CN100588711C - Method for preparing ethanol by using material conataining cassava residues - Google Patents

Method for preparing ethanol by using material conataining cassava residues Download PDF

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Publication number
CN100588711C
CN100588711C CN200810223312A CN200810223312A CN100588711C CN 100588711 C CN100588711 C CN 100588711C CN 200810223312 A CN200810223312 A CN 200810223312A CN 200810223312 A CN200810223312 A CN 200810223312A CN 100588711 C CN100588711 C CN 100588711C
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manioc waste
raw material
weight
enzymolysis
enzyme
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CN101423848A (en
Inventor
刘文信
刘甲申
袁敬伟
商成祥
李春玲
张宁
姜开荣
王国忠
郝小明
林海龙
邓立康
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Cofco Corp
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Cofco Corp
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Priority to PCT/CN2009/073362 priority patent/WO2010034219A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/22—Processes using, or culture media containing, cellulose or hydrolysates thereof
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Abstract

The invention relates to a method for preparing ethanol by adopting a raw material containing cassava residue, wherein the method comprises the following steps: mixing the raw material containing thecassava residue and enzyme, enzymolyzing cellulose in the raw material containing the cassava residue to obtain an enzymolysis product, and fermenting the enzymolysis product, wherein the method of mixing the raw material containing the cassava residue and the enzyme comprises mixing the raw material containing the cassava residue in batch and the enzyme, the batch times are at least two times, and the amount of the raw material containing the cassava residue added in each batch is at most 60 percent of the weight of the total raw material containing the cassava residue. The method has the advantages that the method can improve utilization rate of the raw material containing the cassava residue, and the yield of the prepared ethanol is higher.

Description

Employing contains the feedstock production alcoholic acid method of manioc waste
Technical field
The invention relates to the alcoholic acid preparation method, contain the feedstock production alcoholic acid method of manioc waste more specifically about employing.
Background technology
At present, general tapioca (flour) factory or cassava alcohol factory after producing starch or alcohol, all can produce a large amount of manioc waste refuses, use as feed usually because of it contains a certain amount of starch.
For example, " utilize the research of manioc waste fermentative production cereuisiae fermentum single cell protein " (Tang Yanhua, Xie Bifeng, biotechnology institute of Fujian Normal University, " medicine biotechnology ", 2006,13 (1), 51-54 page or leaf) mentions in, utilize aspergillus niger and cereuisiae fermentum under suitable condition, manioc waste to be carried out the miscegenation fermentation, improve the protein content of manioc waste, in producing and raise livestock, and make waste obtain recycling treatment as a kind of yeast single cell protein feed applications.
" pilot plant conditions of manioc waste solid state fermentation phytase " (Zhong Qiuping, Zhou Wenhua, Li Jianna, Li Meiqiu, Jiang Fangjun, Wu Jianxiong, Food Science and Engineering system of Polytehnic College, Huanan Tropical Agriculture Univ., " tropical crops journal ", 2004,25 (1), the 45-48 page or leaf) studied culture condition to manioc waste influence with aspergillus niger PD strain fermentation production phytase in box solid-state fermentation reactor.The result shows: the single factor experiment top condition is a quality mark 75%, bed thickness 5-10 centimetre, and inoculum size 1.5%, culture temperature 32-34 ℃, incubation time 6-8 days.The phytase vigor is up to 4189 micromoles/(minute gram) with this understanding.
" utilize manioc waste carry out cellulose-decomposing bacterium mixed fermentation technology research " (Lin Jie, Tan Zhaozan, Luo Weicheng, Agricultural University Of South China's Foodstuffs Academy, resource environment institute of Agricultural University Of South China, life science institute of Agricultural University Of South China, " safety and environment journal ", 2005,5 (6), the 26-29 page or leaf) introduced and filter out 6 strains from occurring in nature and do not have the cellulose-decomposing bacterium of antagonistic action each other and carry out mixed fermentation, having studied this, to mix bacterium be that the best condition of enzyme production of fermenting raw materials is with the trade waste manioc waste: culture medium prescription is manioc waste 4 grams, analysis for soybean powder 0.75 gram, potassium primary phosphate 0.2 gram, sodium-chlor 0.02 gram, magnesium sulfate heptahydrate 0.015 gram, 1000 milliliters of distilled water; Zymotechnique is initial pH7.0,40 ℃ of culture temperature, and 140 milliliters of the bottled nutrient solutions of per 250 milliliters of tapers, inoculum size 3% was 140 rev/mins of rotating speed bottom fermentations 72 hours.The result shows, the fermentation of mixed bacterium can increase substantially cellulase activity and to the tolerance of reducing sugar.
By foregoing description as can be known, manioc waste is mainly used in and produces feed, phytase etc. at present.In recent years, raw material as Alcohol Production is also arranged.
For example, CN101195836A discloses a kind of novel technique for producing manioc waste ethanol, this method is that manioc waste and living cassava or tapioca slice are separated, carry out boiling and saccharification respectively, manioc waste carries out pulp water after saccharification separates, Gu slag is directly as feed or fertilizer, isolating manioc waste saccharification filtrate is used to do sizing mixing of living cassava or tapioca slice and uses liquid;
In the method, the boiling and the Mashing process of manioc waste are, manioc waste is through screening, and fineness is below 1.8 millimeters, delivers in the storage pool between alcohol-fueled car, add waste water---the yellow seriflux that produces in the Starch Production, per 1 kilogram of manioc waste adds yellow seriflux 2-5 liter, puts into the jar of sizing mixing, and adds high temperature resistant α-Dian Fenmei, the enzyme addition is the 5-15U/ gram, add interchanger, carry out heat exchange with the digester discharging, temperature liquefies when being raised to 90-110 ℃, enter steam cooker again, temperature is controlled at 90-95 ℃ during boiling, and it is faint yellow or light brown for best that the boiling mash is, and the boiling mash is after vapor-liquid separation, enter in the saccharifying tank, and add saccharifying enzyme when in brew kettle, being cooled to 55-65 ℃ and carry out saccharification, consumption is a 100-150 units/gram raw material, saccharification time 20-60 minute, remove solid slag, manioc waste saccharification filtrate is entered in the storage pool;
In the method, the boiling and the Mashing process of living cassava or tapioca slice are, the manioc waste saccharification filtrate that the boiling and the Mashing process of above-mentioned manioc waste obtained joins in the living cassava or tapioca slice through pulverizing, the per kilogram solid starch adds filtrate 2-4 kilogram, and adding high-temperature resistant alpha-amylase, stir, enter digester after the 90-110 ℃ of liquefaction, boiling 40-60 minute, temperature 90-95 ℃, enter saccharifying tank afterwards, add saccharifying enzyme when cooling to 55-65 ℃, consumption is a 100-150 units/gram raw material, saccharification time 20-60 minute, cools to 30-35 ℃ through interchanger, enter ferment tank, time 50-70 hour, wine part reached more than 8.5%, sent distillation workshop to and distilled according to a conventional method.This method is to mix saccharification once more, alcohol prepared by fermenting after residual starch enzymolysis contained in the manioc waste, the saccharification and with fresh cassava or dried cassava, and still, the utilization ratio of manioc waste is not high in this method, and therefore, ethanol yield is restricted.
Summary of the invention
The utilization ratio that the objective of the invention is to overcome manioc waste in the feedstock production alcoholic acid method that existing employing contains manioc waste is not high, the defective that the alcohol yied for preparing is lower, a kind of manioc waste utilization ratio height is provided, and the higher employing of alcohol yied contains the feedstock production alcoholic acid method of manioc waste.
The present inventor finds that the main component of manioc waste comprises water, starch (butt), Mierocrystalline cellulose, hemicellulose, xylogen, crude protein, crude fat and coarse ash and inorganic salt.(Mierocrystalline cellulose: 25-30 weight %, hemicellulose: 10-12 weight %, xylogen: 30-32 weight %, crude protein: 4-6 weight %, crude fat: 2-4 weight %, starch: 8-10 weight %, crude ash content and inorganic salt: 10-15 weight %) wherein, the content of Mierocrystalline cellulose, hemicellulose and xylogen is higher.In addition, the present inventor finds, though manioc waste also can be called the raw material of cellulose, but different with the raw material of the cellulose of routine such as stalk is, xylogen in the manioc waste and Mierocrystalline cellulose and hemicellulose are to be in looser bonding state, but not the structure that the xylogen in the stalk tightly wraps up Mierocrystalline cellulose, therefore, the present inventor attempts Mierocrystalline cellulose and the hemicellulose in the enzymolysis manioc waste and the enzymolysis product that ferments prepares ethanol and obtained good effect, the more important thing is, before enzymolysis, need not manioc waste is carried out pre-treatment, and can directly manioc waste be carried out enzymolysis and ferment preparing ethanol.
In addition, the present inventor also finds, adopt the method for batch charging, the raw material batch-wise that will contain manioc waste mixes with enzyme, make the raw material that contains manioc waste of every batch of adding mix and fully to contact, and in reaction process, the concentration of enzyme remain on higher level always with enzyme, therefore, the raw material that contains manioc waste can fully react with enzyme.Thereby improved the sugared transformation efficiency of raw material effectively, therefore can effectively improve ethanol yield.
The invention provides the feedstock production alcoholic acid method that a kind of employing contains manioc waste, wherein, this method comprises that the raw material that will contain manioc waste mixes with enzyme, enzymolysis contains the Mierocrystalline cellulose in the raw material of manioc waste, obtain enzymolysis product, and this enzymolysis product that ferments, wherein, the described raw material that will contain manioc waste comprises that with enzyme blended method the raw material that will contain manioc waste mixes with enzyme in batches, described number of times in batches is at least 2 times, the amount of the raw material that contains manioc waste of every batch of adding be at most the raw material that all contains manioc waste weight 60%.
Method of the present invention is mixed with enzyme by the raw material that will contain manioc waste in batches, make the manioc waste of every batch of adding mix and fully to contact with enzyme, and in reaction process, the concentration of enzyme remains on higher level always, therefore, the raw material that contains manioc waste can fully react with enzyme, thereby has improved the sugared transformation efficiency of raw material effectively, and the utilization ratio of raw materials that has effectively improved ethanol yield and contained manioc waste.For example, the alcohol yied of embodiment 1 reaches 14.6%, and the alcohol yied of Comparative Examples 1 only is 11.6%, and increase rate is up to 25.9%; The alcohol yied of embodiment 6 reaches 18.6%, and the alcohol yied of Comparative Examples 2 only is 15.4%, and increase rate is up to 20.8%.
Embodiment
According to the present invention, this method comprises that the raw material that will contain manioc waste mixes with enzyme, enzymolysis contains the Mierocrystalline cellulose in the raw material of manioc waste, obtain enzymolysis product, and this enzymolysis product that ferments, wherein, the described raw material that will contain manioc waste comprises that with enzyme blended method the raw material that will contain manioc waste mixes with enzyme in batches, described number of times in batches is at least 2 times, the amount of the raw material that contains manioc waste of every batch of adding be at most the raw material that all contains manioc waste weight 60%.
Under the preferable case, for enhance productivity and can guarantee simultaneously to contain the raw material of manioc waste and enzyme thorough mixing, obtain the enzyme speed of action of the concentration and the higher level of the higher raw material that contains manioc waste, the amount that first and enzyme blended contain the raw material of manioc waste is the 20-60% of the weight of the raw material that all contains manioc waste, more preferably 20-40%; The amount that contains the raw material of manioc waste with the enzyme blended after first is the 40-80% of weight that all contains the raw material of manioc waste, more preferably 60-80%.
More preferably under the situation, described number of times in batches is 3 times, and the amount that first and enzyme blended contain the raw material of manioc waste is the 20-60% that all contains the raw material weight of manioc waste, more preferably 20-40%; Second batch contains the amount of raw material of manioc waste for all containing the 20-50% of the raw material weight of manioc waste, more preferably 20-40% with the enzyme blended; The 3rd batch contains the amount of raw material of manioc waste for all containing the 20-40% of the raw material weight of manioc waste, more preferably 20-30% with the enzyme blended.
In order to guarantee that enzyme has best reactive behavior, with before enzyme mixes, the pH value of regulating the raw material that contains manioc waste is greater than 3 less than 7, is preferably 4-5.5 at the raw material that first is contained manioc waste, so that the raw material that contains manioc waste 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 described enzymolysis can be regulated before adding enzyme according to this area method commonly used, and the method for described conditioned reaction material pH value can adopt and well known to a person skilled in the art the whole bag of tricks.The raw material that for example will contain manioc waste earlier and water or substratum (enzyme-addedly generally mix with water, add the general substratum with this microorganism of microbes producing cellulase) mix, according to the pH value of gained mixture, and adding acidic substance or alkaline matter in this mixture.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, also comprise the timed interval between described two adjacent batch mixing steps.In order to make the abundant hydrolysis of enzyme energy contain the raw material of manioc waste, improve sugared transformation efficiency, the described timed interval is preferably 10-120 minute, more preferably 30-60 minute.
In order to make enzyme and reaction raw materials blended more even, the described enzymolysis that contains the raw material of manioc waste preferably carries out under the condition that water exists, and more preferably carries out in containing the suspension of water and the raw material that contains manioc waste.That is to say, when the raw material that the present invention preferably contains first manioc waste mixes with enzyme, add entry, after first is contained the raw material of manioc waste and water and be mixed with suspension, again the required whole enzymes of enzymolysis are mixed with suspension, and then continue to add the raw material that contains manioc waste in batches according to method of the present invention.The adjustable extent broad of the amount of the water that adds, under the preferable case, the add-on of described water is 2-5 with the weight ratio that all contains the raw material of manioc waste: 1.More preferably under the situation, the mixture that obtains after mixing with the raw material that all contains manioc waste, water and enzyme is a benchmark, and with the dry weight basis of the raw material that contains manioc waste, first content that contains the raw material of manioc waste is at least 1 weight %, be preferably 1-20 weight %, more preferably 1.2-10 weight %.
The present inventor finds, when enzymolysis, control the raw material that first contains manioc waste, the concentration that contains the raw material of manioc waste in water and the mixture that obtains after enzyme mixes, contain the raw material of manioc waste with first, water is benchmark with the mixture that obtains after enzyme mixes, dry weight basis with the raw material that contains manioc waste, make the content of the raw material that contains manioc waste be at least 1 weight %, be preferably 1-20 weight %, 1.2-10 weight % more preferably, carrying out enzymolysis with raw material that once will all contain manioc waste and enzyme mixing compares, the cycle that can either guarantee enzymolysis is short, guarantees to have high production efficiency, i.e. the productive rate height of monose simultaneously.
According to the present invention, the described raw material that contains manioc waste can be that manioc waste also can be the mixture of the raw material of manioc waste and other cellulose, for example, the mixture of manioc waste and stalk, being used for the manioc waste of enzymolysis, alcohol prepared by fermenting and the mixture manioc waste of stalk and the weight ratio of stalk can adjust in the scope of broad, under the preferable case, gross weight with the mixture of manioc waste and stalk is a benchmark, the content of described manioc waste is 40-90 weight %, and the content of described stalk is 10-60 weight %; More preferably under the situation, be benchmark with the gross weight of the mixture of manioc waste and stalk, the content of described manioc waste is 50-70 weight %, and the content of described stalk is 30-50 weight %.
According to method of the present invention, in order can fully to contact with enzyme, the particle diameter of described manioc waste is preferably the 0.3-5 millimeter, more preferably the 0.5-3 millimeter.The particle diameter that makes manioc waste is the 0.3-5 millimeter, more preferably the method for 0.5-3 millimeter can adopt and well known to a person skilled in the art the whole bag of tricks, for example, can adopt dry type to pulverize or case of wet attrition, the difference between two kinds of grinding modes mainly is whether manioc waste is mixed with water.Case of wet attrition comprises mixes manioc waste with water, carry out one or many then and pulverize.The weight ratio of manioc waste and water can be 1: 0.2-5 is preferably 1: 0.5-2.Can use conventional various pulverizers, for example SFSP series beater disintegrating machine.
The present invention has no particular limits other step of the feedstock production alcoholic acid method that contains manioc waste, can be with reference to the feedstock production alcoholic acid method that adopts cellulose.
Because the Mierocrystalline cellulose in the cassava, the structure of hemicellulose and xylogen is tight firm unlike stalk, and manioc waste generally is the final by product that cassava produces the alcohol process, in its production process, passed through pyroprocessing, thereby make the xylogen in the manioc waste no longer Mierocrystalline cellulose tightly be wrapped up, but be to be in looser bonding state with Mierocrystalline cellulose and hemicellulose, thereby before enzymolysis, need not to make Mierocrystalline cellulose, the pre-treatment that hemicellulose and lignin separation are opened, therefore, be fermenting raw materials adopting manioc waste, can directly manioc waste be carried out enzymolysis and alcohol prepared by fermenting during preparation ethanol.
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, for cost consideration, preferably contains the dry weight basis of the raw material of manioc waste with every gram, and the consumption of described cellulase is the 10-25 enzyme activity unit, more preferably the 15-18 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, considers plant factor, and the time of preferred described enzymolysis is 25-48 hour, more preferably 30-40 hour.Described enzymolysis time begins when enzyme is mixed with the raw material that contains manioc waste to calculate, and therefore, according to the present invention, the timed interval between described adjacent two batch mixing steps is also included within the described enzymolysis time.
According to of the present invention one preferred embodiment, the described method that will contain the raw material enzymolysis of manioc waste comprises that earlier first raw material that contains manioc waste is mixed the back adds in the enzymatic vessel with water, adjusting contain manioc waste raw material the pH value for greater than 3 to less than 7, and be warming up to 45-55 ℃, be preferably 48-52 ℃ to reach the optimum response reactive conditions of enzyme, then, under agitation, in enzymatic vessel, add enzyme, stir after 30-60 minute, add second batch of raw material that contains manioc waste again, after stirring reaction 30-60 minute, add the 3rd batch of raw material that contains manioc waste.The amount of described water is 2-5 with the weight ratio that all contains the raw material of manioc waste: 1; It is described that first amount that contains the raw material of manioc waste is the 20-60% that all contains the raw material weight of manioc waste with the enzyme blended; The second batch of amount that contains the raw material of manioc waste that adds is the 20-50% that all contains the raw material weight of manioc waste; Add the amount of the 3rd batch of raw material that contains manioc waste for all containing the 20-40% of the raw material weight of manioc waste.
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.Contain the dry weight basis of the raw material of manioc waste with every gram, the consumption of described hemicellulase is the 4.0-8.0 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 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 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:
Figure C20081022331200131
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.
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 water content of manioc waste can record by variety of way.Unless specified otherwise, the water content of manioc waste of the present invention all is meant the initial weight W1 of manioc waste and dries poor to the dried meter weight W 2 of the manioc waste of constant weight under 70-100 ℃, with the weight percent of the initial weight W1 of manioc waste, i.e. water content (weight %)=(W1-W2)/W1 * 100%.
According to the present invention, under the preferable case, when the described raw material that contains manioc waste was the mixture of manioc waste and stalk, wherein, described stalk was the stalk and/or the acid-treated stalk of steam explosion.Cellulosic crystalline texture in the stalk is difficult to destroyed, xylogen in the stalk 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.
The method of described acid treatment or steam explosion can adopt method known in those skilled in the art.For example, described acid treatment method comprises stalk and acid mixed and makes it to separate with hemicellulose with Mierocrystalline cellulose with dissolved lignin.The kind of the acid of the described xylogen that is used for dissolving cellulose containing raw material 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 dissolved stalk.
Described steam explosion method comprise with stalk with carry out steam explosion or directly stalk placed steam blasting device to carry out steam explosion after water mixes.Because steam explosion method more helps destroying the reticulated structure between Mierocrystalline cellulose in the stalk, hemicellulose and the xylogen, 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 preferred described stalk of the present invention is a steam explosion method.
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 can be 180-200 ℃, the pressure of described steam explosion can be the 1.4-1.8 MPa, and holding time of described steam explosion pressure can be 3-7 minute.More preferably the temperature of described steam explosion can be 185-195 ℃, and the pressure of described steam explosion can be the 1.4-1.6 MPa, and holding time of described steam explosion pressure can be 4-5 minute.
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 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 mixture, enzyme and the water of steam explosion products and manioc waste, 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 stalk 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 steam explosion stalk, before the steam explosion products that enzymolysis obtains and the mixture of manioc waste, 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 washing is added 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 every gram stalk, 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 stalk, can cause damage to steam explosion equipment, before can also being included in steam explosion stalk is removed stone deironing routine operation, such as removing the stone deironing with the method for " wind send " stalk and 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 stalk itself tangles and the occluding device pipeline easily, therefore, before entering steam explosion equipment, the size that preferably makes described stalk is 0.2-1 centimetre of a 0.5-3 cm x 0.2-1 cm x, and the size that more preferably makes described stalk 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 that employing of the present invention contains the feedstock production alcoholic acid method of manioc waste.
(1) to the mensuration of Mierocrystalline cellulose gross weight in the raw material that contains manioc waste and hemicellulose gross weight:
Discarded manioc waste (water content is 10%) behind the 3000 gram fermenting alcohols is pulverized, being crushed to particle diameter is 3 millimeters, 5 grams of getting are wherein dried under 45 ℃ to constant weight 4 grams, and 300.0 milligrams of these dried manioc wastes 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 1 * 10 -6D-(+) glucose and 1 * 10 of-4.0 mg/ml concentration ranges -6-4.0 mg/ml concentration range D-(+) wood sugars are as standard model.HPLC analyzes and to obtain that glucose concn is 0.00112 mg/ml in the manioc waste acid hydrolysis liquid, calculating can get the described manioc waste acid hydrolysis of 1 gram can obtain the glucose that weight is 0.283 gram, because being the sulphuric acid soln of 72 weight %, concentration the Mierocrystalline cellulose of manioc waste all can be hydrolyzed into glucose, therefore the weight of gained glucose is 1.11 times of cellulose in the manioc waste, promptly cellulose 0.255 gram in the described manioc waste of 1 gram then is total to cellulose 764.1 grams in the 3000 gram manioc wastes.HPLC analyzes and to obtain that xylose concentration is 0.00045 mg/ml in the manioc waste acid hydrolysis liquid, calculating can get the described manioc waste acid hydrolysis of 1 gram can obtain the wood sugar that weight is 0.114 gram, because being the sulphuric acid soln of 72 weight %, concentration the hemicellulose in the manioc waste all can be hydrolyzed into wood sugar, therefore the weight of gained wood sugar is 1.14 times of hemicellulose weight in the manioc waste, promptly contain hemicellulose 0.10 gram in the described manioc waste of 1 gram, then contain hemicellulose 302.4 grams altogether in the 3000 gram manioc wastes.
(2) enzymolysis
In enzymatic vessel, add entry, and under agitation, add remaining manioc waste behind first step (1) sampling and testing, the amount of the manioc waste of first adding be behind step (1) sampling and testing remaining whole manioc waste weight 35%, regulating the pH value then is 4, after being heated to 52 ℃, the cellulase calculating that adds 15 enzyme activity units with the dry weight basis of every gram manioc waste, 2700 gram manioc wastes (dry weight), the cellulase (jade of the He family Bioisystech Co., Ltd) that adds 40500 enzyme activity units (about 270 grams) altogether, and insulation mixed 30 minutes under 52 ℃; Add second batch of manioc waste then, the amount of the manioc waste of second batch of adding be behind step (1) sampling and testing remaining whole manioc waste weight 35%, and 52 ℃ down insulation mixed 50 minutes; And then add the 3rd batch of manioc waste, the amount of the manioc waste of the 3rd batch of adding be behind step (1) sampling and testing remaining whole manioc waste weight 30%, and 52 ℃ down the insulation enzymolysis obtain enzymolysis product after 40 hours.The add-on of described water is 3: 1 with the weight ratio of whole manioc wastes.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 773.8 restrains totally.The glucose weight that described enzymolysis obtains promptly totally 697.1 is restrained by the cellulosic weight of enzymolysis in the manioc waste 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 16.9 grams.(with the dry weight basis of manioc waste, be benchmark with the weight of the mixture that obtains, the content of first manioc waste is 9.2 weight %.)
Cellulose conversion rate=100% * by the cellulosic weight of enzymolysis/cellulosic gross weight
Glucose weight/manioc waste 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 395.2 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/manioc waste dry weight
Comparative Examples 1
The feedstock production alcoholic acid reference method that contains manioc waste 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 manioc waste behind step (1) sampling and testing in the aqueous enzymatic vessel, mix (all the weight ratio of manioc waste and water is 3: 1) then with enzyme, enzymolysis time is 41 hours 20 minutes; According to the described high performance liquid phase condition of embodiment 1 step (1), the glucose of measuring and calculating in the enzymolysis product 643.7 restrains totally.The glucose weight that described enzymolysis obtains promptly totally 580 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 14 grams; Second distillation obtains ethanol 312.7 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 that employing of the present invention contains the feedstock production alcoholic acid method of manioc waste.
Method according to embodiment 1 prepares ethanol, and different is, in enzymolysis step, manioc waste is mixed with enzyme in three batches, the amount of the manioc waste of first adding be behind the sampling and testing remaining whole manioc waste weight 60%, 50 ℃ down insulation mixed 100 minutes; Add second batch of manioc waste then, the amount of the manioc waste of second batch of adding be behind the sampling and testing remaining whole manioc waste weight 20%, 50 ℃ down insulation mixed 50 minutes; And then add the 3rd batch of manioc waste, the amount of the manioc waste of the 3rd batch of adding be behind the sampling and testing remaining whole manioc waste weight 20%, 50 ℃ down the insulation enzymolysis obtain enzymolysis product after 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 757.4 restrains totally.The glucose weight that described enzymolysis obtains promptly totally 682.3 is restrained by the cellulosic weight of enzymolysis in the manioc waste divided by 1.11, and per hour the cellulosic amount of enzymolysis is 16.2 grams; Fermentation step is with embodiment 1, and second distillation obtains ethanol 387.3 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.(with the dry weight basis of manioc waste, be benchmark with the gross weight of the mixture that obtains, the content of first manioc waste is 14.6 weight %.)
Embodiment 3
Present embodiment is used to illustrate that employing of the present invention contains the feedstock production alcoholic acid method of manioc waste.
Method according to embodiment 1 prepares ethanol, and different is, in enzymolysis step, manioc waste is mixed with enzyme in three batches, the amount of the manioc waste of first adding be behind the sampling and testing remaining whole manioc waste weight 20%, 52 ℃ down insulation mixed 20 minutes; Add second batch of manioc waste then, the amount of the manioc waste of second batch of adding be behind the sampling and testing remaining whole manioc waste weight 40%, 52 ℃ down insulation mixed 40 minutes; And then add the 3rd batch of manioc waste, the amount of the manioc waste of the 3rd batch of adding be behind the sampling and testing remaining whole manioc waste weight 40%, 52 ℃ down the insulation enzymolysis obtain enzymolysis product after 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 784.5 restrains totally.The glucose weight that described enzymolysis obtains promptly totally 706.8 is restrained by the cellulosic weight of enzymolysis in the manioc waste divided by 1.11, and per hour the cellulosic amount of enzymolysis is 16.8 grams; 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 401.2 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.(with the dry weight basis of manioc waste, be benchmark with the gross weight of the mixture that obtains, the content of first manioc waste is 5.5 weight %.)
Embodiment 4
Present embodiment is used to illustrate that employing of the present invention contains the feedstock production alcoholic acid method of manioc waste.
Method according to embodiment 1 prepares ethanol, and different is, in enzymolysis step, manioc waste is mixed with enzyme in three batches, the amount of the manioc waste of first adding be behind the sampling and testing remaining whole manioc waste weight 10%, 52 ℃ down insulation mixed 10 minutes; Add second batch of manioc waste then, the amount of the manioc waste of second batch of adding be behind the sampling and testing remaining whole manioc waste weight 40%, 52 ℃ down insulation mixed 50 minutes; And then add the 3rd batch of manioc waste, the amount of the manioc waste of the 3rd batch of adding be behind the sampling and testing remaining whole manioc waste weight 50%, 52 ℃ down the insulation enzymolysis obtain enzymolysis product after 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 760.8 restrains totally.The glucose weight that described enzymolysis obtains promptly totally 685.5 is restrained by the cellulosic weight of enzymolysis in the manioc waste divided by 1.11, and per hour the cellulosic amount of enzymolysis is 16.3 grams; Fermentation step is with embodiment 1, and second distillation obtains ethanol 388.8 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.(with the dry weight basis of manioc waste, be benchmark with the gross weight of the mixture that obtains, the content of first manioc waste is 2.8 weight %.)
Embodiment 5
Present embodiment is used to illustrate that employing of the present invention contains the feedstock production alcoholic acid method of manioc waste.
It is 5 millimeters that 3250 gram manioc wastes (water content 15.1 weight %) are crushed to particle diameter, analyze the method for sulphuric acid hydrolysis manioc waste according to embodiment 1 described HPLC, obtain that glucose concn is 0.00108 mg/ml in the manioc waste acid hydrolysis liquid, the manioc waste acid hydrolysis that calculating can get after the described washing of 1 gram can obtain the glucose that weight is 0.259 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 manioc waste, cellulose 0.233 gram in the manioc waste after the i.e. described washing of 1 gram then is total to cellulose 757.25 grams in the 3250 gram manioc wastes.HPLC analyzes and to obtain that xylose concentration is 0.00048 mg/ml in the manioc waste acid hydrolysis liquid, calculating can get the described manioc waste acid hydrolysis of 1 gram can obtain the wood sugar that weight is 0.115 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 manioc waste, contain hemicellulose 0.101 gram in the manioc waste after the i.e. described washing of 1 gram, then contain hemicellulose 328.4 grams altogether in the 3250 gram manioc wastes.
(2) enzymolysis
In enzymatic vessel, add entry, and under agitation, add remaining manioc waste behind first step (1) sampling and testing, the amount of the manioc waste of first adding be behind step (1) sampling and testing remaining whole manioc waste weight 60%, the pH value of conditioned reaction material is 5 then, and after being heated to 50 ℃, dry weight basis with every gram manioc waste, the Mierocrystalline cellulose that adds 12 enzyme activity units, the hemicellulase of 6 enzyme activity units calculates, 2760 gram manioc wastes (dry weight), add the cellulase (jade of the He family Bioisystech Co., Ltd) of 33000 enzyme activity units (about 220 grams) and the hemicellulase (Beijing chemical reagents corporation) of 16560 enzyme activity units (about 220 grams) altogether, and insulation mixed 80 minutes; Add second batch of manioc waste then, the amount of the manioc waste of second batch of adding be behind step (1) sampling and testing remaining whole manioc waste weight 40%, the insulation enzymolysis obtain enzymolysis product after 42 hours.The add-on of described water is 3.5: 1 with the weight ratio of whole manioc wastes.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 714.5 restrains totally, the weight of the glucose that described enzymolysis obtains is divided by 1.11, be totally 643.7 to be restrained by the cellulosic weight of enzymolysis in the manioc waste, the wood sugar of measuring and calculating in the enzymolysis product 283 restrains totally, the weight of the wood sugar that described enzymolysis obtains is divided by 1.14, be totally 248.3 to be restrained by the weight of the hemicellulose of enzymolysis in the manioc waste, per hour the total amount of the Mierocrystalline cellulose of enzymolysis and hemicellulose is 20.6 grams; And calculate Mierocrystalline cellulose, hemicellulose transformation efficiency and monose productive rate according to the following equation, calculation result sees Table 1.(with the dry weight basis of manioc waste, be benchmark with the gross weight of the mixture that obtains, the content of first manioc waste is 12.0 weight %.)
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)/manioc waste 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 365.4 grams at 78.3 ℃ of following second distillations, and calculates alcohol yied according to the following equation, and calculation result sees Table 1.
The dry weight of alcohol yied=100% * ethanol weight/manioc waste
Embodiment 6
Present embodiment is used to illustrate that employing of the present invention contains the feedstock production alcoholic acid method of manioc waste.
(1) pre-treatment of stalk
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 1504 grams, keep the pressure 5 minutes of 1.6 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 at 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 4510 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 1.6 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 0.134 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 0.121 gram in the steam explosion products after the i.e. described washing of 1 gram then is total to cellulose 544.5 grams in the 4500 gram steam explosion products.HPLC analyzes and to obtain that xylose concentration is 0.4 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 0.029 gram in the steam explosion products after the i.e. described washing of 1 gram, then contain hemicellulose 130.5 grams altogether in the 4500 gram steam explosion products.
(2) enzymolysis
Method according to embodiment 1 is carried out enzymolysis, different is that the raw material of adding is the mixture that the manioc waste (water content 10 weight %) and 1500 among the 1500 gram embodiment 1 restrains the 4500 gram steam explosion products (water content 70 weight %) that obtain after the pre-treatment of stalks (water content 10 weight %) process.
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 942.4 restrains totally, the glucose weight that described enzymolysis obtains is divided by 1.11, be totally 849 to be restrained by the cellulosic weight of enzymolysis in steam explosion products and the manioc waste, calculate cellulose conversion rate and monose productive rate according to following formula, calculation result sees Table 1.Per hour the cellulosic amount of enzymolysis is 20.5 grams.(with the dry weight basis of the mixture of first steam explosion products and manioc waste, gross weight with the mixture of the mixture that contains first steam explosion products and manioc waste, enzyme and the water that obtain is a benchmark, and the content of the mixture of first steam explosion products and manioc waste is 9.2 weight %.)
Cellulose conversion rate=100% * by the cellulosic weight of enzymolysis/cellulosic gross weight
Glucose weight/the manioc waste that monose productive rate=100% * enzymolysis obtains and the gross dry weight of stalk
(3) fermentation
According to the method fermenting enzyme hydrolysis products of embodiment 1, at 100 ℃ of distillation gained tunnings, the gained distillation fraction can get ethanol 502.2 grams at 78.3 ℃ of following second distillations, calculates alcohol yied according to following formula, and calculation result sees Table 1.
The gross dry weight of alcohol yied=100% * ethanol weight/manioc waste and stalk
Comparative Examples 2
The feedstock production alcoholic acid reference method that contains manioc waste is adopted in this Comparative Examples explanation.
Method according to embodiment 6 prepares ethanol, different is, mixture with remaining manioc waste and steam explosion products behind step (1) sampling and testing in enzymolysis step (2) once all joins in the aqueous enzymatic vessel, mix (all the weight ratio of mixture and water is 3: 1) then with enzyme, enzymolysis time is 41 hours 20 minutes; According to the described high performance liquid phase condition of embodiment 1 step (1), the glucose of measuring and calculating in the enzymolysis product 777.6 restrains totally.The glucose weight that described enzymolysis obtains promptly totally 700.5 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 16.9 grams; Second distillation obtains ethanol 415.8 grams.And calculate cellulose conversion rate, monose productive rate and alcohol yied according to the method for embodiment 6 and formula, calculation result sees Table 1.
Embodiment 7
Present embodiment is used to illustrate that employing of the present invention contains the feedstock production alcoholic acid method of manioc waste.
Method according to embodiment 6 prepares ethanol, and different is that in the pre-treatment step of stalk, described steam explosion products is without washing.
Behind the enzymolysis, the glucose of measuring and calculating in the enzymolysis product 933.8 restrains totally, the glucose weight that described enzymolysis obtains is divided by 1.11, be totally 841.3 to be restrained by the cellulosic weight of enzymolysis in steam explosion products and the manioc waste mixture, formula according to embodiment 6 calculates cellulose conversion rate and monose productive rate, and calculation result sees Table 1.Per hour the cellulosic amount of enzymolysis is 20.3 grams.(with the dry weight basis of the mixture of first steam explosion products and manioc waste, gross weight with the mixture of the mixture that contains first steam explosion products and manioc waste, enzyme and the water that obtain is a benchmark, and the content of the mixture of first steam explosion products and manioc waste is 9.2 weight %.) second distillation can get ethanol 470.1 gram, and calculate alcohol yied according to method and the formula of embodiment 6, calculation result sees Table 1.
Embodiment 8
Present embodiment is used to illustrate that employing of the present invention contains the feedstock production alcoholic acid method of manioc waste.
Method according to embodiment 6 prepares ethanol, different is, during enzymolysis, the raw material of adding is the mixture that 2100 gram manioc wastes (water content 10 weight %) and 900 restrain the 2700 gram steam explosion products (water content 70 weight %) that obtain after the pre-treatment of stalks (water content 10 weight %) process; In enzymolysis step, the mixture of manioc waste and steam explosion products is mixed with enzyme in three batches, the amount of the manioc waste of first adding and the mixture of steam explosion products be behind the sampling and testing remaining whole mixture weight 20%, the pH value of conditioned reaction material is 5 then, and after being heated to 50 ℃, dry weight basis with every gram mixture, the cellulase that adds 18 enzyme activity units, the hemicellulase of 5 enzyme activity units calculates, the dry weight of mixture is totally 1980 grams, add the cellulase (jade of the He family Bioisystech Co., Ltd) of 35640 enzyme activity units (about 237 grams) and the hemicellulase (Beijing chemical reagents corporation) of 9900 enzyme activity units (about 132 grams) altogether, and insulation mixed 20 minutes; The mixture that adds second batch of manioc waste and steam explosion products then, the amount of the manioc waste of second batch of adding and the mixture of steam explosion products be behind the sampling and testing remaining whole mixture weight 40%, 52 ℃ down insulation mixed 40 minutes; The add-on of described water is 4: 1 with the weight ratio of whole mixtures.And then add the mixture of the 3rd batch of manioc waste and steam explosion products, the amount of the manioc waste of the 3rd batch of adding and the mixture of steam explosion products be behind the sampling and testing remaining whole mixture weight 40%, 52 ℃ down the insulation enzymolysis obtain enzymolysis product after 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 876.8 restrains totally, the glucose weight that described enzymolysis obtains is divided by 1.11, be totally 789.9 to be restrained by the cellulosic weight of enzymolysis in the mixture, the wood sugar of measuring and calculating in the enzymolysis product 248.1 restrains totally, the weight of the wood sugar that described enzymolysis obtains is divided by 1.14, be totally 217.6 to be restrained by the weight of the hemicellulose of enzymolysis in the mixture, per hour the gross weight of the Mierocrystalline cellulose of enzymolysis and hemicellulose is 23.4 grams; And calculate Mierocrystalline cellulose, hemicellulose transformation efficiency and monose productive rate according to the following equation, calculation result sees Table 1.(with the dry weight basis of the mixture of first steam explosion products and manioc waste, gross weight with the mixture of the mixture that contains first steam explosion products and manioc waste, enzyme and the water that obtain is a benchmark, and the content of the mixture of first steam explosion products and manioc waste is 4.2 weight %.)
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
The gross dry weight of monose productive rate=100% * (the wood sugar weight that glucose weight+enzymolysis that enzymolysis obtains obtains)/manioc waste and stalk
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 574.5 grams at 78.3 ℃ of following second distillations.And calculate alcohol yied according to the following equation, calculation result sees Table 1.
The gross dry weight of alcohol yied=100% * ethanol weight/manioc waste and stalk
Embodiment 9
Present embodiment is used to illustrate that employing of the present invention contains the feedstock production alcoholic acid method of manioc waste.
Method according to embodiment 6 prepares ethanol, different is, described stalk is acid-treated stalk, this method comprises, at 140 ℃, under the 0.06 MPa pressure, with 1203 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 at 1: 3 30 minutes according to mass ratio, 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 4330 gram acid-treated products (water content is 75 weight %) altogether.
Analyze the method for sulphuric acid hydrolysis steam explosion products according to embodiment 6 described HPLC, getting 10 gram acid-treated products tests, obtain that glucose concn is 1.8 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 0.126 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 0.114 gram in the acid-treated product of the i.e. described washing of 1 gram back then is total to cellulose 490.4 grams in the 4320 gram acid-treated products.HPLC analyzes and to obtain that xylose concentration is 0.8 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 0.057 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 0.050 gram in the acid-treated product of the i.e. described washing of 1 gram back, then contain hemicellulose 217.9 grams altogether in the 4320 gram acid-treated products.
And carrying out enzymolysis according to the method for embodiment 6, the raw material of adding is 4320 mixtures that restrain acid-treated products (water content 75 weight %) that manioc waste (water content 10 weight %) among the 1800 gram embodiment 1 and 1200 gram maize straws (water content is 10%) obtain after peracid treatment.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 899.5 restrains totally, the glucose weight that described enzymolysis obtains is divided by 1.11, be by the cellulosic weight of enzymolysis totally 810.4 grams in the mixture of acid-treated product and manioc waste, calculate cellulose conversion rate and monose productive rate according to the formula among the embodiment 6, calculation result sees Table 1.(with the dry weight basis of the mixture of first acid-treated product and manioc waste, the mixture that contains acid-treated product and manioc waste with first that obtains, the gross weight of the mixture of enzyme and water is a benchmark, and the content of the mixture of first acid-treated product and manioc waste is 9.2 weight %.)
The method of fermentation obtains ethanol 458.7 grams with embodiment 6, and calculates alcohol yied according to the formula of embodiment 6, and the result is as shown in table 1.
Table 1
Embodiment or Comparative Examples Embodiment 1 Comparative Examples 1 Embodiment 2 Embodiment 3 Embodiment 4 Embodiment 5 Embodiment 6 Comparative Examples 2 Embodiment 7 Embodiment 8 Embodiment 9
Cellulose conversion rate (%) 91.2 75.9 89.3 92.5 89.7 85.0 91.6 75.6 90.8 91.7 85.4
Hemicellulose transformation efficiency (%) - - - - - 75.6 - - - 75.0 -
Monose productive rate (%) 28.7 23.8 28.1 29.1 28.2 36.1 34.9 28.8 34.6 41.7 33.3
Alcohol yied (%) 14.6 11.6 14.3 14.9 14.4 13.2 18.6 15.4 17.4 21.3 16.9
Data from last table 1 adopt the productive rate of ethanol yield that the feedstock production alcoholic acid method that contains manioc waste of the present invention obtains and monose all higher as can be seen, illustrate that the utilization ratio of raw materials that contains manioc waste is significantly improved.

Claims (15)

1, a kind of employing contains the feedstock production alcoholic acid method of manioc waste, it is characterized in that, this method comprises that the raw material that will contain manioc waste mixes with enzyme and water, enzymolysis contains the Mierocrystalline cellulose in the raw material of manioc waste, obtain enzymolysis product, and this enzymolysis product that ferments, the described raw material that will contain manioc waste comprises that with enzyme blended method the raw material that will contain manioc waste mixes with enzyme in batches, described number of times in batches is at least 2 times, the amount of the raw material that contains manioc waste of every batch of adding be at most the raw material that all contains manioc waste weight 60%; The described raw material that contains manioc waste is the mixture of manioc waste or manioc waste and stalk.
2, method according to claim 1, wherein, first amount that contains the raw material of manioc waste is the 20-60% of the weight of the raw material that all contains manioc waste with the enzyme blended, and the amount of the raw material that contains manioc waste that adds after first is the 40-80% of the weight of the raw material that all contains manioc waste.
3, method according to claim 1, wherein, described number of times in batches is 3 times, first amount that contains the raw material of manioc waste is the 20-60% of the weight of the raw material that all contains manioc waste with the enzyme blended; The amount of the raw material that contains manioc waste of second batch of adding is the 20-50% of the weight of the raw material that all contains manioc waste; The amount of the raw material that contains manioc waste of the 3rd batch of adding is the 20-40% of the weight of the raw material that all contains manioc waste.
4, according to claim 2 or 3 described methods, wherein, when first raw material that contains manioc waste is mixed with enzyme, with the dry weight basis of the raw material that contains manioc waste, weight with the mixture that obtains is benchmark, and first content that contains the raw material of manioc waste is at least 1 weight %.
5, method according to claim 4 wherein, with the dry weight basis of the raw material that contains manioc waste, is a benchmark with the weight of the mixture that obtains, and first content that contains the raw material of manioc waste is 1.2-10 weight %.
6, method according to claim 1 wherein, also comprises the timed interval between two adjacent batch mixing steps.
7, method according to claim 6, wherein, the described timed interval is 10-120 minute.
8, method according to claim 1, wherein, the enzyme that described enzymolysis uses is cellulase, contains the dry weight basis of the raw material of manioc waste with every gram, the consumption of described cellulase is the 10-25 enzyme activity unit; The temperature of enzymolysis is 45-55 ℃, and the time of enzymolysis is 25-48 hour, and the pH value of enzymolysis is 3-7.
9, method according to claim 1, wherein, the enzyme that described enzymolysis uses is the mixture of cellulase and hemicellulase, described hemicellulase is used for the hemicellulose that enzymolysis contains the raw material of manioc waste, contain the dry weight basis of the raw material of manioc waste with every gram, the consumption of described cellulase is the 10-25 enzyme activity unit; Contain the dry weight basis of the raw material of manioc waste with every gram, the consumption of described hemicellulase is the 4-8 enzyme activity unit; The temperature of enzymolysis is 45-55 ℃, and the time of enzymolysis is 25-48 hour, and the pH value of enzymolysis is 3-7.
10, 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 fermentation are 30-36 ℃, and the time of fermentation is 32-48 hour.
11, method according to claim 1, wherein, the described raw material that contains manioc waste is the mixture of manioc waste and stalk, is benchmark with the gross weight of the mixture of manioc waste and stalk, the content of described manioc waste is 40-90 weight %, and the content of described stalk is 10-60 weight %.
12, method according to claim 11 wherein, is a benchmark with the gross weight of the mixture of manioc waste and stalk, and the content of described manioc waste is 50-70 weight %, and the content of described stalk is 30-50 weight %.
13, according to claim 1,11 or 12 described methods, wherein, described stalk is the stalk and/or the acid-treated stalk of steam explosion.
14, according to claim 1,11 or 12 described methods, wherein, the particle diameter of described manioc waste is the 0.3-5 millimeter.
15, method according to claim 1, wherein, the add-on of described water is 2-5 with the weight ratio that all contains the raw material of manioc waste: 1.
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