CN101376898A - Method for preparing ethanol from raw material containing cellulose - Google Patents
Method for preparing ethanol from raw material containing cellulose Download PDFInfo
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- CN101376898A CN101376898A CNA2007101211230A CN200710121123A CN101376898A CN 101376898 A CN101376898 A CN 101376898A CN A2007101211230 A CNA2007101211230 A CN A2007101211230A CN 200710121123 A CN200710121123 A CN 200710121123A CN 101376898 A CN101376898 A CN 101376898A
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Abstract
The invention relates to a method for preparing ethanol with a raw material containing cellulose, which comprises the following steps: the raw material containing cellulose is mixed with enzyme to have zymohydrolysis, and a zymohydrolysis product is obtained and fermented; wherein, the method for mixing the raw material containing cellulose and the enzyme comprises the following steps: first, the first batch of raw material containing cellulose is mixed with enzyme to obtain a mixture; and then, the rest raw material containing cellulose is continuously added into the mixture to be mixed; the adding speed of the rest raw material containing cellulose leads the concentration of the raw material containing cellulose in the reaction system to be no more than 150g/l; the weight of the first batch of raw material containing cellulose mixed with enzyme is 10 percent to 30 percent of the weight of all the raw material containing cellulose. The method can lead the raw materials containing cellulose to evenly and quickly contact and react with the enzyme, so that the reaction system is more stable, thereby effectively improving the sugar conversion rate of the raw material and the yield of ethanol. In addition, the method can also greatly shorten the zymohydrolysis time and improve the zymohydrolysis efficiency.
Description
Technical field
The invention relates to the alcoholic acid preparation method, more specifically about adopting the feedstock production alcoholic acid method of cellulose.
Background technology
The raw material by cellulose of prior art prepares the alcoholic acid method as stalk and mainly comprises three steps: 1, the pre-treatment of the raw material of cellulose, 2, cellulosic enzymolysis; 3, Tang fermentation system ethanol.
The raw material of cellulose is generally root, stem and the leaf of plant, comprises stalk.Described stalk is root, stem and the leaf that stays behind farm crop (being generally herbaceous plant) the results fruit, is mainly stem and leaf, as stalk, the wheat straw of maize straw, broomcorn straw, cereal grass.The main component of stalk is a lignocellulose.Described lignocellulose is the mixture of Mierocrystalline cellulose, hemicellulose and xylogen.The wood fibre that Mierocrystalline cellulose, hemicellulose and xylogen are combined closely and formed by covalent linkage or non covalent bond in the stalk accounts for the 70-90% of stalk gross weight.The content of Mierocrystalline cellulose, hemicellulose 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, hemicellulose 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 be broken, and it can be degraded into glucose fully, becomes ethanol by glucose fermentation then.But cellulosic crystalline texture is difficult to destroyed.In addition, xylogen is a kind of non-polysaccharide material that is polymerized by phenyl-propane, by the derivative of aromatic hydrocarbon with-C-C-key,-O-key is crosslinked together in length and breadth, its side chain again with hemicellulose 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 with hemicellulase, therefore, need earlier stalk to be carried out pre-treatment, contact with cellulase and/or hemicellulase again after steam explosion or the acid treatment, Mierocrystalline cellulose is hydrolyzed as stalk is carried out.
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 filtrate 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 filtrate are the liquid fuel of this method preparation.
The preparation method of described 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, keeps 1-2 hour in the pressure range of 1.5-2.0 MPa.
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 produced the alcoholic acid method from the lignin fiber cellulosic biomass, and this method may further comprise the steps:
It is the 15-30 millimeter that the lignin fiber cellulosic biomass is ground into granular size; The lignin fiber cellulosic biomass of pulverizing is carried out the vapor explosion pre-treatment, keep the pressure of 1-3 MPa and 190-230 ℃ temperature 1-10 minute time, cause explosive decompression afterwards according to the type of material therefor; 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 international unit/restrain; Give fermentation storage inoculation Marx Crewe Vickers yeast CECT the suspension of 10875 heat resistant type yeast cultures; Mixture was shaken under 42 ℃ 72 hours.
The employing aforesaid method all can be realized the feedstock production alcoholic acid purpose by cellulose, but in the feedstock production monose by cellulose, the productive rate of enzymolysis efficiency and monose is lower, and therefore, ethanol yield is lower, generally is no more than 20%.
Summary of the invention
The objective of the invention is to overcome the defective that alcohol yied is lower, enzymolysis efficiency is lower that the feedstock production alcoholic acid method of existing employing cellulose prepares, the feedstock production alcoholic acid method of the employing cellulose that a kind of alcohol yied is higher, enzymolysis efficiency is higher is provided.
The present inventor finds, in the method for the feedstock production alcohol fuel of the employing cellulose of prior art, the raw material of cellulose, normally exist as stalk with the form of liquid slurry, when the whole stalks with the slurries shape directly mix with enzyme, enzyme concn is reduced, enzyme activity is descended.Moreover, to be generally in the mixed solution that stalk particle with fragmentation once all joins enzyme and water through the pretreated stalk and the method for enzyme mixed enzymolysis and mix 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 once 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, the ethanol yield that sugar-fermenting obtains is lower.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.
The invention provides a kind of feedstock production alcoholic acid method that adopts cellulose, this method comprises that the raw material with cellulose mixes with enzyme, enzymolysis, obtain enzymolysis product, and this enzymolysis product that ferments, wherein, described raw material with cellulose and enzyme blended method comprise earlier that the raw material with first cellulose mixes with enzyme and obtain mixture, then the raw material of remaining cellulose is mixed with described mixture continuous the adding, the adding speed of the raw material of described remaining cellulose makes the concentration of raw material in reaction system of cellulose not be higher than 150 grams per liters; The weight of the raw material of described first and enzyme blended cellulose is the 10-30 weight % of the raw material weight of whole cellulose.
Method of the present invention obtains mixture for first raw material with a collection of cellulose mixes with enzyme, then the raw material of remaining cellulose is mixed with described mixture continuous the adding, it is the method for continuous feeding, the raw material that this method can make cellulose evenly, contact, mix and react with enzyme fast, make the reaction of the raw material of cellulose and enzyme more even, more abundant, reaction system is more stable, and the concentration of enzyme can remain on higher level always, thereby improved the sugared transformation efficiency of raw material effectively, and improved ethanol yield greatly.In addition, the method for described continuous feeding can also shorten enzymolysis time greatly, improves enzymolysis efficiency.For example, under the raw materials pretreatment condition situation all identical with other enzymatic hydrolysis condition, in the method for embodiment 1, per hour the cellulosic amount of enzymolysis is 9.86 grams, and alcohol yied reaches 23.6%, and in the method for Comparative Examples 1, per hour the cellulosic amount of enzymolysis is about 6 grams, alcohol yied only is 18.6%, and the increase rate of enzymolysis efficiency reaches 64.3%, and the increase rate of alcohol yied is up to 26.9%.
Embodiment
According to the present invention, this method comprises that the raw material with cellulose mixes with enzyme, enzymolysis, obtain enzymolysis product, and this enzymolysis product that ferments, wherein, described raw material with cellulose and enzyme blended method comprise earlier that the raw material with first cellulose mixes with enzyme and obtain mixture, then the raw material of remaining cellulose is mixed with described mixture continuous the adding, the adding speed of the raw material of described remaining cellulose makes the concentration of raw material in reaction system of cellulose not be higher than 150 grams per liters; The weight of the raw material of described first and enzyme blended cellulose is the 10-30 weight % of the raw material weight of whole cellulose.
Under the preferable case, in order to enhance productivity and can to guarantee simultaneously thorough mixing, the reaction of the raw material and the enzyme of cellulose, obtain the material concentration of higher cellulose and the enzyme speed of action of higher level, it is the 100-150 grams per liter that the adding speed of the raw material of described remaining cellulose makes the concentration of raw material in reaction system of cellulose.
In order to guarantee that enzyme has best reactive behavior, with the raw material of first cellulose with before enzyme mixes, the pH value of regulating the raw material of cellulose is greater than 3 less than 7, is preferably 4.0-5.5, so that the raw material of cellulose is with after enzyme mixes, enzyme has best reactive behavior.The method of described conditioned reaction material pH value can adopt and well known to a person skilled in the art the whole bag of tricks.As pH value, in the raw material of this cellulose, add acidic substance or alkaline matter according to the raw material of gained cellulose.For example, described acidic substance can be one or more in sulfuric acid, hydrochloric acid and the phosphoric acid; Described alkaline matter can be sodium hydroxide and/or potassium hydroxide.
Under the preferable case, with the raw material of first cellulose with after enzyme mixes, before the continuous adding of the raw material of remaining cellulose, also comprise the timed interval, for the raw material that makes first cellulose fully contacts with enzyme and fully hydrolysis, improve the transformation efficiency of sugar, the described timed interval is preferably 10-30 minute.
Described enzymolysis time begins when enzyme is mixed with the raw material of cellulose to calculate, promptly, used time when described enzymolysis time refers to that the raw material with cellulose mixes beginning to the content that cellulose containing raw material is converted into sugar back sugar and no longer increases with enzyme, described raw material with remaining cellulose adds the 15-35% that accounts for enzymolysis time with the described mixture blended time continuously, is preferably 20-30%.In addition, according to the present invention, described with the raw material of first cellulose with after enzyme mixes, the timed interval before the raw material of remaining cellulose added continuously is also included within the described enzymolysis time.
In order to make enzyme and reaction raw materials blended more even, the enzymolysis of the raw material of described cellulose preferably carries out under the water existence condition, more preferably carries out in the suspension of the raw material that contains water and cellulose.That is to say, the present invention is preferred earlier the raw material of first cellulose is mixed with suspension with water after, the more required whole enzymes of enzymolysis are mixed with suspension, and then add the raw material of remaining cellulose according to method continuation successive of the present invention.Amount to the water that added is not particularly limited, and under the preferable case, the add-on of described water is 1-4:1 with whole weight ratios of the raw material of cellulose.More preferably under the situation, the mixture that obtains after mixing with the raw material of first cellulose, water and enzyme is a benchmark, and with the dry weight basis of the raw material of cellulose, the content of the raw material of first cellulose is at least 1 weight %, be preferably 1-20 weight %, more preferably 1.2-10 weight %.In existing feedstock production alcoholic acid method by cellulose, when enzymolysis,, cause the production cycle long because the concentration of the raw material of cellulose is too rare, production efficiency is low.And the present inventor finds, the concentration of cellulosic raw material in the mixture of when enzymolysis, controlling raw material, the water of first cellulose and obtaining after enzyme mixes, with first cellulosic raw material, water and the mixture that obtains after enzyme mixes be benchmark, dry weight basis with cellulosic raw material, make the content of cellulosic raw material be at least 1 weight %, be preferably 1-20 weight %, 1.2-10 weight % more preferably, the cycle that can either guarantee enzymolysis is short, further guarantee to have high production efficiency, i.e. the productive rate height of monose simultaneously.
According to the present invention, the concentration of the raw material of described cellulose in reaction system is meant, ratio * 100% of the gross weight of the weight of the water that preferably contains in the weight of the weight of the raw material of total cellulose, enzyme and the reaction system in the weight of the fibrous raw material of be not liquefied (not with enzyme reaction) and the reaction system.
Because the present invention only relates to improvement to the raw materials mode of cellulose, therefore other step with the feedstock production alcoholic acid method of cellulose is had no particular limits.
The enzyme that described enzymolysis uses comprises cellulase.Described cellulase can obtain by variety of way, for example is commercially available, perhaps by 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 described enzyme is The more the better, and for cost consideration, preferably with the dry weight basis of the raw material of every gram cellulose, the consumption of described cellulase is the 8-20 enzyme activity unit, more preferably the 10-15 enzyme activity unit.The enzyme activity of cellulase of the present 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 described cellulase is under the condition determination of this standard method regulation, in 1 minute 1 gram Whatman No.1 filter paper is converted into the micrograms of the required enzyme of glucose.
The temperature of described 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 described enzymolysis, but, because the present invention adopts the method for continuous feeding to guarantee the uniform mixing of raw material and enzyme after feeding in raw material for the first time, therefore, can effectively shorten enzymolysis time, can guarantee sugared transformation efficiency again, therefore, the time of described enzymolysis is preferably 32-40 hour.
According to of the present invention one preferred embodiment, the method of described raw material enzymolysis with cellulose comprises that earlier the raw material with first cellulose mixes with water afterwards in the adding enzymatic vessel, the pH value of regulating the raw material of cellulose is to less than 7 greater than 3, and be 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 the raw material of remaining cellulose more continuously, the adding speed of the raw material of described remaining cellulose makes the concentration of raw material in reaction system of cellulose not be higher than 150 grams per liters.The amount of described water is 1-4:1 with the weight ratio of the raw material of whole cellulose; The amount of the raw material of described first and enzyme blended cellulose is the 10-30% of the raw material weight of whole cellulose.And with first cellulosic raw material, water and the mixture that obtains after enzyme mixes be benchmark, with the dry weight basis of cellulosic raw material, the content of first cellulosic raw material is 1.2-10 weight %.
Described 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 cellobiase.
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.
Cellobiase then acts on cellobiose, generates glucose.
The enzyme that preferred described enzymolysis uses also comprises hemicellulase.Because hemicellulase can degradation of hemicellulose become water-soluble wood sugar, so the enzyme that enzymolysis uses comprises hemicellulase, one side is the exposed fibers element more fully, increase the contact probability of Mierocrystalline cellulose and cellulase, hemicellulose degraded product wood sugar can be generated ethanol by the pichia stipitis fermentation on the other hand, and two aspect effects can increase alcohol yied.With the dry weight basis of the raw material of every gram cellulose, the consumption of described hemicellulase is the 4.4-8.8 enzyme activity unit.
The enzyme activity unit of hemicellulase of the present 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 per minute decomposes concentration.
The vigor of hemicellulase of the present 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 described 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 as follows:
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 gram Rochelle salts and put into 500 milliliters of Erlenmeyer flasks, after adding 16 gram NaOH, add 50 ml deionized water, dissolve, add 1 gram 3 with 5 ℃/minute speed heating in water bath to solid matters, 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 2 milliliters of wood sugar gradient standardized solution (0.1 mg/ml of 60 minutes of 50 ℃ of heating in water bath, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml and 0..5 mg/ml, described 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, cooling, behind 15 milliliters of the deionized water constant volumes, absorbance value with spectrophotometer wood sugar gradient standardized solution behind difference 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 the 0.1 mol acetate-sodium acetate buffer (xylan blank) of 0.2 milliliter of enzyme liquid to be measured and 1.8 milliliters of described 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 calculate the vigor of hemicellulase according to following formula:
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, and 60 times that are expressed as enzymatic reaction were 60 minutes, and 5 are sampling multiple (having taken out 0.2 milliliter here from 1 milliliter of enzyme liquid to be measured tests).
Can determine the vigor of concrete hemicellulase according to aforesaid method, and then calculate the consumption of hemicellulase.
The raw material of described cellulose can be the stalk of various farm crop, herbal cane etc., as maize straw, straw, wheat stalk, sorghum stalk etc.Because the main component of stalk comprises Mierocrystalline cellulose, hemicellulose and xylogen, for can make when the enzymolysis cellulase and hemicellulase fully with stalk in Mierocrystalline cellulose contact with hemicellulose and carry out abundant enzymolysis, before enzymolysis, preferably also the raw material of cellulose is carried out pre-treatment so that Mierocrystalline cellulose, hemicellulose and lignin separation are opened.
The pretreatment process of the raw material of described cellulose can be the method that well known to a person skilled in the art, for example, and acid treatment method and/or steam explosion method.Described acid treatment method comprises that the raw material with cellulose makes it to separate with hemicellulose with Mierocrystalline cellulose with dissolved lignin with the acid mixing.The kind of the acid of the xylogen of the described raw material that is used for dissolving cellulose is conventionally known to one of skill in the art, as: as described in acid can be the various acid of this area routine, as, be selected from sulfuric acid, hydrochloric acid and the phosphoric acid one or more; The consumption and the concentration of described acid are not particularly limited, as required the consumption and the concentration of the amount of xylogen control acid in the raw material of dissolved cellulose.
Described steam explosion method comprise with the raw material of cellulose with carry out steam explosion after water mixes or directly place steam blasting device to carry out steam explosion the raw material of cellulose.Because steam explosion method more helps destroying the reticulated structure between Mierocrystalline cellulose, hemicellulose and the xylogen in the raw material of cellulose, Mierocrystalline cellulose can fully be separated, help the effect of cellulase, improve the productive rate of cellulosic percent hydrolysis and sugar at cellulose surface.Therefore, the pretreatment process of the raw material of the preferred described cellulose of the present invention is a steam explosion method, and the raw material of described cellulose is preferably the raw material of cellulose of steam explosion and/or the raw material of acid-treated cellulose.
The steam explosion that the steam explosion condition of employing prior art routine is carried out can both reach goal of the invention of the present invention, for example, the temperature of described steam explosion is 180-200 ℃, and the pressure of described steam explosion is the 1.4-1.8 MPa, and holding time of described steam explosion pressure is 3-7 minute.More preferably the temperature of described steam explosion is 185-195 ℃, and the pressure of described steam explosion is the 1.4-1.7 MPa, and holding time of described steam explosion pressure is 4-5 minute.
Can ferment pentoses and/or the microorganism of hexose may be used to fermenting process of the present invention because yeast saccharomyces cerevisiae is the microorganism of the zymohexose that ethanol-tolerant, by product are few, alcohol yied is high of widespread usage on the wine industry; Pichia stipitis for both can ferment pentoses also can zymohexose microorganism (referring to " pichia stipitis continuously ferment pentose and hexose generate alcohol ", Ji Gengsheng etc., Nanjing Forestry University's journal natural science edition, the 28th the 3rd phase of volume, the 9-13 page or leaf, 2004), therefore the employed yeast of preferred described fermentation is pichia stipitis and/or yeast saccharomyces cerevisiae.In every gram enzymolysis product, the employed zymic inoculum size of described fermentation is 10
3-10
8Colony-forming unit, more preferably 10
4-10
6Colony-forming unit.Employed yeast is fermented in the present invention can be for being purchased yeast solids preparation (such as dried yeast powder) or barms (such as the cereuisiae fermentum of ATCC numbering 2601).Described zymic colony-forming unit can be measured by means commonly known in the art, such as the methylene blue staining viable bacteria counting method.The concrete grammar of methylene blue staining viable bacteria counting method is as follows:
1 gram dried yeast powder is dissolved in 10 ml sterile waters, or 1 milliliter of actication of culture liquid is diluted to 10 milliliters with sterilized water, add 0.5 milliliter of 0.1 weight % methylene blue, be incubated 30 minutes down at 35 ℃.Under 10 times of opticmicroscopes,, can get the number of viable bacteria in 1 gram dry yeast or the 1 milliliter of actication of culture liquid, i.e. colony forming single-digit with the number (dead bacterium dyeing, viable bacteria is not dyeed) of viable bacteria in the solution after the blood counting chamber counting insulation.
Described yeast can adopt conventional method inoculation, for example adds the seed liquor of 5-15 volume % in enzymolysis product.Described seed liquor can be the aqueous solution or the culture medium solution of dry yeast, also can or be purchased the activated seed liquid of bacterial classification for dry yeast.The temperature of described fermentation can be any temperature that is suitable for yeast growth, is preferably 30-36 ℃, more preferably 32-35 ℃.The time of described fermentation can be for beginning from inoculation to occur to the decline phase of yeast growth the time of (being that fermentation time is that lag phase, logarithmic phase add stationary phase), and the time of preferred fermentation is 32-48 hour, more preferably 32-40 hour.Tunning ethanol can be with conventional method, according to requirement (requiring alcoholic acid purity to reach more than 99% such as the fuel alcohol) separation and refining of different Industrial products, such as distilling, concentrate, dewatering.
In addition, the pressure and temperature of steam explosion can be sterilized, and acid treatment also can be played germ-resistant effect.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 added the bacteria antibiotic such as industrial penicillin before enzymolysis, described microbiotic does not have effect to yeast but the growth of the bacterium that can suppress to mix.With every milliliter of enzymolysis solution or fermented liquid is benchmark, and described antibiotic add-on is a 1-10 unit.Described enzymolysis solution comprises steam explosion products, enzyme and water, and described fermented liquid comprises the yeast of enzymolysis product and inoculation.
Because under the high-temperature and high-pressure conditions of steam explosion, hemicellulose in the raw material of cellulose can produce the inhibition such as furfural, furans, described inhibition can reduce the vigor and the zymogenic activity of enzyme, therefore described method also is included in after the raw material of steam blasting cellulose-containing, before the steam explosion products that enzymolysis obtains, wash described steam explosion products.Described washing is that steam explosion products is mixed with water, stirs, and above-mentioned inhibition is dissolved in the water, centrifugation steam explosion products then, thus from steam explosion products, remove inhibition.The temperature of described washing is high more in theory, and is can the dissolved inhibition many more, considers energy consumption, and the temperature of preferred described 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 centrifugation, and preferably with the dry weight basis of the raw material of every gram cellulose, the amount of water of described 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 preparation alcoholic acid method of the present invention, can be included in steam explosion and before the raw material of cellulose be 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 enter in the steam explosion equipment with raw material yet, thereby can finish except that the stone deironing.In addition, because the raw material of cellulose itself tangles and the occluding device pipeline easily, therefore, before entering steam explosion equipment, the size that preferably makes the raw material of described 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 described cellulose is 0.5-1 centimetre of a 1-2 cm x 0.4-0.6 cm x.
The present invention will be described in more detail below in conjunction with embodiment.
Embodiment 1
Present embodiment is used to illustrate the feedstock production alcoholic acid method of employing cellulose of the present invention.
(1) pre-treatment of the raw material of cellulose
Do not have the maize straw (water content 10%) of impurity to be cut into the segment that is no more than 1.0 centimetres of 1.2 cm x, 0.5 cm x 1000 grams, keep the pressure 5 minutes of 1.70 MPas under 195 ℃, steam explosion is finished in pressure release then.Gained steam explosion products and 70 ℃ of water were mixed 30 minutes according to mass ratio 1:3, use LW400 type horizontal screw centrifuge (Zhangjiagang Huada Centrifugal Manufacturing Co., Ltd.) under 900 rev/mins rotating speed, to carry out solid-liquid separation then, obtain 3000 gram washing steam explosion products (water content is 70 weight %) altogether.
The mensuration of Mierocrystalline cellulose gross weight and hemicellulose gross weight in the gained solid steam explosion products:
Get the above-mentioned washing steam explosion products of 10 grams and dry under 45 ℃ to constant weight 3 grams, 300.0 milligrams of these dried steam explosion products of weighing are positioned in 100 milliliters of dry Erlenmeyer flasks of heavy 80 grams.Adding 3.00 ml concns in described Erlenmeyer flask is the sulphuric acid soln of 72 weight %, stirs 1 minute.Then Erlenmeyer flask was placed 60 minutes in 30 ℃ water-bath, stirred once to guarantee even hydrolysis every 5 minutes.Hydrolysis makes the vitriolic concentration dilution to 4 weight % with deionized water after finishing, and filters with B then, obtains 84 milliliters of filtrates altogether.20 milliliters of filtrates are transferred in the triangular flask of 50 milliliters of exsiccant.Use 2.5 gram lime carbonate to regulate this pH value of filtrate to 5.5, left standstill 5 hours, collect supernatant liquid.With the supernatant liquid that 0.2 micron membrane filtration is collected, gained filtrate is analyzed with Biorad Aminex HPX-87P high performance liquid chromatography (HPLC).HPLC condition: sample size 20 microlitres; Moving phase is the HPLC ultrapure water of the 0.2 micron membrane filtration and 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 1.67 mg/ml in the steam explosion products acid hydrolysis liquid, the steam explosion products acid hydrolysis that calculating can get after the described washing of 1 gram can obtain the glucose that weight is 0.140 gram, because being the sulphuric acid soln of 72 weight %, concentration the Mierocrystalline cellulose of the product of steam explosion all can be hydrolyzed into glucose, therefore the weight of gained glucose is 1.11 times of cellulose in the steam explosion products, cellulose 0.126 gram in the steam explosion products after the i.e. described washing of 1 gram then is total to cellulose 378.4 grams in the 3000 gram steam explosion products.HPLC analyzes and to obtain that xylose concentration is 0.42 mg/ml in the steam explosion products acid hydrolysis liquid, the steam explosion products acid hydrolysis that calculating can get after the described washing of 1 gram can obtain the wood sugar that weight is 0.036 gram, because being the sulphuric acid soln of 72 weight %, concentration the hemicellulose of the product of steam explosion all can be hydrolyzed into wood sugar, therefore the weight of gained wood sugar is 1.14 times of hemicellulose weight in the steam explosion products, contain hemicellulose 0.032 gram in the steam explosion products after the i.e. described washing of 1 gram, then contain hemicellulose 94.7 grams altogether in the 3000 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, after being heated to 52 ℃, dry weight basis with every gram stalk, the enzyme that adds 20 enzyme activity units calculates, all the dry weight of stalk is 900 grams, the cellulase (jade of the He family Bioisystech Co., Ltd) that adds 18000 enzyme activity units (about 120 grams) altogether, and insulation mixed 20 minutes under 52 ℃; Add remaining washing steam explosion products then continuously, it is 140 grams per liters that the adding speed of the raw material of described remaining cellulose makes the concentration of raw material in reaction system of cellulose, 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 described water is 2:1 with the weight ratio of all washing steam explosion products.Enzymolysis product is filtered with B, 20 milliliters of filtrates 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 described high performance liquid phase condition of above-mentioned steps (1), the glucose of measuring and calculating in the enzymolysis product 394 restrains totally.The glucose weight that described enzymolysis obtains promptly totally 354.9 is restrained by the cellulosic weight of enzymolysis in the steam explosion products divided by 1.11, calculates cellulose conversion rate and monose productive rate according to following formula, and calculation result sees Table 1.Per hour the cellulosic amount of enzymolysis is 9.86 grams.(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 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
(3) fermentation
Make the temperature of enzymolysis product reduce to 35 ℃, in the weight of every gram enzymolysis product, inoculation 10
5The yeast saccharomyces cerevisiae of colony-forming unit (the super highly active dry yeast in Angel, Hubei Angel Yeast stock company), gained mixture under 32 ℃ in fermentor tank stir culture 40 hours.At 100 ℃ of distillation gained tunnings, the gained distillation fraction can get ethanol 212.8 grams at 78.3 ℃ of following second distillations, calculates alcohol yied according to following formula, and calculation result sees Table 1.
Alcohol yied=100% * ethanol weight/stalk dry weight
Comparative Examples 1
The feedstock production alcoholic acid reference method of cellulose is adopted in the explanation of this Comparative Examples.
Method according to embodiment 1 prepares ethanol, different is, in enzymolysis step (2), once all join remaining washing steam explosion products behind step (1) sampling and testing in the aqueous enzymatic vessel, mix (weight ratio of all washing steam explosion products and water is 2:1) then with enzyme, enzymolysis time needs 42 hours; According to the described high performance liquid phase condition of embodiment 1 step (1), the glucose of measuring and calculating in the enzymolysis product 304.5 restrains totally.The glucose weight that described enzymolysis obtains promptly totally 274.3 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 6.5 grams; Second distillation obtains ethanol 167.4 grams.And calculate cellulose conversion rate, monose productive rate and alcohol yied according to the method for embodiment 1 and formula, calculation result sees Table 1.
Embodiment 2
Present embodiment is used to illustrate the feedstock production alcoholic acid method of employing cellulose of the present invention.
Method according to embodiment 1 prepares ethanol, and different is, in the pre-treatment step of the raw material of cellulose, described steam explosion products is without washing; Analyze the method for the steam explosion products of sulphuric acid hydrolysis according to the described HPLC of embodiment 1, record in the 3000 gram steam explosion products cellulose 370.4 grams altogether, hemicellulose 88 grams.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, after being heated to 52 ℃, after adding cellulase, add remaining washing steam explosion products continuously, it is 100 grams per liters that the adding speed of the raw material of described remaining cellulose makes the concentration of raw material in reaction system of cellulose, 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 described high performance liquid phase condition of embodiment 1 step (1), the glucose of measuring and calculating in the enzymolysis product 371.3 restrains totally.The glucose weight that described enzymolysis obtains is divided by 1.11, be totally 334.5 to be restrained by the cellulosic weight of enzymolysis in the steam explosion products, per hour the cellulosic amount of enzymolysis is that 8.70 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 %.); Fermentation step is with embodiment 1, and second distillation obtains ethanol 189.0 grams.And calculate cellulose conversion rate, monose productive rate and alcohol yied according to the method for embodiment 1 and formula, calculation result sees Table 1.
Embodiment 3
Present embodiment is used to illustrate the feedstock production alcoholic acid method of employing cellulose of the present invention.
Method according to embodiment 1 prepares ethanol, 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, after being heated to 50 ℃, behind the 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 the raw material of described remaining cellulose makes the concentration of raw material in reaction system of cellulose, 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 described high performance liquid phase condition of embodiment 1 step (1), the glucose of measuring and calculating in the enzymolysis product 386.4 restrains totally.The glucose weight that described enzymolysis obtains is divided by 1.11, be totally 348.1 to be restrained by the cellulosic weight of enzymolysis in the steam explosion products, per hour the cellulosic amount of enzymolysis is that 9.30 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 %); In fermentation step, different is, makes the temperature of enzymolysis product reduce to 32 ℃, in the weight of every gram enzymolysis product, and inoculation 10
6The yeast saccharomyces cerevisiae of colony-forming unit (the super highly active dry yeast in Angel, Hubei Angel Yeast stock company), gained mixture under 32 ℃ in fermentor tank stir culture 36 hours; At 100 ℃ of distillation gained tunnings, the gained distillation fraction can get ethanol 202.5 grams at 78.3 ℃ of following second distillations.And calculate cellulose conversion rate, monose productive rate and alcohol yied according to the method for embodiment 1 and formula, calculation result sees Table 1.
Embodiment 4
Present embodiment is used to illustrate the feedstock production alcoholic acid method of employing cellulose of the present invention.
(1) pre-treatment of the raw material of cellulose
There is not the maize straw (water content 10%) of impurity to be cut into the segment that is no more than 1.0 centimetres of 1.5 cm x, 0.4 cm x 1000 grams, with the stalk section water logging after 5 minutes (weight ratio of stalk and water is 2.5:1) under 190 ℃, keep the pressure 5 minutes of 1.5 MPas, steam explosion is finished in pressure release then.Gained steam explosion products and 65 ℃ of water were mixed 30 minutes according to mass ratio 1:3, use LW400 type horizontal screw centrifuge (Zhangjiagang Huada Centrifugal Manufacturing Co., Ltd.) under 900 rev/mins rotating speed, to carry out solid-liquid separation then.Obtain 3250 gram steam explosion products (water content is 72.3 weight %) altogether.
Analyze the method for sulphuric acid hydrolysis steam explosion products according to embodiment 1 described HPLC, obtain that glucose concn is 1.62 mg/ml in the steam explosion products acid hydrolysis liquid, the steam explosion products acid hydrolysis that calculating can get after the described washing of 1 gram can obtain the glucose that weight is 0.136 gram, because being the sulphuric acid soln of 72 weight %, concentration the Mierocrystalline cellulose of the product of steam explosion all can be hydrolyzed into glucose, therefore the weight of gained glucose is 1.11 times of cellulose in the steam explosion products, cellulose 0.123 gram in the steam explosion products after the i.e. described washing of 1 gram then is total to cellulose 399.75 grams in the 3250 gram steam explosion products.HPLC analyzes and to obtain that xylose concentration is 0.41 mg/ml in the steam explosion products acid hydrolysis liquid, the steam explosion products acid hydrolysis that calculating can get after the described washing of 1 gram can obtain the wood sugar that weight is 0.034 gram, because being the sulphuric acid soln of 72 weight %, concentration the hemicellulose of the product of steam explosion all can be hydrolyzed into wood sugar, therefore the weight of gained wood sugar is 1.14 times of hemicellulose weight in the steam explosion products, contain hemicellulose 0.030 gram in the steam explosion products after the i.e. described washing of 1 gram, then contain hemicellulose 97.5 grams altogether in the 3250 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 20%, the pH value of conditioned reaction material is 5 then, and after being heated to 50 ℃, dry weight basis with every gram stalk, the cellulase that adds 17.8 enzyme activity units, the hemicellulase of 5 enzyme activity units calculates, all the dry weight of stalk is 900 grams, add the cellulase (jade of the He family Bioisystech Co., Ltd) of 16000 enzyme activity units (about 105 grams) and the hemicellulase (Beijing chemical reagents corporation) of 4500 enzyme activity units (about 62 grams) altogether, and be incubated mixing 25 minutes down at 50 ℃; Add remaining washing steam explosion products then continuously, it is 150 grams per liters that the adding speed of the raw material of described remaining cellulose makes the concentration of raw material in reaction system of cellulose, 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 described water is 3:1 with the weight ratio of all washing steam explosion products.And according to the described high performance liquid phase condition of embodiment 1 step (1), the glucose of measuring and calculating in the enzymolysis product 412.6 restrains totally, the weight of the glucose that described enzymolysis obtains is divided by 1.11, be totally 371.7 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 80.9 restrains totally, the weight of the wood sugar that described enzymolysis obtains is divided by 1.14, be totally 71.0 to be restrained by the weight of the hemicellulose of enzymolysis in the steam explosion products, per hour the gross weight of the Mierocrystalline cellulose of enzymolysis and hemicellulose is that 11.7 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 Mierocrystalline cellulose, hemicellulose transformation efficiency and monose productive rate according to the following equation, calculation result sees Table 1.
Cellulose conversion rate=100% * by the cellulosic weight of enzymolysis/cellulosic gross weight
Hemicellulose transformation efficiency=100% * by the gross weight of the weight/hemicellulose of the hemicellulose of enzymolysis
Monose productive rate=100% * (the wood sugar weight that glucose weight+enzymolysis that enzymolysis obtains obtains)/stalk dry weight
(3) fermentation
Make the temperature of enzymolysis product reduce to 34 ℃, in the weight of every gram enzymolysis product, inoculation is 10
4The yeast saccharomyces cerevisiae of colony-forming unit (the super highly active dry yeast in Angel, Hubei Angel Yeast stock company) and 10
4The pichia stipitis of colony-forming unit, gained mixture under 33 ℃ in fermentor tank stir culture 37 hours.At 100 ℃ of distillation gained tunnings, the gained distillation fraction can get ethanol 252.0 grams at 78.3 ℃ of following second distillations, and calculates alcohol yied according to the formula of embodiment 1, and calculation result sees Table 1.
Embodiment 5
Present embodiment is used to illustrate the feedstock production alcoholic acid method of employing cellulose of the present invention.
Method according to embodiment 1 prepares ethanol, different is, in the pre-treatment step of the raw material of cellulose, adopt the raw material of acid treatment cellulose, this method comprises, under 140 ℃, 0.06 MPa pressure, with 1000 gram maize straws (water content is 10%), with 250 ml concns is that the dilute sulphuric acid insulation of 2 weight % mixed 40 minutes, obtains acid-treated product.The gained acid-treated product is filtered, and mixed 30 minutes according to mass ratio 1:3, use LW400 type horizontal screw centrifuge (Zhangjiagang Huada Centrifugal Manufacturing Co., Ltd.) under 900 rev/mins rotating speed, to carry out solid-liquid separation then with water.Obtain the raw material product (water content is 75 weight %) of 3600 gram acid treatment cellulose altogether.
Analyze the method for sulphuric acid hydrolysis steam explosion products according to embodiment 1 described HPLC, obtain that glucose concn is 1.75 mg/ml in the acid-treated product acid hydrolysis liquid, the acid-treated product acid hydrolysis that calculating can get after the described washing of 1 gram can obtain the glucose that weight is 0.120 gram, because being the sulphuric acid soln of 72 weight %, concentration the Mierocrystalline cellulose in the acid-treated product all can be hydrolyzed into glucose, therefore the weight of gained glucose is 1.11 times of cellulose in the acid-treated product, cellulose 0.108 gram in the acid-treated product of the i.e. described washing of 1 gram back then is total to cellulose 387.0 grams in the 3600 gram acid-treated products.HPLC analyzes and to obtain that xylose concentration is 0.76 mg/ml in the acid-treated product acid hydrolysis liquid, calculating can get the described washing of 1 gram back acid-treated product acid hydrolysis can obtain the wood sugar that weight is 0.051 gram, because being the sulphuric acid soln of 72 weight %, concentration the hemicellulose of acid-treated product all can be hydrolyzed into wood sugar, therefore the weight of gained wood sugar is 1.14 times of hemicellulose weight in the acid-treated product, contain hemicellulose 0.045 gram in the acid-treated product of the i.e. described washing of 1 gram back, then contain hemicellulose 162.2 grams altogether in the 3600 gram acid-treated products.
In enzymolysis step, different is, remaining acid-treated product after in the enzymatic vessel that contains water, adding first step (1) sampling and testing, the amount of the acid-treated product of first adding be behind step (1) sampling and testing remaining whole acid-treated product weight 15%, the pH value that adds NaOH adjusting material then is 5, after being heated to 49 ℃, dry weight basis with every gram stalk, the cellulase that adds 20 enzyme activity units calculates, all the dry weight of stalk is 900 grams, the cellulase (jade of the He family Bioisystech Co., Ltd) that adds 18000 enzyme activity units (about 120 grams) altogether, and insulation mixed 20 minutes under 49 ℃; And then according to the remaining acid-treated product of the continuous adding of the method in the enzymolysis step of embodiment 1, the add-on of described water is 3.8:1 with the weight ratios of all washing steam explosion products.Other step is identical with the method for embodiment 1 with condition.According to the described high performance liquid phase condition of embodiment 1 step (1), the glucose of measuring and calculating in the enzymolysis product 384.3 restrains totally, and the weight of the glucose that described enzymolysis obtains promptly totally 346.2 is restrained by the cellulosic weight of enzymolysis in the acid-treated product divided by 1.11; Per hour the cellulose of enzymolysis is 9.6 grams (with the dry weight basis of acid-treated product, be benchmark with the weight of the mixture that obtains, the content of first acid-treated product is 0.9 weight %).
In fermentation step, different is, makes the temperature of enzymolysis product reduce to 33 ℃, in the weight of every gram enzymolysis product, and inoculation 10
6The yeast saccharomyces cerevisiae of colony-forming unit (the super highly active dry yeast in Angel, Hubei Angel Yeast stock company), gained mixture under 33 ℃ in fermentor tank stir culture 36 hours.At 100 ℃ of distillation gained tunnings, the gained distillation fraction can get ethanol 200.7 grams at 78.3 ℃ of following second distillations.Method and formula according to embodiment 1 calculate cellulose conversion rate, monose productive rate and alcohol yied, and calculation result sees Table 1.
Table 1
| Embodiment or Comparative Examples | Embodiment 1 | Comparative Examples 1 | Embodiment 2 | Embodiment 3 | Embodiment 4 | Embodiment 5 |
| Cellulose conversion rate (%) | 93.8 | 72.5 | 90.3 | 92.0 | 93.0 | 89.5 |
| Hemicellulose transformation efficiency (%) | - | - | - | - | 72.8 | - |
| Monose productive rate (%) | 43.8 | 33.8 | 41.3 | 42.9 | 54.8 | 42.7 |
| Alcohol yied (%) | 23.6 | 18.6 | 21.0 | 22.5 | 28.0 | 22.3 |
Data from last table 1 adopt the method for the productivity ratio Comparative Examples 1 of ethanol yield that the feedstock production alcoholic acid method of cellulose of the present invention obtains and monose all to be significantly improved as can be seen.And method enzymolysis efficiency height of the present invention, but the mixture 8.7-11.7 of enzymolysis Mierocrystalline cellulose or Mierocrystalline cellulose and hemicellulose gram per hour.
Claims (11)
1, a kind of feedstock production alcoholic acid method that adopts cellulose, this method comprises that the raw material with cellulose mixes with enzyme, enzymolysis, obtain enzymolysis product, and this enzymolysis product that ferments, it is characterized in that, described raw material with cellulose and enzyme blended method comprise earlier that the raw material with first cellulose mixes with enzyme and obtain mixture, then the raw material of remaining cellulose is mixed with described mixture continuous the adding, the adding speed of the raw material of described remaining cellulose makes the concentration of raw material in reaction system of cellulose not be higher than 150 grams per liters; The weight of the raw material of described first and enzyme blended cellulose is the 10-30 weight % of the raw material weight of whole cellulose.
2, method according to claim 1, wherein, it is the 100-150 grams per liter that the adding speed of the raw material of described remaining cellulose makes the concentration of raw material in reaction system of cellulose.
3, method according to claim 1, wherein, when the raw material of first cellulose is mixed with enzyme, this method also comprises and adds entry, with the dry weight basis of the raw material of cellulose, be benchmark with the weight of the mixture that obtains, the content of the raw material of first cellulose is at least 1 weight %.
4, method according to claim 3 wherein, with the dry weight basis of the raw material of cellulose, is a benchmark with the weight of the mixture that obtains, and the content of the raw material of first cellulose is 1.2-10 weight %.
5, method according to claim 1, wherein, the enzyme that described enzymolysis uses comprises cellulase, with the dry weight basis of the raw material of every gram cellulose, the consumption of described cellulase is the 8-20 enzyme activity unit; The temperature of described enzymolysis is 45-55 ℃, and the time of described enzymolysis is 32-40 hour, and the pH value of described enzymolysis is 3.0-7.0.
6, method according to claim 5, wherein, the enzyme that described enzymolysis uses also comprises hemicellulase, with the dry weight basis of the raw material of every gram cellulose, the consumption of described hemicellulase is the 4.4-8.8 enzyme activity unit.
7, method according to claim 1, wherein, the raw material of remaining cellulose is added the 15-35% that accounts for enzymolysis time with the described mixture blended time continuously, and described enzymolysis time refers to that the raw material with cellulose mixes beginning used time when cellulose containing raw material being converted into sugared content no longer increases after the sugar with enzyme.
8, method according to claim 1, wherein, with the raw material of first cellulose with after enzyme mixes, before the raw material of remaining cellulose added continuously, also comprise the timed interval, the described timed interval is 10-30 minute.
9, method according to claim 1, wherein, the raw material of cellulose is the raw material of cellulose of steam explosion and/or the raw material of acid-treated cellulose.
10, according to claim 1 or 9 described methods, wherein, the raw material of described cellulose is a stalk.
11, method according to claim 1, wherein, the employed yeast of described fermentation is pichia stipitis and/or yeast saccharomyces cerevisiae; In every gram enzymolysis product, the employed zymic inoculum size of described fermentation is 10
3-10
8Colony-forming unit, the temperature of described fermentation are 30-36 ℃, and the time of fermentation is 32-48 hour.
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