explosion products weight.And with first steam explosion products, water and the mixture that obtains after enzyme mixes be benchmark, with the dry weight basis of steam explosion products, the content of first steam explosion products is 1.2-10 weight %.
Said cellulase is a prozyme, comprises C at least
1The plain enzyme of fiber type, the plain enzyme of Cx fiber type and three kinds of enzymes of cellose.
C
1Enzyme can make the crystalline Mierocrystalline cellulose change amorphous Mierocrystalline cellulose into.
C
xThe plain enzyme of fiber type is divided into C again
X1Plain enzyme of fiber type and C
X2Two kinds of the plain enzymes of fiber type.C
X1The plain enzyme of fiber type is the endo-type cellulase, can be from the plain intramolecular action of hydration amorphous cellulose in β-1, and the 4-glycosidic link generates cellodextrin and cellobiose.C
X2The plain enzyme of fiber type is a kind of circumscribed-type cellulase, can act on β-1 from the non-reducing end of hydration non-cellulose molecule, and the 4-glycosidic link cuts off β-1 one by one, and the 4-glycosidic link generates glucose.
Cellose then acts on cellobiose, generates glucose.
The enzyme that preferred said enzymolysis uses also comprises hemicellulase.Because hemicellulase can degradation of hemicellulose become water-soluble wood sugar; So the enzyme that enzymolysis uses comprises hemicellulase; Exposed fibers is plain more fully on the one hand; Increase the contact probability of Mierocrystalline cellulose and cellulase, semicellulose degraded product wood sugar also is important monose product on the other hand.With the dry weight basis of every gram steam explosion products, the consumption of said hemicellulase is the 4.4-8.8 enzyme activity unit.
The enzyme activity unit of hemicellulase according to the invention (U) is under 50 ℃, pH=4.8 condition, and it is that 1 weight % xylan solution produces the required enzyme amount of 1 microgram reducing sugar (in wood sugar) that PM decomposes concentration.
The vigor of hemicellulase according to the invention refers to the unit of activity that every gram hemicellulase is had.It is that hydrolysis 1 weight % xylan produces reducing sugar (in wood sugar) under 4.8 the condition at 50 ℃, pH that the vigor of said hemicellulase utilizes hemicellulase; Gained reducing sugar and excessive 3; Color reaction takes place in 5-dinitrosalicylic acid (DNS), records the proportional mensuration of growing amount of the absorbance value and the reducing sugar (in wood sugar) of reaction solution 550 nanometers with spectrophotometer.Concrete measuring method is following:
Accurately take by weighing 1.000 gram xylans, dissolve, use deionized water constant volume to 100 milliliter then, obtain 1 weight % xylan solution with the 0.1 mol acetate of 0.5 milliliter of pH=4.8-sodium acetate buffer solution;
Take by weighing 30 the gram Rochelle salts put into 500 milliliters of Erlenmeyer flasks, add 16 the gram NaOH after, add 50 ml deionized water; Speed heating in water bath to solid matter with 5 ℃/minute dissolves, and adds 1 gram 3, the 5-dinitrosalicylic acid; To dissolving, be cooled to room temperature, be settled to 100 milliliters with deionized water; Can get 3,5-dinitrosalicylic acid (DNS) solution;
Wood sugar is dried to constant weight for 80 ℃, accurately take by weighing 1.000 grams and be dissolved in 1000 ml waters, it is anticorrosion to add 10 milligrams of sodiumazide, obtains the normal wood sugar soln of 1 mg/ml;
Accurately take by weighing 1.000 gram solid hemicellulases or pipette 1 milliliters of liquid hemicellulase stoste; 0.1 mole of acetic acid-sodium acetate buffer solution dissolving with 0.5 milliliter of pH=4.8; Use deionized water constant volume to 100 milliliter then, obtain diluting 100 times enzyme liquid to be measured;
Respectively will be in 50 ℃ of heating in water bath 2 milliliters of wood sugar gradient standardized solution (0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml and 0..5 mg/ml of 60 minutes; Said wood sugar gradient standardized solution is mixed with the normal wood sugar soln of deionized water and 1 mg/ml) or deionized water (wood sugar blank); With 2 milliliters of DNS mixing boiling water baths 5 minutes; After the cooling, 15 milliliters of deionized water constant volumes, with the absorbance value of spectrophotometer wood sugar gradient standardized solution after distinguishing assaying reaction under 550 nanometers; With the absorbance value is X-coordinate, and xylose concentration is an ordinate zou drawing standard curve.Can get regression equation y=bx+a by this typical curve, wherein, x is an absorbance value, and y is an xylose concentration, and a is the intercept of gained straight-line equation, and b is the slope of gained straight-line equation;
Get 0.1 mol acetate-sodium acetate buffer solution (xylan blank) of 0.2 milliliter of enzyme liquid to be measured and 1.8 milliliters of said 1 weight % xylan solutions or pH=4.8, according to the step test light absorption value identical with above-mentioned wood sugar gradient standardized solution.And according to the vigor of computes hemicellulase:
X is the absorbance value of enzyme liquid to be measured in the formula; B is consistent to b in the regression equation of absorbance value and a with xylose concentration with a; N is the extension rate of enzyme, 60 be expressed as enzymatic reaction time be 60 minutes, 5 for the sampling multiple (from 1 milliliter of enzyme liquid to be measured, having taken out 0.2 milliliter here tests).
Can determine the vigor of concrete hemicellulase according to aforesaid method, and then calculate the consumption of hemicellulase.
In addition, the pressure and temperature of steam explosion can be sterilized.For preventing that in enzymolysis process assorted bacterium (mainly being bacterium) pollutes the toxin of the enzyme activity that exerts an influence; And prevent that living contaminants influences the zymic growth during the fermentation; Preferably before enzymolysis, add the bacteria antibiotic such as industrial penicillium mould, said microbiotic does not have effect to yeast but the growth of the bacterium that can suppress to mix.With every milliliter of enzymolysis solution is benchmark, and said antibiotic add-on is a 1-10 unit.Said enzymolysis solution comprises steam explosion products, enzyme and water.
Because under the high-temperature and high-pressure conditions of steam explosion; Semicellulose in the raw material of cellulose can produce the inhibition such as furfural, furans; Said inhibition can reduce the vigor and the zymogenic activity of enzyme; Therefore said method also is included in after the raw material of steam blasting cellulose-containing, before the steam explosion products that enzymolysis obtains, washes said steam explosion products.Said washing is that steam explosion products is mixed with water, stirs, and above-mentioned inhibition is dissolved in the water, spinning steam explosion products then, thus from steam explosion products, remove inhibition.The temperature of said washing is high more in theory, and is can the dissolved inhibition many more, considers energy consumption, and the temperature of preferred said washing is at least 60 ℃, more preferably 60-80 ℃.Because the water that added of washing is many more, the solvent that then dissolves inhibition is many more, but considers the energy consumption of spinning, and preferably with the dry weight basis of the raw material of every gram cellulose, the amount of water of said washing is the 2-10 gram, more preferably the 2-3 gram.
Owing to may contain sandstone impurity and iron contamination in the raw material of cellulose; Can cause damage to steam explosion equipment; So method for preparing monose of the present invention; Can be included in steam explosion and before the raw material of cellulose removed stone deironing routine operation, such as removing the stone deironing with the raw material of " wind send " cellulose and the method for attraction.Sandstone can not be delivered in the steam explosion equipment by wind because quality is big, iron contamination since the attraction of magnet can not get in the steam explosion equipment with raw material yet, thereby can accomplish except that the stone deironing.In addition; Because the raw material of cellulose itself tangles and the occluding device pipeline easily; Therefore; Before getting into steam explosion equipment, the size that preferably makes the raw material of said cellulose is 0.2-1 centimetre of a 0.5-3 cm x 0.2-1 cm x, and the size that more preferably makes the raw material of said cellulose is 0.5-1 centimetre of a 1-2 cm x 0.4-0.6 cm x.
Monose in the enzymolysis product can be through sugar industry method separation and purification commonly used.For example will remove the solid matter (for example comprising the cellulosic xylogen of not degraded) in the enzymolysis product through filtration, centrifugation method.Collect filtrating or supernatant, evaporation is removed the portion water branch and is obtained the supersaturation syrup that contents of monosaccharides is 60-65 weight %.In the vacuum crystallization jar, through transpiring moisture the monose crystallization is separated out, crystal and syrup form massecuite; When the crystal volume meets the requirements, massecuite is transferred to crystallizer from the vacuum crystallization jar, cooling is further separated out uncrystallized monose gradually.Carry out solid-liquid separation with whizzer, crystallization monose and molasses get product.Molasses are repeated said process crystallization 2-4 time, can fully extract monose.Crystallisation process can adopt nature graining method, stimulate graining method and nucleus graining method graining, preferably in syrup, adds crystallization with the nucleus graining method graining of kind of paste as crystal seed.
The present invention will be described in more detail below in conjunction with embodiment.
Embodiment 1
Present embodiment is used to explain the method for the feedstock production monose of employing cellulose of the present invention.
(1) steam explosion
Test corn straw raw material as shown in Figure 1; The water cut that records corn straw according to above-mentioned method is 20 weight %; Raw material is cut into the segment of 0.5 centimetre of 1.5 cm x, 0.2 cm x; With vertical steeping cell (the auspicious mechanical ltd in river in Shangdong Province, Shandong) and weight is after 1/3rd water of said corn straw raw material weight mixes, and can get the corn straw of water cut 40 weight %.Getting 1000 gram water cut is the corn straw of 40 weight %, under 195 ℃, keeps 5 minutes with the pressure of 1.6 MPas, and steam explosion is accomplished in pressure release then.The water of gained steam explosion products and 70 ℃ was mixed 30 minutes according to weight ratio at 1: 3; Use LW400 type horizontal screw centrifuge (Zhangjiagang Huada Centrifugal Manufacturing Co., Ltd.) under 900 rev/mins of rotating speeds, to carry out solid-liquid separation then, obtain 2000 gram solid steam explosion products (water cut is 70 weight %) altogether.
The mensuration of Mierocrystalline cellulose gross weight and semicellulose gross weight in the gained solid steam explosion products:
Get the steam explosion products of 5 grams after the said washing and under 45 ℃, dry to constant weight 1.5 grams, 300.0 milligrams of this dried steam explosion products of weighing are positioned in heavy 80 100 milliliters of dry Erlenmeyer flasks restraining.Add the sulphuric acid soln that 3.00 ml concns are 72 weight % to said Erlenmeyer flask, stirred 1 minute.Then Erlenmeyer flask was heated 60 minutes in 30 ℃ of water-baths, every at a distance from 5 minutes with stirring once to guarantee even hydrolysis.Water-bath makes the vitriolic concentration dilution to 4 weight % with deionized water after finishing.Filter the steam explosion products acid hydrolysis liquid after the said dilution with B, obtain altogether filtrating 84 milliliters.20 milliliters of filtratings are transferred in the triangular flask of 50 milliliters of dryings.Use 2.5 gram lime carbonate to regulate this pH value of filtrate to 5.5, left standstill 5 hours, collect supernatant liquid.0.2 the supernatant liquid that the micron membrane filtration is collected, the filtrating of gained supernatant liquid is analyzed with Biorad Aminex HPX-87P performance liquid chromatography (HPLC).HPLC condition: sample size 20 microlitres; Moving phase is 0.2 micron membrane filtration, and the HPLC ultrapure water of the sonic oscillation degassing; Flow velocity is 0.6 ml/min; Column temperature 80-85 ℃; Detector temperature 80-85 ℃; Detector is a refractive index detector; Be 35 minutes working time.With D-(+) glucose of 0.1-4.0 mg/ml concentration range and 0.1-4.0 mg/ml concentration range D-(+) wood sugar as standard model.HPLC analyzes and to obtain that glucose concn is 1.77 mg/ml in the steam explosion products acid hydrolysis liquid; The steam explosion products acid hydrolysis that calculating can get after the said washing of 1 gram can obtain the glucose that weight is 0.149 gram; Because being the sulphuric acid soln of 72 weight %, concentration can the Mierocrystalline cellulose of the product of steam explosion all be hydrolyzed into glucose; Therefore the weight of gained glucose is 1.11 times of cellulosic weight in the steam explosion products; Cellulose 0.134 gram in the steam explosion products after the i.e. said washing of 1 gram then is total to cellulose 267 grams in the 2000 gram steam explosion products.HPLC analyzes and to obtain that xylose concentration is 0.49 mg/ml in the steam explosion products acid hydrolysis liquid; The steam explosion products acid hydrolysis that calculating can get after the said washing of 1 gram can obtain the wood sugar that weight is 0.041 gram; Because being the sulphuric acid soln of 72 weight %, concentration can semicellulose in the product of steam explosion all be hydrolyzed into wood sugar; Therefore the weight of gained wood sugar is 1.14 times of weight of semicellulose in the steam explosion products; Promptly contain semicellulose 0.036 gram in the 1 gram steam explosion products, then contain semicellulose 72 grams altogether in the 2000 gram steam explosion products.
(2) enzymolysis
In enzymatic vessel, add entry, and under agitation, add remaining washing steam explosion products behind first step (1) sampling and testing; The amount of the washing steam explosion products of first adding be behind step (1) sampling and testing remaining whole washing steam explosion products weight 15%; Regulating the pH value then is 4, be heated to 52 ℃ after, with the dry weight basis of every gram steam explosion products; The enzyme that adds 20 enzyme activity units calculates; All the dry weight of steam explosion products is 600 grams, add the cellulase (jade of the He family Bioisystech Co., Ltd) of 12000 enzyme activity units (about 80 grams) altogether, and insulation mixes 20 minutes under 52 ℃; Add remaining washing steam explosion products then continuously; It is 140 grams per liters that the adding speed of said remaining washing steam explosion products makes the concentration of steam explosion products in reaction system; Behind reinforced the end; 52 ℃ down the insulation enzymolysis obtain enzymolysis product to enzyme-added back 36 hours (time that adds remaining washing steam explosion products continuously account for enzymolysis time 30%).The add-on of said water is 2: 1 with the weight ratio of all washing steam explosion products.Enzymolysis product is filtered with B, 20 milliliters of filtratings are transferred in the triangular flask of 50 milliliters of dryings, left standstill 5 hours, collect supernatant liquid.0.2 the supernatant liquid that the micron membrane filtration is collected, according to the said performance liquid condition of above-mentioned steps (1), the glucose of measuring and calculating in the enzymolysis product 279.6 restrains totally.The glucose weight that said enzymolysis obtains promptly totally 251.9 is restrained by the cellulosic weight of enzymolysis in the steam explosion products divided by 1.11, and according to computes cellulose conversion rate and monose productive rate, calculation result is seen table 1.Per hour the cellulosic amount of enzymolysis is 7.0 grams.(with the dry weight basis of steam explosion products, be benchmark with the gross weight of the mixture that obtains, the content of first steam explosion products is 2.1 weight %.)
Cellulose conversion rate=100% * by the cellulosic weight of enzymolysis/cellulosic gross weight
Glucose weight/stalk dry weight that monose productive rate=100% * enzymolysis obtains
Comparative Examples 1
The reference method of the feedstock production monose of cellulose is adopted in the explanation of this Comparative Examples.
Method according to embodiment 1 prepares monose, and different is that (Zhang Xuefeng, orchid are kindly helped secure the success of according to document " to the introduction of straw biomass to alcohol conversion process "; Wine brewing; The 29th the 4th phase of volume, 37-38 page or leaf, in July, 2002) disclosed method; Stalk was soaked in water earlier before steam explosion 40 minutes, and the water cut that records the corn straw raw material after the immersion reaches 83 weight %; Getting 3250 these water cut of gram is that the corn straw of 83 weight % carries out steam explosion according to the condition of embodiment 1, and obtains 4300 gram steam explosion products (water cut is 86%) after the washing.Analyze the method for the steam explosion products of sulphuric acid hydrolysis according to the said HPLC of embodiment 1, record in the 4300 gram steam explosion products cellulose 267 grams altogether, semicellulose 70 grams.In enzymolysis step (2), once all join remaining washing steam explosion products behind step (1) sampling and testing in the aqueous enzymatic vessel; Mix (amount of water is 2: 1 with the weight ratio of all washing steam explosion products) then with enzyme, enzymolysis time needs 42 hours; According to the said performance liquid condition of embodiment 1 step (1), the glucose of measuring and calculating in the enzymolysis product 221.2 restrains totally.The glucose weight that said enzymolysis obtains promptly totally 199.3 is restrained by the cellulosic weight of enzymolysis in the steam explosion products divided by 1.11, and per hour the cellulosic amount of enzymolysis is 4.75 grams.And calculate cellulose conversion rate and monose productive rate according to the method for embodiment 1 and formula, calculation result is seen table 1.
Comparative Examples 2
The reference method of the feedstock production monose of cellulose is adopted in the explanation of this Comparative Examples.
Method according to embodiment 1 prepares monose, and different is that using 750 gram water cut is that 20 weight % corn straws directly carry out steam explosion, and obtains 1500 gram steam explosion products (water cut is 60%) after the washing.Analyze the method for the steam explosion products of sulphuric acid hydrolysis according to the said HPLC of embodiment 1, record in the 1500 gram steam explosion products cellulose 266 grams altogether, semicellulose 72 grams.And according to the method enzymolysis steam explosion products of Comparative Examples 1, fermenting enzyme hydrolysis products; According to the said performance liquid condition of embodiment 1 step (1), the glucose of measuring and calculating in the enzymolysis product 217.9 restrains totally.The glucose weight that said enzymolysis obtains promptly totally 196.3 is restrained by the cellulosic weight of enzymolysis in the steam explosion products divided by 1.11, and per hour the cellulosic amount of enzymolysis is 4.67 grams.And calculate cellulose conversion rate and monose productive rate according to the method for embodiment 1 and formula, calculation result is seen table 1.
The electron microscopic observation result:
Amplify 1000 times of steam explosion products of observing Comparative Examples 1, Comparative Examples 2 and embodiment 1 with the JSM-35CF ESEM, the result is respectively like Fig. 2, Fig. 3 and shown in Figure 4.As can beappreciated from fig. 2, because stalk inside does not have moisture can be gasificated into water vapor, so steam explosion can not fully destroy stalk from inside to outside; As can beappreciated from fig. 3, the effect of Comparative Examples 2 corn straw steam explosions and bad, Mierocrystalline cellulose still is pencil, and cellulose surface is residual that the xylogen sheath arranged; And the steam explosion effect of embodiment 1 as shown in Figure 4 is fine, can see the free Mierocrystalline cellulose.
Embodiment 2
Present embodiment is used to explain the method for the feedstock production monose of employing cellulose of the present invention.Method according to embodiment 1 prepares monose; Different is that getting 1000 gram water cut is the corn straw of 50 weight %, is cut into the segment of 0.8 centimetre of 1.3 cm x, 0.4 cm x; Under 190 ℃, kept 4 minutes with the pressure of 1.8 MPas; Steam explosion is accomplished in pressure release then, and the water of gained steam explosion products and 65 ℃ was mixed 40 minutes according to mass ratio at 1: 2; Use impeller pump LW400 type horizontal screw centrifuge (Zhangjiagang Huada Centrifugal Manufacturing Co., Ltd.) under 900 rev/mins of rotating speeds, to carry out solid-liquid separation then, obtain 2000 gram steam explosion products (water cut is 75 weight %) altogether.
Analyze the method for the steam explosion products of sulphuric acid hydrolysis according to the said HPLC of embodiment 1, record in the 2000 gram steam explosion products cellulose 220 grams altogether, semicellulose 60 grams.
And steam explosion products being carried out enzymolysis according to the method for embodiment 1, the consumption of enzyme is about 65 grams, according to the said performance liquid condition of embodiment 1 step (1), measures and calculate glucose totally 226.9 grams in the enzymolysis product.The glucose weight that said enzymolysis obtains is divided by 1.11; Be totally 204.4 to be restrained by the cellulosic weight of enzymolysis in the steam explosion products; Per hour the cellulosic amount of enzymolysis is that 5.68 grams are (with the dry weight basis of steam explosion products; Weight with the mixture that obtains is benchmark, and the content of first steam explosion products is 1.7 weight %).And calculate cellulose conversion rate and monose productive rate according to the method for embodiment 1 and formula, calculation result is seen table 1.
Embodiment 3
Present embodiment is used to explain the method for the feedstock production monose of employing cellulose of the present invention.
Method according to embodiment 1 prepares monose, and different is that getting 1000 gram water cut is the corn straw of 35 weight %; Be cut into the segment of 0.7 centimetre of 1.2 cm x, 0.4 cm x; Under 200 ℃, kept 6 minutes with the pressure of 1.7 MPas, steam explosion is accomplished in pressure release then.The water of gained steam explosion products and 75 ℃ was mixed 50 minutes according to weight ratio at 1: 2.5; Use impeller pump LW400 type horizontal screw centrifuge (Zhangjiagang Huada Centrifugal Manufacturing Co., Ltd.) under 900 rev/mins of rotating speeds, to carry out solid-liquid separation then, obtain 2000 gram solid steam explosion products (water cut is 67.5 weight %) altogether.
Analyze the method for the steam explosion products of sulphuric acid hydrolysis according to the said HPLC of embodiment 1, record in the 2000 gram steam explosion products cellulose 288 grams altogether, semicellulose 78 grams.
And steam explosion products being carried out enzymolysis according to the method for embodiment 1, the consumption of enzyme is about 85 grams, according to the said performance liquid condition of embodiment 1 step (1), measures and calculate glucose totally 293.4 grams in the enzymolysis product.The glucose weight that said enzymolysis obtains is divided by 1.11; Be totally 264.3 to be restrained by the cellulosic weight of enzymolysis in the steam explosion products; Per hour the cellulosic amount of enzymolysis is that 7.3 grams are (with the dry weight basis of steam explosion products; Weight with the mixture that obtains is benchmark, and the content of first steam explosion products is 2.2 weight %).And calculate cellulose conversion rate and monose productive rate according to the method for embodiment 1 and formula, calculation result is seen table 1.
Embodiment 4
Present embodiment is used to explain the method for the feedstock production monose of employing cellulose of the present invention.
Method according to embodiment 1 prepares monose, and different is that getting 1400 gram water cut is the corn straw of 55 weight %; Be cut into the segment of 0.8 centimetre of 2.0 cm x, 0.6 cm x; Under 190 ℃, kept 7 minutes with the pressure of 1.55 MPas, steam explosion is accomplished in pressure release then.Obtain 1700 gram solid steam explosion products (water cut is 63 weight %) altogether.
Analyze the method for the steam explosion products of sulphuric acid hydrolysis according to the said HPLC of embodiment 1, record in the 1700 gram steam explosion products cellulose 280 grams altogether, semicellulose 75.5 grams.
And steam explosion products being carried out enzymolysis according to the method for embodiment 1, the consumption of enzyme is about 84 grams, according to the said performance liquid condition of embodiment 1 step (1), measures and calculate glucose totally 279.1 grams in the enzymolysis product.The glucose weight that said enzymolysis obtains is divided by 1.11; Be totally 251.4 to be restrained by the cellulosic weight of enzymolysis in the steam explosion products; Per hour the cellulosic amount of enzymolysis is that 6.9 grams are (with the dry weight basis of steam explosion products; Weight with the mixture that obtains is benchmark, and the content of first steam explosion products is 2.5 weight %).And calculate cellulose conversion rate and monose productive rate according to the method for embodiment 1 and formula, calculation result is seen table 1.
Embodiment 5
Present embodiment is used to explain the method for the feedstock production monose of employing cellulose of the present invention.
Method according to embodiment 1 prepares monose, and different is, in enzymolysis step; The amount of the steam explosion products of first adding be behind the sampling and testing remaining whole steam explosion products weight 20%, regulating the pH value is 4, be heated to 52 ℃ after; After adding cellulase; Add remaining washing steam explosion products continuously, it is 100 grams per liters that the adding speed of said remaining washing steam explosion products makes the concentration of steam explosion products in reaction system, behind reinforced the end; 52 ℃ down the insulation enzymolysis obtain enzymolysis product to enzyme-added back 38.5 hours (time that adds remaining washing steam explosion products continuously account for enzymolysis time 13%).According to the said performance liquid condition of embodiment 1 step (1), the glucose of measuring and calculating in the enzymolysis product 278.2 restrains totally.The glucose weight that said enzymolysis obtains is divided by 1.11; Be totally 250.6 to be restrained by the cellulosic weight of enzymolysis in the steam explosion products; Per hour the cellulosic amount of enzymolysis is that 6.5 grams are (with the dry weight basis of steam explosion products; Weight with the mixture that obtains is benchmark, and the content of first steam explosion products is 2.7 weight %).And calculate cellulose conversion rate and monose productive rate according to the method for embodiment 1 and formula, calculation result is seen table 1.
Embodiment 6
Present embodiment is used to explain the method for the feedstock production monose of employing cellulose of the present invention.
Method according to embodiment 1 prepares monose, and different is that different is; In enzymolysis step; The amount of the washing steam explosion products of first adding be behind the sampling and testing remaining whole steam explosion products weight 30%, regulating the pH value is 5, be heated to 50 ℃ after; After adding cellulase, after 50 ℃ insulation mixes 30 minutes down; Add remaining washing steam explosion products continuously; It is 130 grams per liters that the adding speed of said remaining washing steam explosion products makes the concentration of steam explosion products in reaction system; Behind reinforced the end; 52 ℃ down the insulation enzymolysis obtain enzymolysis product to enzyme-added back 37.5 hours (time that adds remaining washing steam explosion products continuously account for enzymolysis time 25%).According to the said performance liquid condition of embodiment 1 step (1), the glucose of measuring and calculating in the enzymolysis product 276 restrains totally.The glucose weight that said enzymolysis obtains is divided by 1.11; Be totally 248.7 to be restrained by the cellulosic weight of enzymolysis in the steam explosion products; Per hour the cellulosic amount of enzymolysis is that 6.6 grams are (with the dry weight basis of steam explosion products; Weight with the mixture that obtains is benchmark, and the content of first steam explosion products is 3.8 weight %).And calculate cellulose conversion rate and monose productive rate according to the method for embodiment 1 and formula, calculation result is seen table 1.
Embodiment 7
Present embodiment is used to explain the method for the feedstock production monose of employing cellulose of the present invention.
Method according to embodiment 1 prepares monose, and different is in enzymatic vessel, to add entry; And under agitation, add remaining washing steam explosion products behind first step (1) sampling and testing, the amount of the washing steam explosion products of first adding be behind step (1) sampling and testing remaining whole washing steam explosion products weight 10%; The pH value of conditioned reaction material is 5 then; And after being heated to 50 ℃,, add the cellulase of 12 enzyme activity units with the dry weight basis of every gram steam explosion products; The hemicellulase of 5 enzyme activity units calculates; All the dry weight of steam explosion products is 600 grams, add the cellulase (jade of the He family Bioisystech Co., Ltd) of 7200 enzyme activity units (about 50 grams) and the hemicellulase (Beijing chemical reagents corporation) of 3000 enzyme activity units (about 40 grams) altogether, and insulation mixes 25 minutes under 50 ℃; Add remaining washing steam explosion products then continuously; It is 150 grams per liters that the adding speed of said remaining washing steam explosion products makes the concentration of steam explosion products in reaction system; Behind reinforced the end; 52 ℃ down the insulation enzymolysis obtain enzymolysis product to enzyme-added back 38 hours (time that adds remaining washing steam explosion products continuously account for enzymolysis time 20%).The add-on of said water is 3.5: 1 with the weight ratio of all washing steam explosion products.And according to the said performance liquid condition of embodiment 1 step (1); The glucose of measuring and calculating in the enzymolysis product 278.7 restrains totally; The weight of the glucose that said enzymolysis obtains is divided by 1.11; Be totally 251.1 to be restrained by the cellulosic weight of enzymolysis in the steam explosion products, the wood sugar of measuring and calculating in the enzymolysis product 53.4 restrains totally, and the weight of the wood sugar that said enzymolysis obtains is divided by 1.14; Be totally 46.8 to be restrained by the weight of the semicellulose of enzymolysis in the steam explosion products; Per hour the gross weight of the Mierocrystalline cellulose of enzymolysis and semicellulose is 7.8 grams (with the dry weight basis of steam explosion products, be benchmark with the gross weight of the mixture that obtains, the content of first steam explosion products is 0.8 weight %).And calculate Mierocrystalline cellulose, semicellulose transformation efficiency and monose productive rate according to the following equation, calculation result is seen table 1.
Cellulose conversion rate=100% * by the cellulosic weight of enzymolysis/cellulosic gross weight
Semicellulose transformation efficiency=100% * by the gross weight of the weight/semicellulose of the semicellulose of enzymolysis
Monose productive rate=100% * (the wood sugar weight that glucose weight+enzymolysis that enzymolysis obtains obtains)/stalk dry weight
Table 1
| Embodiment or Comparative Examples | Cellulose conversion rate (%) | Semicellulose transformation efficiency (%) | Monose productive rate (%) |
| Embodiment 1 | 94.3 | - | 46.6 |
| Comparative Examples 1 | 74.6 | - | 36.9 |
| Comparative Examples 2 | 73.8 | - | 36.3 |
| Embodiment 2 | 92.9 | - | 45.4 |
| Embodiment 3 | 91.8 | - | 45.1 |
| Embodiment 4 | 89.8 | - | 44.3 |
| Embodiment 5 | 93.9 | - | 46.4 |
| Embodiment 6 | 93.1 | - | 46.0 |
| Embodiment 7 | 94.0 | 65.0 | 55.4 |
Data from last table 1 can find out, adopt the method for the productivity ratio Comparative Examples 1 of the monose that the method for the feedstock production monose of cellulose of the present invention obtains all to be significantly improved.And method enzymolysis efficiency of the present invention is high, but per hour the mixture 5.7-7.8 of enzymolysis Mierocrystalline cellulose or Mierocrystalline cellulose and semicellulose restrains.
Claims (9)
1. method that adopts the feedstock production monose of cellulose, this method comprises the raw material of steam blasting cellulose-containing; With the steam explosion products that obtains mix with enzyme, enzymolysis; It is characterized in that; Said steam explosion products and enzyme method of mixing are comprised earlier first steam explosion products being mixed with enzyme obtain mixture; Then remaining steam explosion products is added continuously and mix with said mixture, the adding speed of said remaining steam explosion products makes the concentration of steam explosion products in reaction system not be higher than 150 grams per liters; The weight of said first and enzyme blended steam explosion products is the 10-30 weight % of whole steam explosion products weight; Remaining steam explosion products is added continuously the 15-35% that accounts for enzymolysis time with the said mixture blended time, and said enzymolysis time refers to steam explosion products is mixed beginning used time when steam explosion products being converted into sugared content no longer increases after the sugar with enzyme; The water cut of the raw material of said cellulose is 30-60 weight %.
2. method according to claim 1, wherein, it is the 100-150 grams per liter that the adding speed of said remaining steam explosion products makes the concentration of steam explosion products in reaction system.
3. method according to claim 1, wherein, the water cut of the raw material of said cellulose is 35-50 weight %.
4. method according to claim 1, wherein, when first steam explosion products is mixed with enzyme; This method also comprises and adds entry; With the dry weight basis of steam explosion products, be benchmark with the weight of the mixture that obtains, the content of first steam explosion products is at least 1 weight %.
5. method according to claim 4 wherein, with the dry weight basis of steam explosion products, is a benchmark with the weight of the mixture that obtains, and the content of first steam explosion products is 1.2-10 weight %.
6. method according to claim 1, wherein, the enzyme that said enzymolysis uses comprises cellulase, with the dry weight basis of every gram steam explosion products, the consumption of said cellulase is the 8-20 enzyme activity unit; The temperature of said enzymolysis is 45-55 ℃, and the time of said enzymolysis is 30-40 hour, and the pH value of said enzymolysis is 3-7.
7. method according to claim 6, wherein, the enzyme that said enzymolysis uses also comprises hemicellulase, with the dry weight basis of every gram steam explosion products, the consumption of said hemicellulase is the 4.4-8.8 enzyme activity unit.
8. method according to claim 1, wherein, with first steam explosion products with after enzyme mixes, before remaining steam explosion products added continuously, also comprise the timed interval, the said timed interval is 10-30 minute.
9. method according to claim 1, wherein, the raw material of said cellulose is a stalk.
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| WO2015085219A1 (en) * | 2013-12-05 | 2015-06-11 | Watt Gerald D | Metal catalyzed hydrolysis of cellulosic biomass |
| US9809613B2 (en) | 2013-12-05 | 2017-11-07 | Brigham Young University | Metal catalyzed hydrolysis of cellulose and hemicellulose to produce monomeric carbohydrates for transportation fuel and electrical production |
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