CN101289678A - Process for preparing ethanol by using cellulose-containing raw material - Google Patents
Process for preparing ethanol by using cellulose-containing raw material Download PDFInfo
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- CN101289678A CN101289678A CNA2007100984034A CN200710098403A CN101289678A CN 101289678 A CN101289678 A CN 101289678A CN A2007100984034 A CNA2007100984034 A CN A2007100984034A CN 200710098403 A CN200710098403 A CN 200710098403A CN 101289678 A CN101289678 A CN 101289678A
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Abstract
The invention provides a method of adopting material containing cellulose to prepare ethanol; the method comprises the steps of exploding the material containing the cellulose via steam, zymohydrolyzing and fermenting the obtained steam explosion product to obtain zymohydrolysis product, wherein the water content of the material containing the cellulose is between thirty and sixty weight percent. According to the method of adopting the material containing the cellulose to prepare the ethanol, the water content of the material containing the cellulose is strictly controlled around between thirty and sixty weight percent to guarantee that the water of the material containing the cellulose is changed to steam, under the condition of steam explosion and the steam dashes out of the material containing the cellulose when pressure is relieved to destroy lignin sheath embedding the cellulose and semi cellulose thoroughly in the material containing the cellulose, so as to increase the proportion of the cellulose which can contact with enzyme in the material containing the cellulose and finally increase the productivity of the ethanol.
Description
Technical field
The invention relates to a kind of preparation alcoholic acid method, more specifically, is about adopting the feedstock production alcoholic acid method of cellulose.
Background technology
Prior art can be used the feedstock production ethanol of cellulose, and the raw material of described cellulose is generally the root of plant, stem and leaf, comprises stalk.Described stalk is remaining, stem and a leaf behind farm crop (being generally herbaceous plant) results seed or the fruit, be mainly stem and leaf, as comprise the cereal grass of maize straw, jowar stalk, rice straw, wheat straw stalk, comprise stalk and the cotton stalk of the oilseed plant of beanstalk.Mierocrystalline cellulose, hemicellulose and xylogen are combined closely by covalent linkage and/or non covalent bond in the stalk, and the three accounts for the 70-90 weight % of stalk dry-matter 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 can be degraded into glucose under the effect of cellulase, hemicellulose can be degraded into 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.But xylogen is a kind of non-polysaccharide material that is polymerized by phenyl-propane, thereby water insoluble being difficult to degrades.Since xylogen by the derivative of aromatic hydrocarbon with-C-C-key ,-the O-key is crosslinked in length and breadth, side chain and hemicellulose are with covalent bonds, form fine and close network structure---xylogen sheath embedding Mierocrystalline cellulose and hemicellulose, thereby making cellulase and hemicellulase be difficult for contacting with hemicellulose with Mierocrystalline cellulose, the result causes stalk also to be difficult to degraded.
Existing feedstock production alcoholic acid method with cellulose generally adopts steam explosion and/or acid hydrolysis to carry out pre-treatment, to destroy the embedding of xylogen to Mierocrystalline cellulose and hemicellulose.Though acid hydrolysis can destroy xylogen, also can destroy Mierocrystalline cellulose, reduce the productive rate of glucose then, and also need after the acid hydrolysis to add in a large amount of alkali and could carry out enzymolysis and fermentation.Therefore more tendency is only finished preprocessing process by steam explosion, reaches the embedding effect of xylogen to Mierocrystalline cellulose and hemicellulose that destroy.
Document " to the introduction of straw biomass to alcohol conversion process " for example, Zhang Xuefeng, orchid is kindly helped secure the success of, wine brewing, the 29th the 4th phase of volume, 37-38 page or leaf, in July, 2002, disclose a kind of method of utilizing straw to produce alcohol, this method comprises to be removed maize straw after the stone deironing cleans, and cuts into 1.5 centimetres segment, water logging 40 minutes, feed 2.5 MPa steam, be incubated 8 minutes, pressure release, the steam explosion products of washing 1/10th, and this wooden enzyme produces cellulase in cultivating in described 1/10th steam explosion products, and the gained culture is mixed with other 9/10ths steam explosion products, is incubated 50 ℃ of hydrolysis 24 hours, obtain 6% liquid glucose after filtration, mineral membrane is condensed into the liquid glucose more than 20%, adds Xiu Hata yeast (0.8-1.2 hundred million/milliliter), ferments 24 hours, produce wine part 10%, distill 99.5% alcohol.
But existing with in the feedstock production alcoholic acid method of cellulose, the steam explosion pre-treatment destroys inadequately the xylogen sheath of embedding Mierocrystalline cellulose and hemicellulose in the raw material of cellulose, thereby causes final alcohol yied low.
Summary of the invention
The objective of the invention is to overcome the existing low shortcoming of feedstock production alcoholic acid method alcohol yied, the feedstock production alcoholic acid method of the high usefulness cellulose of a kind of alcohol yied is provided with cellulose.
During the raw material of prior art steam blasting cellulose-containing, the raw material that it has been generally acknowledged that described cellulose is maize straw for example, fully water logging, can obtain the effect of better steam explosion, because fully water logging can make the raw material of described cellulose for example contain a large amount of water in maize straw inside, when steam explosion, the raw material of the described cellulose for example water of maize straw inside can be gasificated into water vapor, for example go out maize straw inside by the raw material of described cellulose during pressure release, with the raw material that fully the destroys described cellulose from inside to outside xylogen sheath of maize straw embedding Mierocrystalline cellulose and hemicellulose for example, such as at above-mentioned " to the introduction of straw biomass to alcohol conversion process " (Zhang Xuefeng, orchid is kindly helped secure the success of, wine brewing, the 29th the 4th phase of volume, the 37-38 page or leaf, in July, 2002) disclosed method in need just be carried out steam explosion with the maize straw water logging after 40 minutes.But the present inventor is surprised to find that, with maize straw (sapphire rice stalk and the dry maize straw) water logging of various water content after 40 minutes, the water content of maize straw is all up to more than the 80 weight %, and the water content of the raw material of the cellulose of steam explosion is low, it is no doubt bad for example to be lower than 30 weight %, if but the water content of the raw material of cellulose, as be higher than 60 weight %, also can reduce the alcoholic acid yield.
The invention provides a kind of feedstock production alcoholic acid method that adopts cellulose, this method comprises the raw material of steam blasting cellulose-containing, the steam explosion products that enzymolysis obtains, the enzymolysis product that fermentation obtains, wherein, the water content of the raw material of described cellulose is 30-60 weight %.
According to preparation alcoholic acid method of the present invention, the strict water content of controlling the raw material of the cellulose that is used for steam explosion is 30-60 weight %, can guarantee under the steam explosion condition, to make the aqueous vapor of the raw material inside of cellulose to change into water vapor, water vapour is gone out by the raw material inside of cellulose when pressure release, the xylogen sheath of embedding Mierocrystalline cellulose and hemicellulose in the raw material of abundant destruction cellulose, with the cellulose ratios that can contact with enzyme in the raw material that has increased cellulose, thereby can improve ethanol yield.For example, under all identical situation of other steam explosion condition, the alcohol yied of embodiment 1 reaches 21.5%, and the alcohol yied of Comparative Examples 1 only is 18.2%, and increase rate is up to 18.1%.
Description of drawings
Fig. 1 is the electron scanning micrograph of natural corn stalk;
Fig. 2 is the electron scanning micrograph according to stalk behind the method steam explosion of Comparative Examples 1 of the present invention;
Fig. 3 is the electron scanning micrograph according to stalk behind the method steam explosion of Comparative Examples 2 of the present invention;
Fig. 4 is the electron scanning micrograph according to stalk behind the method steam explosion of the embodiment of the invention 1.
Embodiment
The raw material that comprises steam blasting cellulose-containing with the feedstock production alcoholic acid method of cellulose provided by the invention, the steam explosion products that enzymolysis obtains, the enzymolysis product that fermentation obtains, wherein, the water content of the raw material of described cellulose is 30-60 weight %, is preferably 35-50 weight %.
The raw material water content difference of the cellulose of different sources is very big, water content difference such as the maize straw raw material of different times purchase is just very big, harvesting is no more than the fresh straw water content in a week and reaches more than the 80 weight %, but may have only 10% through the air-dry storage stalk water content of airing.Existing method all adopts the similarity condition water logging to handle for the raw material of the cellulose of different moisture content without exception, cause the water content of raw material of cellulose excessive, under the steam explosion condition, the moisture of the raw material inside of cellulose is gone out with liquid form during pressure release, does not reach the effect of the xylogen sheath of embedding Mierocrystalline cellulose and hemicellulose in the raw material that destroys cellulose from inside to outside.
According to method provided by the invention, can obtain the raw material that water content is the cellulose of 30-60 weight % by variety of way.The raw material of the different batches cellulose that there are differences for water content can carry out different processing, such as for the high stalk of water content, can 45-90 ℃ of oven dry part moisture, and make it water content and be reduced to 30-60 weight %; For the low stalk of water content, can adopt control water logging time method, the water content that increases stalk is to 30-60 weight %; Just in time at stalk, can directly carry out steam explosion for water content to 30-60 weight %.The water content of the raw material of the preferred described cellulose that is used for steam explosion is 35-50 weight %.Unless specified otherwise, the water content of the raw material of cellulose of the present invention all is meant the initial weight W1 of cellulose containing raw material and dries poor to the dried meter weight W 2 of the cellulose containing raw material of constant weight under 70-100 ℃, with the weight percent of the initial weight W1 of cellulose containing raw material, i.e. water content (weight %)=(W1-W2)/W1 * 100%.
According to the feedstock production alcoholic acid method with cellulose of the present invention, can adopt the steam explosion condition of prior art routine to finish steam explosion to the raw material of cellulose, for example, the temperature of described steam explosion can be 180-200 ℃, the pressure of described steam explosion can be the 1.4-2.0 MPa, and holding time of described steam explosion pressure can be 3-7 minute.More preferably the temperature of described steam explosion is 185-195 ℃, and the pressure of described steam explosion is the 1.5-1.6 MPa, and holding time of described steam explosion pressure is 4-5 minute.Under above-mentioned steam explosion condition, can both fully destroy the xylogen sheath of stalk embedding Mierocrystalline cellulose and hemicellulose, realize purpose of the present invention.
Described enzymolysis step can be finished by this area method commonly used, such as adding microbes producing cellulase and/or enzyme in steam explosion products, is incubated under the growth temperature of microbes producing cellulase and/or the great-hearted temperature of enzyme and finishes.Described microbes producing cellulase be can eccrine fiber the microbes producing cellulase of plain enzyme.Described enzyme comprises cellulase.
Because microorganism growth can produce by product, therefore the preferred enzyme that directly adds, the consumption of described enzyme is The more the better, 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 with the dry weight basis of the raw material of every gram cellulose, and the 10-15 enzyme activity unit.The enzyme activity of cellulase of the present invention is according to American National renewable energy source laboratory (National Renewable 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, and transforming 1 gram Whatman No.1 filter paper in 1 minute is 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.The pH value of described enzymolysis can be generally 3.0-7.0 for the suitableeest any action pH of cellulase, and more preferably the pH value is 4.5-5.5.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, for example earlier steam explosion products and water or substratum (are enzyme-addedly generally mixed with water, add microbes producing cellulase generally with the substratum of this microorganism) mix, the solid content that generally makes the gained mixture is 50-70 weight %, pH value according to the gained mixture, the mixture pH regulator that to treat enzymolysis with sulphuric acid soln or sodium hydroxide more preferably is adjusted to the pH value and is 4.5-5.5 to 3.0-7.0.
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, makes the cellobiose enzymolysis generate glucose.
The enzyme that preferred described enzymolysis uses also comprises hemicellulase.Because hemicellulase is degraded into water-soluble wood sugar with hemicellulose, 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 mol acetate-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:
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 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, 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, 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 centrifugation steam explosion products then owing to above-mentioned inhibition easily is dissolved in the water, thereby can be removed inhibition by washing from steam explosion products.The temperature of described washing is high more, and is can the dissolved inhibition many more, considers energy consumption, and the temperature of preferred described washing is 60-90 ℃, more preferably 60-80 ℃.Because the water that washing is added is many more, the inhibition that then dissolves is also many more, but consider the energy consumption of centrifugation, preferably with the dry weight basis of the described cellulose of every gram, the amount of water of described washing is the 2-10 gram, more preferably with the dry weight basis of the raw material of every gram cellulose, the amount of water of described washing is the 2-3 gram.
Owing to may contain sandstone impurity and iron contamination in the raw material of cellulose such as the maize straw, can cause damage to steam explosion equipment, therefore the present invention prepares the alcoholic acid method, can comprise the existing stone deironing routine operation that removes, 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 is entangled with and the occluding device pipeline easily, therefore the size of the raw material of preferred described cellulose is 0.2-1 centimetre of a 0.5-3 cm x 0.2-1 cm x, and more preferably the size of 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 explanation the present invention feedstock production alcoholic acid method of cellulose.
(1) steam explosion
Test maize straw raw material as shown in Figure 1, the water content that records maize straw according to above-mentioned method is 20 weight %, raw material is cut into the segment of 0.5 centimetre of 1.5 cm x, 0.2 cm x, with vertical steeping cell (the auspicious mechanical company limited in river in Shangdong Province, Shandong) and weight is after 1/3rd water of described maize straw raw material weight mixes, and can get the maize straw of water content 40 weight %.Getting 1000 gram water content is the maize straw of 40 weight %, keeps 5 minutes with the pressure of 1.6 MPas under 195 ℃, and steam explosion is finished in pressure release then.The water of gained steam explosion products and 70 ℃ was mixed 30 minutes according to weight ratio at 1: 3, use LW400 type horizontal screw centrifuge (Zhangjiagang Huada Centrifugal Manufacturing Co., Ltd.) under 900 rev/mins of rotating speeds, to carry out solid-liquid separation then, obtain 2000 gram solid steam explosion products (water 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 steam explosion products of 5 grams after the described washing and dry under 45 ℃ to constant weight 1.5 grams, 300.0 milligrams of this dried steam explosion products of weighing are positioned in heavy 80 100 milliliters of dry Erlenmeyer flasks that restrain.Add the sulphuric acid soln that 3.00 ml concns are 72 weight % to described Erlenmeyer flask, stirred 1 minute.Then Erlenmeyer flask was heated 60 minutes in 30 ℃ of water-baths, every 5 minutes with stirring once to guarantee even hydrolysis.Water-bath makes the vitriolic concentration dilution to 4 weight % with deionized water after finishing.Filter steam explosion products acid hydrolysis liquid after the described dilution with B, obtain 84 milliliters of filtrates altogether.20 milliliters of filtrates are transferred in the triangular flask of 50 milliliters of dryings.Use 2.5 gram lime carbonate to regulate this pH value of filtrate to 5.5, left standstill 5 hours, collect supernatant liquid.0.2 the supernatant liquid that the micron membrane filtration is collected, the filtrate of gained supernatant liquid is analyzed with Biorad Aminex HPX-87P high performance liquid chromatography (HPLC).HPLC condition: sample size 20 microlitres; Moving phase is 0.2 micron membrane filtration, and the HPLC ultrapure water of the sonic oscillation degassing; Flow velocity is 0.6 ml/min; Column temperature 80-85 ℃; Detector temperature 80-85 ℃; Detector is a refractive index detector; Be 35 minutes working time.With D-(+) glucose of 0.1-4.0 mg/ml concentration range and 0.1-4.0 mg/ml concentration range D-(+) wood sugar as standard model.HPLC analyzes and to obtain that glucose concn is 1.62 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 cellulosic weight in the steam explosion products, cellulose 0.122 gram in the steam explosion products after the i.e. described washing of 1 gram then is total to cellulose 245 grams in the 2000 gram steam explosion products.HPLC analyzes and to obtain that xylose concentration is 0.38 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 0.0319 gram, because being the sulphuric acid soln of 72 weight %, concentration hemicellulose in the product of steam explosion all can be hydrolyzed into wood sugar, therefore the weight of gained wood sugar is 1.14 times of weight of hemicellulose in the steam explosion products, promptly contain hemicellulose 0.028 gram in the 1 gram steam explosion products, then contain hemicellulose 56 grams altogether in the 2000 gram steam explosion products.
(2) enzymolysis
With remaining washing steam explosion products behind step (1) sampling and testing, mix with 1000 gram water, regulate pH value to 5.0, after being heated to 50 ℃, with the dry weight basis of every gram stalk, add the cellulase (jade of the He family Bioisystech Co., Ltd) of 20 enzyme activity units, and be incubated 40 hours down at 50 ℃.Enzymolysis product is filtered with B, 20 milliliters of filtrates are transferred in 50 milliliters of triangular flasks of exsiccant, 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 step (1), the glucose of measuring and calculating in the enzymolysis product 248 restrains totally.The glucose weight that described enzymolysis obtains promptly totally 223.4 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.
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 every gram enzymolysis product, inoculation is 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 35 ℃ in fermentor tank stir culture 40 hours.At 100 ℃ of distillation gained tunnings, the gained distillation fraction can get ethanol 129 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
This Comparative Examples explanation prior art feedstock production alcoholic acid method of cellulose.
Method according to embodiment 1 prepares ethanol, different is that (Zhang Xuefeng, orchid are kindly helped secure the success of according to document " to the introduction of straw biomass to alcohol conversion process ", wine brewing, the 29th the 4th phase of volume, the 37-38 page or leaf, in July, 2002) disclosed method, stalk was soaked in water earlier before steam explosion 40 minutes, the water content that records the maize straw raw material after the immersion reaches 83 weight %, the results are shown in Table 1.
Comparative Examples 2
Method according to embodiment 1 prepares ethanol, and different is with water content is that 20 weight % maize straws directly carry out steam explosion, the results are shown in Table 1.
The electron microscopic observation result:
Amplify 1000 times of steam explosion products of observing Comparative Examples 1, Comparative Examples 2 and embodiment 1 with the JSM-35CF scanning electron microscope, the result is respectively as Fig. 2, Fig. 3 and shown in Figure 4.As can be seen from Figure 2, because stalk inside does not have moisture can be gasificated into water vapor, so steam explosion can not fully destroy stalk from inside to outside; As can be seen from Figure 3, the effect of Comparative Examples 1 maize straw steam explosion and bad, Mierocrystalline cellulose still is pencil, and cellulose surface is residual that the xylogen sheath arranged; And the steam explosion effect of embodiment 1 as shown in Figure 4 is fine, can see the free Mierocrystalline cellulose.
Embodiment 2
The present embodiment explanation prepares the alcoholic acid method with raw material the present invention of cellulose.
(1) steam explosion
Getting 1000 gram water content is the maize straw of 50 weight %, be cut into the segment of 0.8 centimetre of 1.3 cm x, 0.4 cm x, under 190 ℃, kept 4 minutes pressure release then with the pressure of 1.8 MPas, finish steam explosion, obtain 1200 gram steam explosion products (water content is 60 weight %) altogether.
Analyze the method for the steam explosion products of sulphuric acid hydrolysis according to the described HPLC of embodiment 1, record in the 1200 gram steam explosion products cellulose 210 grams altogether, hemicellulose 60 grams.
(2) enzymolysis
With remaining steam explosion products behind step (1) sampling and testing, mixes with 400 gram water, regulate pH value to 6.0, be heated to 55 ℃ after, with the dry weight basis of every gram stalk, add the cellulase of 15 enzyme activity units, and descend to be incubated 30 hours at 55 ℃.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 embodiment 1 step (1), the glucose of measuring and calculating in the enzymolysis product 198.1 restrains totally.The glucose weight that described enzymolysis obtains promptly totally 178.5 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.
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 32 ℃, in every gram enzymolysis product, inoculation is the yeast saccharomyces cerevisiae (the super highly active dry yeast in Angel, Hubei Angel Yeast stock company) of 106 colony-forming units, the gained mixture under 32 ℃ in fermentor tank stir culture 35 hours.At 100 ℃ of distillation gained tunnings, the gained distillation fraction can get ethanol 103 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
Embodiment 3
Present embodiment explanation the present invention feedstock production alcoholic acid method of cellulose.
(1) steam explosion
Getting 1000 gram water content is the maize straw of 35 weight %, is cut into the segment of 0.7 centimetre of 1.2 cm x, 0.4 cm x, keeps 6 minutes with the pressure of 1.7 MPas under 200 ℃, and steam explosion is finished in pressure release then.The water of gained steam explosion products and 75 ℃ was mixed 50 minutes according to weight ratio at 1: 2.5, use impeller pump LW400 type horizontal screw centrifuge (Zhangjiagang Huada Centrifugal Manufacturing Co., Ltd.) under 900 rev/mins of rotating speeds, to carry out solid-liquid separation then, obtain 2000 gram solid steam explosion products (water content is 70 weight %) altogether.
Analyze the method for the steam explosion products of sulphuric acid hydrolysis according to the described HPLC of embodiment 1, record in the 2000 gram steam explosion products cellulose 260 grams altogether, hemicellulose 78 grams.
(2) enzymolysis
With remaining washing steam explosion products behind step (1) sampling and testing, mix with 1000 gram water, regulate pH value to 4.0, after being heated to 45 ℃, dry weight basis with every gram stalk, add the cellulase of 18 enzyme activity units and the hemicellulase of 8.0 enzyme activity units (Beijing chemical reagents corporation), and be incubated 35 hours down at 45 ℃.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 embodiment 1 step (1), the glucose of measuring and calculating in the enzymolysis product 256.6 restrains totally, and wood sugar is totally 62.5 grams.The glucose weight that described enzymolysis obtains is divided by 1.11, be totally 231.1 to be restrained by the cellulosic weight of enzymolysis in the steam explosion products, the wood sugar weight that described enzymolysis obtains is divided by 1.14, be totally 54.8 to be restrained by the weight of the hemicellulose of enzymolysis in the steam explosion products, calculate cellulose conversion rate, hemicellulose transformation efficiency and monose productive rate according to following formula, 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 33 ℃, in every gram enzymolysis product, inoculation is 10
5The yeast saccharomyces cerevisiae of colony-forming unit (ATCC numbering 2601) and 10
3The pichia stipitis of colony-forming unit (ATCC numbering 58376), under 33 ℃ in fermentor tank stir culture 39 hours.At 100 ℃ of distillation gained tunnings, the gained distillation fraction can get ethanol 163.2 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/stalk dry weight
Embodiment 4
Present embodiment explanation the present invention feedstock production alcoholic acid method of cellulose.
(1) steam explosion
Getting 1000 gram water content is the maize straw of 55 weight %, is cut into the segment of 0.8 centimetre of 2.0 cm x, 0.6 cm x, keeps 7 minutes with the pressure of 1.55 MPas under 190 ℃, and steam explosion is finished in pressure release then.The water of gained steam explosion products and 65 ℃ was mixed 40 minutes according to mass ratio at 1: 2, use impeller pump LW400 type horizontal screw centrifuge (Zhangjiagang Huada Centrifugal Manufacturing Co., Ltd.) under 900 rev/mins of rotating speeds, to carry out solid-liquid separation then, obtain 1800 gram solid steam explosion products (water content is 78 weight %) altogether.
Analyze the method for the steam explosion products of sulphuric acid hydrolysis according to the described HPLC of embodiment 1, record in the 1800 gram steam explosion products cellulose 184.5 grams altogether, hemicellulose 56.2 grams.
(2) enzymolysis
With remaining washing steam explosion products behind step (1) sampling and testing, mix with 800 gram water, regulate pH value to 3.5, after being heated to 50 ℃, dry weight basis with every gram stalk, add the cellulase of 10 enzyme activity units and the hemicellulase of 4.5 enzyme activity units (Beijing chemical reagents corporation), and be incubated 48 hours down at 50 ℃.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 embodiment 1 step (1), the glucose of measuring and calculating in the enzymolysis product 178.8 restrains totally, and wood sugar is totally 45.2 grams.The glucose weight that described enzymolysis obtains is divided by 1.11, be totally 161.1 to be restrained by the cellulosic weight of enzymolysis in the steam explosion products, the wood sugar weight that described enzymolysis obtains is divided by 1.14, be totally 39.6 to be restrained by the weight of the hemicellulose of enzymolysis in the steam explosion products, calculate cellulose conversion rate, hemicellulose transformation efficiency and monose productive rate according to following formula, 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 every gram enzymolysis product, inoculation is 10
4The yeast saccharomyces cerevisiae of colony-forming unit and 10
4The pichia stipitis of colony-forming unit, under 34 ℃ in fermentor tank stir culture 37 hours.At 100 ℃ of distillation gained tunnings, the gained distillation fraction can get ethanol 112.1 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
Table 1
| Embodiment or Comparative Examples | Embodiment 1 | Comparative Examples 1 | Comparative Examples 2 | Embodiment 2 | Embodiment 3 | Embodiment 4 |
| Cellulose conversion rate (%) | 91.2 | 76.1 | 74.5 | 85.0 | 88.9 | 87.3 |
| Hemicellulose transformation efficiency (%) | - | - | - | - | 70.3 | 70.5 |
| Monose productive rate (%) | 41.3 | 34.5 | 33.8 | 39.6 | (49.1 glucose 39.5 wood sugars 9.6) | (49.7 glucose 39.7 wood sugars 10.0) |
| Alcohol yied (%) | 21.5 | 18.2 | 17.2 | 20.6 | 25.1 | 24.9 |
As can be seen from Table 1, method cellulose conversion rate, monose productive rate and the alcohol yied of the embodiment of the invention are compared all with the method for Comparative Examples 1 and Comparative Examples 2 and are significantly improved.
Claims (8)
1, a kind of feedstock production alcoholic acid method that adopts cellulose, this method comprises the raw material of steam blasting cellulose-containing, the steam explosion products that enzymolysis obtains, the enzymolysis product that fermentation obtains, it is characterized in that the water content of the raw material of described cellulose is 30-60 weight %.
2, method according to claim 1, wherein, the water content of the raw material of described cellulose is 35-50 weight %.
3, method according to claim 1, wherein, the temperature of described steam explosion is 180-200 ℃, and the pressure of described steam explosion is the 1.4-2.0 MPa, and holding time of described steam explosion pressure is 3-7 minute.
4, 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 25-48 hour, and the pH value of described enzymolysis is 3.0-7.0.
5, method according to claim 4, 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.
6, method according to claim 1, wherein, the employed yeast of described fermentation is pichia stipitis and 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.
7, method according to claim 1, wherein, the size of the raw material of described cellulose is 0.2-1 centimetre of a 0.2-3 cm x 0.2-1 cm x.
8, method according to claim 1, wherein, the raw material of described cellulose is a stalk.
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| WO2009135898A3 (en) * | 2008-05-07 | 2010-01-21 | Novozymes A/S | Fermentation of a lignocellulose-containing material |
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