CN105200094A - Method for producing ethanol from microbial fermentation lignocellulose raw material - Google Patents
Method for producing ethanol from microbial fermentation lignocellulose raw material Download PDFInfo
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- CN105200094A CN105200094A CN201410240486.6A CN201410240486A CN105200094A CN 105200094 A CN105200094 A CN 105200094A CN 201410240486 A CN201410240486 A CN 201410240486A CN 105200094 A CN105200094 A CN 105200094A
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Abstract
The invention provides a method for producing ethanol from a microbial fermentation lignocellulose raw material. The method comprises carrying out enzymolysis on a lignocellulose raw material to obtain a lignocellulose raw material enzymatic hydrolysate, carrying out microbial enlarging cultivation to obtain an enlarging cultivation solution, inoculating the lignocellulose raw material enzymatic hydrolysate with the enlarging cultivation solution, and adjusting fermentation by intermittent oxygen supply. The method greatly improves an ethanol yield.
Description
Technical field
The invention belongs to field of microbial fermentation, be specifically related to a kind of method utilizing fermentable lignocellulosic material producing and ethanol.
Background technology
Many fossil energies as non-renewable in oil etc. are day by day exhausted, make renewable energy source particularly biofuel receive increasing concern, and bring huge business opportunity and social effect.
According to the related definition of International Energy Agency (IEA), standard biologic fuel comprises glycosyl and starch base ethanol, oil crops base biofuel, the vegetables oil that can directly utilize and the biogas obtained by anaerobic digestion; Advanced biofuel technology comprises the transformation technology that finger is also in research and development, pilot scale or demonstration phase, is commonly referred to second-generation technology or third-generation technology.This classification comprise with animal tallow and vegetables oil refining hydrogenated vegetable oil (HVO), and with lignocellulose-containing biomass production biofuel, such as ethanol, fischer-tropsch liquids and synthetic natural gas.
Ethanol is clean regeneratable liquors fuel, and many countries have brought into use the gasoline-gasohol that with the addition of certain proportion ethanol, to replace the consumption of gasoline.This New-type fuel can alleviate the wear rate of oil, can reduce automobile exhaust pollution again, has great application and development potentiality.China starts to promote the use of gasohol from calendar year 2001, and current gasohol accounts for about 20% of gasoline-like fuel total flow, and is in the impetus increased year by year.
The main raw material that production ethanol uses both at home and abroad is at present the food crop such as corn.Along with the continuous growth of world population, grain worsening shortages, therefore in the long run, food crop are not the desirable feedstock of producing ethanol.Meanwhile, there is the improper process of mode of burning etc. of a large amount of agroforestry waste wood filamentary materials (as maize straw etc.) every year, not only cause the waste of raw material, and pollute environment.Lignocellulose is primarily of Mierocrystalline cellulose, hemicellulose, xylogen composition, and in agriculture waste lignocellulosic material, the content of three approximately: Mierocrystalline cellulose accounts for 30 ~ 40%, and hemicellulose accounts for 20 ~ 30%, and xylogen accounts for 10 ~ 25%.Wherein glucose is cellulosic main component units, and wood sugar is the main component units of hemicellulose.In sponge hydrolyzed solution, wood sugar accounts for about 30%, is the sugar that nature is the abundantest after glucose, thus utilizes co-fermentation of glucose and xylose to produce ethanol abundant raw materials, has wide market outlook.
Generally speaking, advanced biofuel technology category is belonged to the production that the biomass of lignocellulose-containing carry out biofuel.Utilize lignocellulose-containing raw material production ethanol usually to adopt the technique of biochemical conversion, comprise the steps such as pre-treatment, hydrolysis, fermentation and recovery.Through pre-treatment, add the accessibility of enzyme and cellulose materials, thus improve raw-material utilizability; After hydrolysis (comprising enzymolysis), hemicellulose main decomposition is C5 sugar, and Mierocrystalline cellulose main decomposition is C6 sugar.Due to the undefined structure of hemicellulose, it is more easily hydrolyzed to C5 sugar.In pretreatment stage and enzymolysis stage, Partial fermentation inhibition can be obtained, such as formic acid, acetic acid, furfural etc.
But the corresponding predicament faced is, the microorganism that occurring in nature efficiently can produce alcohol can only utilize the hexoses such as glucose to produce ethanol usually, can not utilize wood sugar; And minority can utilize wood sugar to produce the microorganism of alcohol, produce alcohol inferior capabilities, which has limited the effective utilization to occurring in nature lignocellulose.By metabolic engineering, yeast is transformed, and utilize its metabolic conversion C5 sugar main through following reaction: Xylose reductase relies on NADPH and wood sugar is reduced to Xylitol, and Xylitol is converted into xylulose again under xylitol dehydrogenase effect; Form X 5P through xylulokinase phosphorylation again, then enter phosphopentose pathway (PPP).The intermediate product G6P of PPP approach and glyceraldehyde 3-phosphate form pyruvic acid by diphosphate pathway, pyruvic acid or be reduced to ethanol through pyruvic carboxylase, ethanol dehydrogenase decarboxylation.
Under the promotion of genetic engineering technique, researchist obtains a collection of restructuring organism of fermentation that xylose metabolism can be utilized to produce ethanol.But, in the suitability for industrialized production of reality, especially the extensive industrialization stage, above-mentioned microorganism enlarged culturing rate of propagation is slower, be difficult to realize large-scale application, and because microbial metabolism is containing the consumption otherness demand supplied fermenting in C5 sugar and the lignocellulosic material enzymolysis solution process of C6 sugar, causes the transformation efficiency in actual production too low always, also becoming one of difficult point limiting this technical development.
Summary of the invention
Therefore, the object of the invention is to overcome microbial metabolism in prior art and contain the consumption otherness demand to fermentation supply in the lignocellulosic material enzymolysis solution process of C5 and C6 sugar, the defect that in the actual production caused, transformation efficiency is too low, thus provide a kind of method of efficiency utilization fermentable lignocellulosic material producing and ethanol, thus improve the transformation efficiency of ethanol.
For this reason, the invention provides a kind of method utilizing fermentable lignocellulosic material producing and ethanol, comprise the steps:
1), by described lignocellulosic material enzymolysis, lignocellulosic material enzymolysis solution is obtained;
2), by described microorganism enlarged culturing, scale-up medium is obtained;
3), described scale-up medium is seeded in described lignocellulosic material enzymolysis solution by 5-20%, control temperature of reaction 25-38 DEG C, pH5.0-7.0 carries out fermentative production, control to carry out intermittent type oxygen supply in described fermenting process, air flow 0.01-0.5mL/L, aeration time 0.5h, interval time is 5-6h, and fermentation obtains fermented liquid;
4), be separated described fermented liquid and obtain ethanol.
Described step 3) in, control air air flow 0.1mL/L-0.2mL/L in fermenting process.
Described lignocellulosic material is selected from one or more in maize straw, corn cob, hardwood, cork, nutshell, grass, paper, barley-straw, wheat straw, leaf, cottonseed wadding, withy, oat shell.
From structure, lignocellulose mainly comprises Mierocrystalline cellulose, hemicellulose and xylogen.Preferably, described lignin-cellulose material comprises at least 30wt%, preferably at least 50wt%, more preferably at least 70wt%, even more preferably the lignocellulose of at least 90wt%.It should be understood that in lignocellulose-containing raw material and can also comprise other component, as protein material, starch, sugar, as fermentable sugar and/or not fermentable sugar.
Described enzyme is selected from cellulase, hemicellulase, amylase, proteolytic enzyme, glucoamylase, lipase.Described cellulase includes but not limited to cellobiohydrolase (cellobiohydrolase I and cellobiohydrolase II) and endo-glucanase and beta-glucosidase enzyme.
In described lignocellulosic material enzymolysis solution also containing one or more in soybean cake powder, corn-dodger powder, corn steep liquor, fish meal or yeast extract paste as nitrogenous source, containing one or more in Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC, potassium primary phosphate, dipotassium hydrogen phosphate, Secondary ammonium phosphate as inorganic salt, and optionally containing urea and/or inhibitor.
The carbon source contained in described fermention medium, by weight ratio, glucose is 5-20%, and wood sugar is 3-8%; As a kind of preferred content in described fermention medium, glucose is 6-12%, and wood sugar is 3-6%.
Also containing as one or both in the protein of nitrogenous source, total free aminoacids in described fermention medium.Wherein protein content is in the fermentation medium 3-4g/L, is mainly used in providing the nitrogenous source needed for described microorganism growth; Described total free aminoacids comprises one or more in L-Ala, proline(Pro), phenylalanine, arginine, Isoleucine, methionine(Met), L-glutamic acid, Threonine, Gelucystine, leucine and α-amino-isovaleric acid.
The inorganic salt contained in described fermention medium are selected from one or more in Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC, potassium primary phosphate, dipotassium hydrogen phosphate, Secondary ammonium phosphate, and the content of described inorganic salt in described fermention medium is 0.5-4g/L, be preferably 1-2.5g/L.
Also containing one or more in VITAMIN, trace element in described fermention medium.Described VITAMIN comprises vitamin B group, and described vitamin B group at least comprises vitamin H, folic acid; Described trace element comprises one or more in copper, calcium, iron, zinc, lead, silver, chromium, Yan acid, pyridoxic acid, thiamines and calcium pantothenate.
Also the fungistat of varied bacteria growing is suppressed containing being used in fermention medium of the present invention, certainly, if there is no miscellaneous bacteria in fermentation culture process, also above-mentioned fungistat can not be added, described fungistat is selected from one or more in penicillin, penbritin, Streptomycin sulphate, paraxin, terramycin, tsiklomitsin, and be preferably penicillin and/or Streptomycin sulphate, in fermention medium, the working concentration of fungistat is 10-50 unit/mL, be preferably 15-25 unit/mL, be more preferably 20 units/mL.
When described fermention medium derives from described lignocellulosic material enzymolysis solution, ratio by weight, the content of its glucose is 1-25%, and the content of wood sugar is 1-10%; Preferably, the content of glucose is 5-20%, and the content of wood sugar is 3-8%; Preferred, the content of glucose is 6-12%, and the content of wood sugar is 3-6%.
Simultaneously, those skilled in the art should understand, although certain density acetic acid may produce restraining effect to described microorganism cells, but still can allow to there is a small amount of acetic acid in lignocellulosic material enzymolysis solution, the content of described acetic acid in described fermention medium is preferably 0.1-1%, is more preferably 0.1-0.8%.
Described enlarged culturing is in enlarged culturing base by described microbial inoculant, by at least one-level enlarged culturing step, enlarged culturing is carried out to described microorganism, in described enlarged culturing step, realize the enlarged culturing to described microorganism by the confession oxygen concn in adjustment enlarged culturing process.
Use one-level enlarged culturing step to carry out enlarged culturing to described microorganism, comprise the steps:
Described seed liquor is spread cultivation in tank according to the inoculum size of the 5-20% one-level be seeded to containing enlarged culturing base, control temperature 25-35 DEG C, pH4-6.5, rotating speed 180-220rpm, air flow 0.08-0.5L/L.min, enlarged culturing 6-12h; Adjust rotating speed 40-100rpm, air flow 0.18-0.5L/L.min subsequently, enlarged culturing to described microorganism concn reaches (0.1-0.5) × 10
9/ mL.
Use secondary enlarged culturing step to carry out enlarged culturing to described microorganism, comprise the steps:
1) one-level that described seed liquor is seeded to containing described enlarged culturing base according to the inoculum size of 5-20% is spread cultivation in tank, control temperature 25-35 DEG C, pH4-6.5, rotating speed 180-220rpm, air flow 0.08-0.5L/L.min, carry out enlarged culturing to described microorganism concn and reach (0.1-0.5) × 10
9/ mL;
2) by step 1) in the secondary that is seeded to containing described enlarged culturing base according to the inoculum size of 5-20% of the microorganism that spreads cultivation that obtains spread cultivation in tank, control temperature 25-35 DEG C, pH4-6.5, rotating speed 40-100rpm, air flow 0.18-0.5L/L.min, carry out enlarged culturing to described microorganism concn and reach (0.1-0.5) × 10
9/ mL.
Use three grades of enlarged culturing steps to carry out enlarged culturing to described microorganism, comprise the steps:
1) one-level that described seed liquor is seeded to containing described enlarged culturing base according to the inoculum size of 5-20% is spread cultivation in tank, control temperature 25-35 DEG C, pH4-6.5, rotating speed 180-220rpm, air flow 0.08-0.5L/L.min, carry out enlarged culturing to described microorganism concn and reach (0.1-0.5) × 10
9/ mL;
2) by step 1) in the secondary that is seeded to containing described enlarged culturing base according to the inoculum size of 5-20% of the microorganism that spreads cultivation that obtains spread cultivation in tank, control temperature 25-35 DEG C, pH4-6.5, rotating speed 40-60rpm, air flow 0.18-0.5L/L.min, carry out enlarged culturing to described microorganism concn and reach (0.1-0.5) × 10
9/ mL;
3) by step 2) in the microorganism that spreads cultivation that obtains three grades of being seeded to containing described enlarged culturing base according to the inoculum size of 5-20% spread cultivation in tank, control temperature 25-35 DEG C, pH4-6.5, rotating speed 40-60rpm, air flow 0.01-0.2L/L.min, carry out enlarged culturing to described microorganism concn and reach (0.1-0.5) × 10
9/ mL.
Use level Four enlarged culturing step to carry out enlarged culturing to described microorganism, comprise the steps:
1) one-level that described seed liquor is seeded to containing described enlarged culturing base according to the inoculum size of 5-20% is spread cultivation in tank, control temperature 25-35 DEG C, pH4-6.5, rotating speed 180-220rpm, air flow 0.08-0.5L/L.min, carry out enlarged culturing to described microorganism concn and reach (0.1-0.5) × 10
9/ mL;
2) by step 1) in the secondary that is seeded to containing described enlarged culturing base according to the inoculum size of 5-20% of the microorganism that spreads cultivation that obtains spread cultivation in tank, control temperature 25-35 DEG C, pH4-6.5, rotating speed 40-60rpm, air flow 0.18-0.5L/L.min, carry out enlarged culturing to described microorganism concn and reach (0.1-0.5) × 10
9/ mL;
3) by step 2) in the microorganism that spreads cultivation that obtains three grades of being seeded to containing described enlarged culturing base according to the inoculum size of 5-20% spread cultivation in tank, control temperature 25-35 DEG C, pH4-6.5, rotating speed 40-60rpm, air flow 0.01-0.2L/L.min, carry out enlarged culturing to described microorganism concn and reach (0.1-0.5) × 10
9/ mL;
4) by step 3) in the level Four that is seeded to containing described enlarged culturing base according to the inoculum size of 5-20% of the microorganism that spreads cultivation that obtains spread cultivation in tank, control temperature 25-35 DEG C, pH4-6.5, air flow 0.0005-0.02L/L.min, carry out enlarged culturing to described microorganism concn and reach (0.1-0.5) × 10
9/ mL.
In described enlarged culturing process at different levels, the one-level tank that spreads cultivation is that 50L spreads cultivation tank, and the secondary tank that spreads cultivation is that 500L spreads cultivation tank, and three grades of tanks that spread cultivation are 5 cubes of tanks that spread cultivation, and the level Four tank that spreads cultivation is 50 cubes of tanks that spread cultivation.
In the process of described enlarged culturing at different levels, described enlarged culturing base independent of each other containing one or more in corn mash, molasses, glucose, sweet sorghum stalk juice or lignocellulosic material enzymolysis solution as carbon source, containing one or more in soybean cake powder, corn-dodger powder, corn steep liquor, fish meal, yeast extract paste as nitrogenous source; And optionally containing urea, inorganic salt, amino acid, trace element and/or inhibitor.
Described enlarged culturing can produce for subsequent fermentation the microbe population that ethanol Step provides abundant, and wherein enlarged culturing base is the source of nutrition that microorganism obtains existence, has direct impact to the activity of microbial growth, enzyme and output.
Described selecting property of scale-up medium base comprise peptone, corn mash, molasses, glucose, sweet sorghum stalk juice or lignocellulosic material enzymolysis solution.
Preferred as one, described enlarged culturing base is one or more in peptone, corn mash, molasses, glucose, sweet sorghum stalk juice or lignocellulosic material enzymolysis solution, and in described enlarged culturing base the content of glucose by weight percentage content be 2-10%, preferred content is 4-6%.
Preferred as another kind, the substratum containing 4% weight percent glucose in described enlarged culturing base, or the corn mash or the molasses that contain that weight percent is 4% glucose.
Alternatively preferred version, described enlarged culturing base can also be that weaker concn is the lignocellulosic material enzymolysis solution of 10-75% by weight percentage, and wherein said lignocellulose raw material enzymolysis solution is not contained glucose 1-25%, wood sugar 1-10% by weight percentage by during dilution; Preferably, glucose content is 5-20%, and Xylose Content is preferably 3-8%; Preferred, glucose content is 6-12%, and Xylose Content is 3-6%.Preferably, when described lignocellulosic material enzymolysis solution is as described enlarged culturing base, its by weight percentage weaker concn be 20-30%.
Preferred as another, the material such as nitrogenous source, trace element, VITAMIN required during for providing described microorganism enlarged culturing, described enlarged culturing base is also the mixture of the corn steep liquor of 40-60wt%, fish meal and/or yeast extract paste containing the soybean cake powder of 5-10g/L, corn-dodger powder, dry matter content, is preferably 8-10g/L; Thus provide the multiple amino acids of the total free aminoacids comprising L-Ala, proline(Pro), phenylalanine, arginine, Isoleucine, methionine(Met), L-glutamic acid, Threonine, Gelucystine, leucine and/or α-amino-isovaleric acid for described microorganism, comprise copper, calcium, iron, zinc, lead, silver, chromium, Yan acid, pyridoxic acid, thiamines and/or calcium pantothenate various trace elements and comprise the various VITAMIN of vitamin B group.
As further preferred, also containing urea and/or inorganic salt in described enlarged culturing base, the content of described urea in described enlarged culturing base is 2-5g/L, is preferably 3-4g/L; Described inorganic salt are selected from one or more in Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC, potassium primary phosphate, dipotassium hydrogen phosphate, Secondary ammonium phosphate, be preferably potassium primary phosphate and/or Secondary ammonium phosphate, content in described enlarged culturing base is 0.5-4g/L, preferred 1-3g/L.Preferred, containing the potassium primary phosphate of 1.5g/L and the Secondary ammonium phosphate of 1.5g/L in described enlarged culturing base.
Under normal circumstances, do not need to add fungistat if do not have miscellaneous bacteria to enter in described enlarged culturing process, if when having miscellaneous bacteria to enter, as further preferred, fungistat can be added in described enlarged culturing base, described fungistat is selected from one or more in penicillin, penbritin, Streptomycin sulphate, paraxin, terramycin, tsiklomitsin, be preferably penicillin or/Streptomycin sulphate, described in described enlarged culturing base fluid, the working concentration of fungistat is 10-50 unit/mL, be preferably 15-25 unit/mL, be more preferably 20 units/mL.
Also comprised before enlarged culturing is by described microorganism and described microorganism cultivated in seed culture medium 12-24h and reach (0.1-0.5) × 10 to described microorganism concn
9the step of/ml.
Seed culture medium is adopted to cultivate described microorganism, object is microorganism described in rapid amplifying, to reach the cell concentration described in enlarged culturing needed for microorganism as early as possible, described seed culture medium can adopt existing seed culture medium in prior art, as YEPD etc.
Described YEPD refers to that medium component is yeast powder 10g/L, peptone 20g/L, glucose 20g/L, is mixed with and obtains after packing sterilizing.
Described microorganism is selected from Saccharomyces Cerevisiae in S accharomycescerevisiae, pachysolen tannophilus Pachysolentannophilus, pichia stipitis Pichiastipitis, shehatae candida Candidashehatae, Brettanomyces Brettanomycesnaardenensis, very thin candiyeast Candidatenuis, candida tropicalis Candidatropicalis, match ditch pichia spp Pichiasegobiensis, Bacteroides polypragmatus Bacteroidespolypragmatus, chrysanthemum Erwinia Erwiniachrysanthem, plant klebsiella spp Klebsiellaplanticola, thermophilic anaerobic ethanol bacterium Thermoanaerobacterethanolicus, spherical spiral body Spirochaetacoccoidessp., plant fermentation clostridium Clostridiumphytofermentassp., Fusarium oxysporum Fusariumoxysporum, Neuraspora crassa Neurosporacrassa, fusarium avenaceum Fusariumavenaceum, zymomonas mobilis Zymomonasmobilis, intestinal bacteria Escherichiacoli and recombinant Saccharomyces cerevisiae S.cerevisiae, restructuring zymomonas mobilis Zymomonasmobilis, recombination bacillus coli Escherichiacoli, and other known bacterial strains that can carry out C5 and C6 co-fermentation producing and ethanol well-known to those skilled in the art.
Mentioned microorganism selected by the method for the invention is separated from nature to obtain, or obtain after genetically engineered, comprises bacterium, fungi and yeast wine brewing.
Wherein, described recombinant Saccharomyces cerevisiae S.cerevisiae is expressed in yeast saccharomyces cerevisiae by the xylose transport enzyme of fungi, xylose isomerase (XI) approach or wood sugar reduction-Xylitol oxidative pathway (XR-XDH) is selected to carry out xylulose conversion, and phosphopentose downstream metabolic path being carried out the ability modifying to strengthen xylose metabolism ethanol conversion, available S. cervisiae is documented in the bacterial strain disclosed in patent WO9742307A, WO9513362A, US20110027847, CN1966694A, CN101205525A.
The xylA of E.coli (xylose isomerase gene), xylB (xylulokinase gene), talB (transketolase gene), tktA (transaldolase gene) can import in Z.mobilis by the transformation of described restructuring zymomonas mobilis Zymomonasmobilis, such as patent US5843760, US5514583, WO200851348A, WO200958927A, WO200944868A or WO200958938A.
Described recombination bacillus coli Escherichiacoli, refer to that the central metabolites thing-pyruvic acid by the Plastid transformation containing PET operon (carrying zymomonas mobilis pyruvic oxidase and alcohol dehydrogenase gene) can make the sugar of restructuring E.coli and pentose metabolism be formed to E.coli bacterial strain turns to alcohol production, the bacterial strain such as, reported in patent CN101875912A.
Preferably, described microorganism is selected from Saccharomyces Cerevisiae in S accharomycescerevisiae, zymomonas mobilis Zymomonasmobilis and/or E. coli.
Preferred further, described microorganism is Saccharomyces Cerevisiae in S accharomycescerevisiae, zymomonas mobilis Zymomonasmobilis.
From fermented liquid, separating alcohol can adopt ordinary method known in the art, such as, adopt distillation, can adopt the conventional distil-lation equipment described in this area, such as, have the distillation tower of double pass tray and cross-current trays.Because the suspended solids content of fermentation spirituosity product is higher, in general, double fluid sieve column plate or crossing current valve column plate are preferred.In various preferred embodiment, the tower comprising crossing current valve column plate is preferred, because often provide higher turndown ratio and the efficiency of Geng Gao by crossing current valve column plate.
A kind of method of fermentable lignocellulosic material producing and ethanol that utilizes of the present invention has the following advantages:
1. the method utilizing fermentable lignocellulosic material producing and ethanol of the present invention, by obtaining lignocellulosic material enzymolysis solution by after lignocellulosic material enzymolysis, then by the microbial inoculant after enlarged culturing to above-mentioned lignocellulosic material enzymolysis solution, take the fermentation strategies of intermittent type oxygen supply during the fermentation, make microorganism fully can fully utilize glucose and xylose in enzymolysis solution, the efficiency of producing and ethanol improves greatly.
2. the method utilizing fermentable lignocellulosic material producing and ethanol of the present invention, the lignocellulosic material used is selected from maize straw, corn cob, hardwood, cork, nutshell, grass, paper, barley-straw, wheat straw, leaf, cottonseed is wadded a quilt with cotton, withy, one or more in oat shell, thus the organic waste being difficult to rationally dispose can not only be utilized in reality in a large number, be conducive to environmental protect, alleviate the pressure processing above-mentioned organic waste, and the ethanol of high level can also be produced, use as clean fuel, thus alleviate the utilization of fossil oil, slow down the speed of Fossil fuel consumption, and protect environment, above-mentioned raw material sources is sufficient, can avoid using grain fermentation producing and ethanol, has saved grain, has reduced the production cost of ethanol.
4. the method utilizing fermentable lignocellulosic material producing and ethanol of the present invention, soybean cake powder can also be contained in above-mentioned lignocellulosic material enzymolysis solution, corn-dodger powder, corn steep liquor, one or more in fish meal or yeast extract paste are as nitrogenous source, containing Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC, potassium primary phosphate, dipotassium hydrogen phosphate, one or more in Secondary ammonium phosphate are as inorganic salt, and optionally containing urea and/or inhibitor etc., various nutritive substances needed for mentioned microorganism can be provided during the fermentation, the fermentation strategies of above-mentioned intermittent type oxygen supply is coordinated to promote the fast breeding of microorganism, improve quality and the speed of fermentation.
5. the method utilizing fermentable lignocellulosic material producing and ethanol of the present invention, by the enlarged culturing step of at least one-level, described microorganism is cultivated, and in described enlarged culturing step, regulate for oxygen concn, thus described microorganism is rised in value fast, and fermentative activity strengthens.
6. the method utilizing fermentable lignocellulosic material producing and ethanol of the present invention, the microorganism used is separated from nature to obtain, or obtain after genetically engineered, comprise bacterium, fungi and yeast wine brewing etc., mentioned microorganism, under the different fermentations stage of the present invention adopts the production method of varying environment condition, the fermentation of efficiency utilization lignocellulosic material enzymolysis solution can produce ethanol.
Embodiment
Below in conjunction with embodiment, more concrete description is done to a kind of method of fermentable lignocellulosic material producing and ethanol that utilizes of the present invention.
Embodiment 1
The present embodiment provides a kind of C5 and C6 that ferment altogether to produce the enlarged culturing method of ethanol microbe, enlarged culturing experiment is carried out for recombinant Saccharomyces cerevisiae S.cerevisiae424A (LNH-ST), described recombinant Saccharomyces cerevisiae S.cerevisiae424A (LNH-ST) is (as CellulosicethanolproductionfromAFEX-treatedcornstoverusi ngSaccharomycescerevisiae424A (LNH-ST), M.W.Lau, B.E.Dale, ProcNatlAcadSciUSA, 106 (2009), pp.1368-1373; Two-stepSSCFtoconvertAFEX-treatedswitchgrasstoethanolusi ngcommercialenzymesandSaccharomycescerevisiae424A (LNH-ST), M.Jin, M.W.Lau, V.Balan, B.E.Dale, BioresourTechnol, 101 (2010), pp.8171 reports such as – 8178 grade), be commercially available acquisition, described enlarged culturing method adopts one-level enlarged culturing step, specifically comprises:
A, seed liquor culturing step
Preparation YEPD substratum, being seeded in YEPD substratum by the above-mentioned thalline preserved, control temperature 25 DEG C, pH6.0, rotating speed 200rpm, air flow 0.2L/L.min, cultivating 12h, is (0.1-0.5) × 10 to above-mentioned bacterial classification concentration
9/ ml;
Described YEPD substratum specifically containing yeast powder 10g/L, peptone 20g/L, glucose 20g/L, and regulates pH6.5.
B, enlarged culturing step
Spread cultivation in tank by the seed liquor after cultivating according to the one-level containing one-level enlarged culturing base that the inoculum size access volume of 5% (v/v) is 50L, control temperature 35 DEG C, pH4, rotating speed 180rpm pass into air, air flow 0.5L/L.min, cultivate 12h; Adjusting rotating speed is subsequently 40rpm, and air flow is 0.18L/L.min, enlarged culturing 12h, reaches (0.1-0.5) × 10 to bacterial classification concentration
9/ ml;
Described one-level enlarged culturing base comprise be diluted with water to 30wt% lignocellulosic material enzymolysis solution, corn steep liquor (dry matter content 40%) 15g/L, KH
2pO
41.5g/L, (NH
4)
2hPO
42.5g/L, uses deionized water or softening water configuration solution, packing sterilizing, mixing after cooling, and adds 2g/L urea.
Embodiment 2
The present embodiment provides a kind of C5 and C6 that ferment altogether to produce the enlarged culturing method of ethanol microbe, enlarged culturing experiment is carried out for candida tropicalis Candidatropicalis, described candida tropicalis Candidatropicalis is commercially available gained, described enlarged culturing method adopts secondary enlarged culturing step, specifically comprises:
A, seed liquor culturing step
Preparation YEPD substratum, is seeded in YEPD substratum by the above-mentioned thalline preserved, control temperature 25 DEG C, pH6.0, rotating speed 200rpm, air flow 0.2L/L.min, is cultured to above-mentioned bacterial classification concentration for (0.1-0.5) × 10
9/ ml;
Described YEPD substratum specifically containing yeast powder 10g/L, peptone 20g/L, glucose 20g/L, and regulates pH6.0.
B, enlarged culturing step
1) seed liquor after cultivation is spread cultivation in tank according to the one-level containing one-level enlarged culturing base that the inoculum size access volume of 20% (v/v) is 50L, control temperature 25 DEG C, pH6.5, rotating speed 220rpm pass into air, air flow 0.08L/L.min, cultivate 16h, reach (0.1-0.5) × 10 to bacterial classification concentration
9/ ml;
2) spread cultivation that to be seeded to volume be that the secondary containing secondary enlarged culturing base of 500L spreads cultivation in tank to liquid according to described one-level to be spread cultivation one-level that step obtains of the inoculum size of 20% (v/v), control temperature 25 DEG C, pH6.5, rotating speed 100rpm pass into air, air flow 0.18L/L.min, cultivate 12h, reach (0.1-0.5) × 10 to bacterial classification concentration
9/ ml;
Described one-level enlarged culturing base is identical with secondary enlarged culturing base, be containing mass percent be 4% glucose enlarged culturing base, and containing corn steep liquor (dry matter content 40%) 15g/L, KH
2pO
41.5g/L, (NH
4)
2hPO
41.5g/L; Deionized water or softening water configuration solution, packing sterilizing, mixing after cooling, and add 3g/L urea.
Embodiment 3
The present embodiment provides a kind of C5 and C6 that ferment altogether to produce the enlarged culturing method of ethanol microbe, enlarged culturing experiment (with embodiment 1) is carried out for recombinant Saccharomyces cerevisiae S.cerevisiae424A (LNH-ST), described enlarged culturing method adopts three grades of enlarged culturing steps, specifically comprises:
A, seed liquor culturing step
Preparation YEPD substratum, being seeded in YEPD substratum by the above-mentioned thalline preserved, control temperature 30 DEG C, pH6.0, air flow 0.2L/L.min, rotating speed 200rpm, cultivating 14h, is (0.2-0.3) × 10 to above-mentioned bacterial classification concentration
9/ ml;
Described YEPD substratum specifically containing yeast powder 10g/L, peptone 20g/L, glucose 20g/L, and regulates pH6.5.
B, enlarged culturing step
1) seed liquor after cultivation is spread cultivation in tank according to the one-level containing one-level enlarged culturing base that the inoculum size access volume of 10% (v/v) is 50L, control temperature 30 DEG C, pH5.0, rotating speed 200rpm pass into air, air flow 0.5L/L.min, cultivates 14h and reaches (0.2-0.3) × 10 to bacterial classification concentration
9/ ml;
2) spread cultivation that to be seeded to volume be that the secondary containing secondary enlarged culturing base of 500L spreads cultivation in tank to liquid according to described one-level to be spread cultivation one-level that step obtains of the inoculum size of 15% (v/v), control temperature 25 DEG C, pH6.0, rotating speed 100rpm pass into air, air flow 0.5L/L.min, cultivates 16h and reaches (0.1-0.5) × 10 to bacterial classification concentration
9/ ml;
3) spread cultivation that to be seeded to volume be that 5 cubic metres three grades containing three grades of enlarged culturing bases spread cultivation in tank to liquid according to be spread cultivation by described secondary secondary that step obtains of the inoculum size of 5% (v/v), control temperature 33 DEG C, pH6.2, rotating speed 60rpm pass into air, air flow 0.01L/L.min, cultivates 16h and reaches (0.2-0.3) × 10 to bacterial classification concentration
9/ ml;
Described one-level enlarged culturing base is identical with secondary enlarged culturing base, is: containing corn mash 4wt%, adds 3g/L urea after sterilizing;
Described three grades of enlarged culturing bases are: be diluted with water to the matter cellulose material enzymolysis liquid of 30wt% wood, corn steep liquor (dry matter content 40%) 15g/L, KH
2pO
41.5g/L, (NH
4)
2hPO
41.5g/L, deionized water or softening water configuration solution, packing sterilizing, mixing after cooling, and add 3g/L urea.
Embodiment 4
The present embodiment provides a kind of C5 and C6 that ferment altogether to produce the enlarged culturing method of ethanol microbe, fermenting experiment (with embodiment 1) is carried out for recombinant Saccharomyces cerevisiae S.cerevisiae424A (LNH-ST), described enlarged culturing method adopts level Four enlarged culturing step, specifically comprises:
A, seed liquor culturing step
Preparation YEPD substratum, being seeded in YEPD substratum by the above-mentioned thalline preserved, control temperature 30 DEG C, pH6.0, air flow 0.2L/L.min, rotating speed 200rpm, cultivating 20h, is (0.2-0.3) × 10 to above-mentioned bacterial classification concentration
9/ ml;
Described YEPD substratum specifically containing yeast powder 5g/L, peptone 15g/L, glucose 15g/L, and regulates pH6.0.
B, enlarged culturing step
1) seed liquor after cultivation is spread cultivation in tank according to the one-level containing one-level enlarged culturing base that the inoculum size access volume of 10% (v/v) is 50L, control temperature 33 DEG C, pH4.8, rotating speed 200rpm pass into air, air flow 0.4L/L.min, cultivates 14h and reaches (0.2-0.3) × 10 to bacterial classification concentration
9/ ml;
2) spread cultivation that to be seeded to volume be that the secondary containing secondary enlarged culturing base of 500L spreads cultivation in tank to liquid according to described one-level to be spread cultivation one-level that step obtains of the inoculum size of 17% (v/v), control temperature 30 DEG C, pH5, rotating speed 60rpm pass into air, air flow 0.3L/L.min, cultivates 14h and reaches (0.2-0.3) × 10 to bacterial classification concentration
9/ ml;
3) spread cultivation that to be seeded to volume be that 5 cubic metres three grades containing three grades of enlarged culturing bases spread cultivation in tank to liquid according to be spread cultivation by described secondary secondary that step obtains of the inoculum size of 18% (v/v), control temperature 35 DEG C, pH6.5, rotating speed 60rpm pass into air, air flow 0.18L/L.min, cultivates 16h and reaches (0.2-0.3) × 10 to bacterial classification concentration
9/ ml;
4) three grades of liquid that spread cultivation that described three grades of steps that spread cultivation obtain being seeded to volume according to the inoculum size of 20% (v/v) is that the level Four containing level Four enlarged culturing base of 30 cubic metres spreads cultivation in tank, control temperature 33 DEG C, pH5.8, air flow 0.01L/L.min, cultivate 16h and reach (0.2-0.3) × 10 to bacterial classification concentration
9/ ml.
Described one-level enlarged culturing base and secondary enlarged culturing base are all identical with secondary enlarged culturing base with the one-level enlarged culturing base in embodiment 3;
Described three grades of enlarged culturing bases and level Four enlarged culturing base are all identical with grade enlarged culturing base of three in embodiment 3.
In embodiment 1,3,4, the raw material of lignocellulosic material enzymolysis solution is selected from maize straw, and its preparation method is as follows:
Step (1): maize straw is broken for the particle that granularity is 10-100mm, raw material is dropped into scroll feeder, extrusion dehydration is carried out to dry 20%-50% to it, form fine and close material plug, while guarantee cellulosic material constantly enters processing vessel, the steam can also resisted in container leaks;
Step (2): the material plug formed in step 1, after scroll feeder process, is mixed into the dilute sulphuric acid solution of its quality 2% in cellulosic material, obtains acid cellulose raw material after entering the mixing of sour mixing tank;
Step (3): the acid cellulose raw material in step 2 is in boiling processing vessel, process with the low pressure steam contacts of 0.6MPa (G), treatment time is between 15-25min, then according to the mode amount discharge container of certain frequency intermittent decompression discharge, this process can open the structure of cellulosic material, hemicellulose fraction is degraded, and portion of cellulose is exposed on surface;
Step (4): biomass processes product is washed, adjust ph is 5.0, after being heated to 50 DEG C, with the dry weight basis of every gram of product, the cellulase adding 20-50 filter paper enzyme activity unit of force calculates, clamp-on cellulase (letter company limited of Novi provides), and be incubated mix and blend 72h at 50 DEG C, obtain described enzymolysis solution.
The enlarged culturing strain liquid obtained will be cultivated in embodiment 1-4, all be connected in the fermentor tank containing fermention medium by 10% inoculum size, control temperature 30 DEG C in fermenting process, pH6.0, rotating speed 200rpm passes into air, air flow 0.2L/L.min, fermentation 16h, detects tunning.
Described fermention medium is: glucose 8wt%, wood sugar 4.5wt%, KH
2pO
40.5g/L, (NH
4)
2hPO
42g/L; Deionized water or softening water configuration solution, packing sterilizing, mixing after cooling.
| Spread cultivation progression | Initial sugared concentration (g/L) | Terminal alcohol concn (g/L) | Total used time (spreads cultivation+ferments (h) | |
| Embodiment 1 | 1 | 77.00+43.42 | 30.96 | 28 |
| Embodiment 2 | 2 | 77.00+43.42 | 41.30 | 44 |
| Embodiment 3 | 3 | 77.00+43.42 | 45.78 | 62 |
| Embodiment 4 | 4 | 77.00+43.42 | 50.35 | 76 |
Embodiment 5
The present embodiment provides a kind of method utilizing fermentable lignocellulosic material producing and ethanol, fermenting experiment is carried out for recombinant Saccharomyces cerevisiae S.cerevisiae424A (LNH-ST), described recombinant Saccharomyces cerevisiae S.cerevisiae424A (LNH-ST) is (as CellulosicethanolproductionfromAFEX-treatedcornstoverusi ngSaccharomycescerevisiae424A (LNH-ST), M.W.Lau, B.E.Dale, ProcNatlAcadSciUSA, 106 (2009), pp.1368-1373, Two-stepSSCFtoconvertAFEX-treatedswitchgrasstoethanolusi ngcommercialenzymesandSaccharomycescerevisiae424A (LNH-ST), M.Jin, M.W.Lau, V.Balan, B.E.Dale, BioresourTechnol, 101 (2010), report such as pp.8171 – 8178 grade), for commercially available acquisition, the described method of fermentable lignocellulosic material producing and ethanol that utilizes specifically comprises: step lignocellulosic material enzymolysis being obtained lignocellulosic material enzymolysis solution, the step of seed culture, the step of strain expanded culture, and the step of fermentation producing and ethanol, specifically comprise:
A, enzymolysis lignocellulosic material obtain the step of lignocellulosic material enzymolysis solution
The raw material of described lignocellulosic material enzymolysis solution is selected from maize straw, and its preparation method is as follows:
Step 1): maize straw is broken for the particle that granularity is 10-100mm, raw material is dropped into scroll feeder, extrusion dehydration is carried out to dry 20%-50% to it, form fine and close material plug, while guarantee cellulosic material constantly enters processing vessel, the steam can also resisted in container leaks;
Step 2): in step 1) in formed material plug after scroll feeder process, in cellulosic material, be mixed into the dilute sulphuric acid solution of its quality 2%, enter sour mixing tank mixing after obtain acid cellulose raw material;
Step 3): step 2) in acid cellulose raw material in boiling processing vessel, process with the low pressure steam contacts of 0.6MPa (G), treatment time is between 15-25min, then according to the mode amount discharge container of certain frequency intermittent decompression discharge, this process can open the structure of cellulosic material, hemicellulose fraction is degraded, and portion of cellulose is exposed on surface;
Step 4): biomass processes product is washed, adjust ph is 5.0, after being heated to 50 DEG C, with the dry weight basis of every gram of product, the cellulase adding 20-50 filter paper enzyme activity unit of force calculates, clamp-on cellulase (letter company limited of Novi provides), and be incubated mix and blend 72h at 50 DEG C, obtain described enzymolysis solution.
B, seed liquor culturing step
Preparation YEPD substratum, being seeded in YEPD substratum by the above-mentioned thalline preserved, control temperature 30 DEG C, pH6.0, rotating speed 200rpm, air flow 0.2L/L.min, cultivating 20h, is (0.2-0.3) × 10 to above-mentioned bacterial classification concentration
9/ ml;
Described YEPD substratum specifically containing yeast powder 10g/L, peptone 20g/L, glucose 20g/L, and regulates pH6.0.
C, enlarged culturing step
1) seed liquor after cultivation is spread cultivation in tank according to the one-level containing one-level enlarged culturing base that the inoculum size access volume of 5% (v/v) is 50L, control temperature 30 DEG C, pH6.0, rotating speed 200rpm pass into air, air flow 0.1L/L.min, cultivates 16h and reaches (0.2-0.3) × 10 to bacterial classification concentration
9/ ml;
2) spread cultivation that to be seeded to volume be that the secondary containing secondary enlarged culturing base of 500L spreads cultivation in tank to liquid according to described one-level to be spread cultivation one-level that step obtains of the inoculum size of 5% (v/v), control temperature 30 DEG C, pH6.0, rotating speed 50rpm pass into air, air flow 0.2L/L.min, cultivates 16h and reaches (0.1-0.5) × 10 to bacterial classification concentration
9/ ml;
Described one-level enlarged culturing base is identical with secondary enlarged culturing base, be containing mass percent be 4% glucose enlarged culturing base, and containing corn steep liquor (dry matter content 40%) 15g/L, KH
2pO
41.5g/L, (NH
4)
2hPO
41.5g/L; Deionized water or softening water configuration solution, packing sterilizing, mixing after cooling, and add 3g/L urea.
D, fermentation producing and ethanol step
Get second stage enlarged culturing terminate after secondary to spread cultivation liquid, be seeded to containing above-mentioned lignocellulosic material enzymolysis solution as in the fermentor tank of fermention medium according to the inoculum size of 10% (v/v), control temperature 28 DEG C, pH6.0, air flow 0.01mL/L, interval ventilation, aeration time is 0.5h, interval time is 6h, and fermentation 72h, obtains tunning.
Embodiment 6
The present embodiment provides a kind of method utilizing fermentable lignocellulosic material producing and ethanol, fermenting experiment (with embodiment 5) is carried out for recombinant Saccharomyces cerevisiae S.cerevisiae424A (LNH-ST), the method of described fermentation producing and ethanol specifically comprises: lignocellulosic material enzymolysis is obtained the step of lignocellulosic material enzymolysis solution, the step of seed culture, the step of strain expanded culture, and the step of fermentation producing and ethanol, specifically comprise:
A, enzymolysis lignocellulosic material obtain the step of lignocellulosic material enzymolysis solution
With embodiment 5, difference is, the lignocellulosic material used is corn cob and grass.
B, seed liquor culturing step
Preparation YEPD substratum, being seeded in YEPD substratum by the above-mentioned thalline preserved, control temperature 25 DEG C, pH6.0, rotating speed 200rpm, air flow 0.2L/L.min, cultivating 12h, is (0.1-0.5) × 10 to above-mentioned bacterial classification concentration
9/ ml;
Described YEPD substratum specifically containing yeast powder 10g/L, peptone 20g/L, glucose 20g/L, and regulates pH6.5.
C, enlarged culturing step
Seed liquor after cultivating is spread cultivation in tank according to the one-level containing one-level enlarged culturing base that the inoculum size access volume of 10% (v/v) is 50L, control temperature 30 DEG C, pH6.0, rotating speed 200rpm pass into air, air flow 0.05L/L.min, cultivates 14h and reaches (0.2-0.3) × 10 to bacterial classification concentration
9/ ml;
According to 10% inoculum size, one-level after spreading cultivation described in the getting secondary that liquid is seeded to 500L that spreads cultivation spreads cultivation in tank, control temperature 30 DEG C, pH6.0, rotating speed 40rpm pass into air, air flow 0.3L/L.min, cultivate 14h and reach (0.2-0.3) × 10 to bacterial classification concentration
9/ ml;
Described one-level enlarged culturing base is lignocellulosic material enzymolysis solution, corn steep liquor (dry matter content 40%) 15g/L, KH of being diluted with water to 30wt%
2pO
41.5g/L, (NH
4)
2hPO
41.5g/L, deionized water or softening water configuration solution, packing sterilizing, mixing after cooling, and add 2g/L urea.
D, fermentation producing and ethanol step
Get above-mentioned enlarged culturing terminate after the liquid that spreads cultivation, be seeded in the fermentor tank containing fermention medium according to the inoculum size of 10% (v/v), control temperature 25 DEG C, pH5, air flow 0.5mL/L, interval ventilation, aeration time is 0.5h, and interval time is 6h, fermentation 72h; Obtain tunning;
Described fermention medium is: be diluted with water to the above-mentioned lignocellulosic material enzymolysis solution of 30wt%, corn steep liquor (dry matter content 45wt%) 7.5g/L, KH
2pO
40.5g/L, (NH
4)
2hPO
42g/L, regulates pH to be 5.0 before fermentation starts.
Embodiment 7
The present embodiment provides a kind of method utilizing fermentable lignocellulosic material producing and ethanol, fermenting experiment (with embodiment 5) is carried out for recombinant Saccharomyces cerevisiae S.cerevisiae424A (LNH-ST), the method of described fermentation producing and ethanol specifically comprises: lignocellulosic material enzymolysis is obtained the step of lignocellulosic material enzymolysis solution, the step of seed culture, the step of strain expanded culture, and the step of fermentation producing and ethanol, specifically comprise:
A, enzymolysis lignocellulosic material obtain the step of lignocellulosic material enzymolysis solution
With embodiment 5, difference is, the lignocellulosic material used is barley-straw and wheat-straw.
B, seed liquor culturing step
Preparation YEPD substratum, being seeded in YEPD substratum by the above-mentioned thalline preserved, control temperature 30 DEG C, pH6.0, rotating speed 200rpm, air flow 0.2L/L.min, cultivating 14h, is (0.2-0.3) × 10 to above-mentioned bacterial classification concentration
9/ ml;
Described YEPD substratum specifically containing yeast powder 10g/L, peptone 20g/L, glucose 20g/L, and regulates pH6.0-6.5.
C, enlarged culturing step
One-level is cultivated: the one-level enlarged culturing base of the present embodiment adopts 4% glucose enlarged culturing base: wherein containing glucose 4wt%, corn steep liquor (dry matter content 40%) 15g/L, KH2PO41.5g/L, (NH4) 2HPO41.5g/L, deionized water or softening water configuration solution, packing sterilizing, after cooling, mixing, adds 5g/L urea; The method and parameter cultivated is identical with embodiment 6;
Secondary is cultivated: identical with embodiment 6.
D, fermentation producing and ethanol step
The liquid that spreads cultivation after enlarged culturing terminates, according to the inoculum size of 10% (v/v) be seeded to volume be 30 cubic metres containing fermention medium fermentor tank in, control temperature 30 DEG C, pH6.5, air flow 0.1mL/L, interval ventilation, aeration time is 0.5h, interval time is 5h, and fermentation 72h, obtains tunning;
Micro-oxygen equipment is adopted to carry out oxygen supply in fermenting process.
Described fermention medium is: be diluted with water to the lignocellulosic material enzymolysis solution of 30wt%, corn steep liquor (dry matter content 40wt%) 7.5g/L, KH
2pO
40.5g/L, (NH
4)
2hPO
42g/L, regulates pH to be 6.0 before fermentation starts.
Embodiment 8
The present embodiment provides a kind of method utilizing fermentable lignocellulosic material producing and ethanol, fermenting experiment (with embodiment 5) is carried out for recombinant Saccharomyces cerevisiae S.cerevisiae424A (LNH-ST), the method of described fermentation producing and ethanol specifically comprises: enzymolysis lignocellulosic material obtains step, the step of seed culture, the step of strain expanded culture of lignocellulosic material enzymolysis solution, and the step of fermentation producing and ethanol, specifically comprise:
A, enzymolysis lignocellulosic material obtain the step of lignocellulosic material enzymolysis solution
With embodiment 5, difference is, the lignocellulosic material used in the present embodiment is paper, leaf and oat shell.
B, seed liquor culturing step
Preparation YEPD substratum, being seeded in YEPD substratum by the above-mentioned Candida tropicalis body preserved, control temperature 30 DEG C, pH6.0, rotating speed 200rpm, air flow 0.2L/L.min, cultivating 20h, is (0.2-0.3) × 10 to above-mentioned bacterial classification concentration
9/ ml;
Described YEPD substratum specifically containing yeast powder 10g/L, peptone 20g/L, glucose 20g/L, and regulates pH6.0-6.5.
C, enlarged culturing step
With embodiment 6;
Difference is: the one-level enlarged culturing base of the present embodiment adopts corn mash: containing corn mash 4wt%, add 3g/L urea after sterilizing.
D, fermentation producing and ethanol step
Get enlarged culturing terminate after the liquid that spreads cultivation, be seeded in the fermentor tank of 5 cubic metres containing fermention medium according to the inoculum size of 8% (v/v), control temperature 37 DEG C, pH4.8, air flow 0.2mL/L, interval ventilation, aeration time is 0.5h, and interval time is 6h;
Micro-oxygen equipment is adopted to carry out oxygen supply in fermenting process; The fermention medium used is identical with embodiment 7.
Embodiment 9
The method that the seed liquor of bacterial classification described in the present embodiment is cultivated, enlarged culturing one grade fermemtation is cultivated and parameter and seed liquor substratum, fermented liquid substratum are all identical with embodiment 7 with the selection of the liquid culture medium that spreads cultivation, its difference is only, in the process of fermentation culture, adopt the condition of continuously fermenting and cultivating, control leavening temperature 28 DEG C, pH6.0, keeps air flow 0.5mL/L to carry out fermentation 72h, detects tunning.
Detect the tunning content of above-described embodiment 5-9, see the following form.
Obviously, above-described embodiment is only for clearly example being described, and the restriction not to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without the need to also giving all embodiments.And thus the apparent change of extending out or variation be still among the protection domain of the invention.
Claims (14)
1. utilize a method for fermentable lignocellulosic material producing and ethanol, it is characterized in that, comprise the steps:
1), by described lignocellulosic material enzymolysis, lignocellulosic material enzymolysis solution is obtained;
2), by described microorganism enlarged culturing, scale-up medium is obtained;
3), described scale-up medium is seeded in described lignocellulosic material enzymolysis solution by 5-20%, control temperature of reaction 25-38 DEG C, pH5.0-7.0 carries out fermentative production, control to carry out intermittent type oxygen supply in described fermenting process, air flow 0.01-0.5mL/L.min, aeration time 10-30min, interval time is 5-6h, and fermentation obtains fermented liquid;
4), be separated described fermented liquid and obtain ethanol.
2. utilize the method for fermentable lignocellulosic material producing and ethanol according to claim 1, it is characterized in that, described step 3) in, control air air flow 0.1-0.2mL/L.min in fermenting process.
3. according to claim 1 or 2, utilize the method for fermentable lignocellulosic material producing and ethanol, it is characterized in that, described lignocellulosic material is selected from one or more in maize straw, corn cob, hardwood, cork, nutshell, grass, paper, barley-straw, wheat straw, leaf, cottonseed wadding, withy, oat shell.
4. utilize the method for fermentable lignocellulosic material producing and ethanol according to claim 3, it is characterized in that, described enzyme is selected from cellulase, hemicellulase, amylase, proteolytic enzyme, glucoamylase, lipase.
5. according to any one of claim 1-4, utilize the method for fermentable lignocellulosic material producing and ethanol, it is characterized in that, in described lignocellulosic material enzymolysis solution also containing one or more in soybean cake powder, corn-dodger powder, corn steep liquor, fish meal or yeast extract paste as nitrogenous source; Containing one or more in Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC, potassium primary phosphate, dipotassium hydrogen phosphate, Secondary ammonium phosphate as inorganic salt, and optionally containing urea and/or inhibitor.
6. according to any one of claim 1-5, utilize the method for fermentable lignocellulosic material producing and ethanol, it is characterized in that, described enlarged culturing step is in enlarged culturing base by described microbial inoculant, by at least one-level enlarged culturing step, enlarged culturing is carried out to described microorganism, in described enlarged culturing step, realize the enlarged culturing to described microorganism by the confession oxygen concn in adjustment enlarged culturing process.
7. utilize the method for fermentable lignocellulosic material producing and ethanol according to claim 6, it is characterized in that, use one-level enlarged culturing step to carry out enlarged culturing to described microorganism, comprise the steps:
Described seed liquor is spread cultivation in tank according to the inoculum size of the 5-20% one-level be seeded to containing enlarged culturing base, control temperature 25-35 DEG C, pH4-6.5, rotating speed 180-220rpm, air flow 0.08-0.5L/L.min, enlarged culturing 6-12h; Adjust rotating speed 40-100rpm, air flow 0.18-0.5L/L.min subsequently, enlarged culturing to described microorganism concn reaches (0.1-0.5) × 10
9/ mL.
8. utilize the method for fermentable lignocellulosic material producing and ethanol according to claim 6, it is characterized in that, use secondary enlarged culturing step to carry out enlarged culturing to described microorganism, comprise the steps:
1) one-level that described seed liquor is seeded to containing described enlarged culturing base according to the inoculum size of 5-20% is spread cultivation in tank, control temperature 25-35 DEG C, pH4-6.5, rotating speed 180-220rpm, air flow 0.08-0.5L/L.min, carry out enlarged culturing to described microorganism concn and reach (0.1-0.5) × 10
9/ mL;
2) by step 1) in the secondary that is seeded to containing described enlarged culturing base according to the inoculum size of 5-20% of the microorganism that spreads cultivation that obtains spread cultivation in tank, control temperature 25-35 DEG C, pH4-6.5, rotating speed 40-100rpm, air flow 0.18-0.5L/L.min, carry out enlarged culturing to described microorganism concn and reach (0.1-0.5) × 10
9/ mL.
9. utilize the method for fermentable lignocellulosic material producing and ethanol according to claim 6, it is characterized in that, use three grades of enlarged culturing steps to carry out enlarged culturing to described microorganism, comprise the steps:
1) one-level that described seed liquor is seeded to containing described enlarged culturing base according to the inoculum size of 5-20% is spread cultivation in tank, control temperature 25-35 DEG C, pH4-6.5, rotating speed 180-220rpm, air flow 0.08-0.5L/L.min, carry out enlarged culturing to described microorganism concn and reach (0.1-0.5) × 10
9/ mL;
2) by step 1) in the secondary that is seeded to containing described enlarged culturing base according to the inoculum size of 5-20% of the microorganism that spreads cultivation that obtains spread cultivation in tank, control temperature 25-35 DEG C, pH4-6.5, rotating speed 40-60rpm, air flow 0.18-0.5L/L.min, carry out enlarged culturing to described microorganism concn and reach (0.1-0.5) × 10
9/ mL;
3) by step 2) in the microorganism that spreads cultivation that obtains three grades of being seeded to containing described enlarged culturing base according to the inoculum size of 5-20% spread cultivation in tank, control temperature 25-35 DEG C, pH4-6.5, rotating speed 40-60rpm, air flow 0.01-0.2L/L.min, carry out enlarged culturing to described microorganism concn and reach (0.1-0.5) × 10
9/ mL.
10. utilize the method for fermentable lignocellulosic material producing and ethanol according to claim 6, it is characterized in that, use level Four enlarged culturing step to carry out enlarged culturing to described microorganism, comprise the steps:
1) one-level that described seed liquor is seeded to containing described enlarged culturing base according to the inoculum size of 5-20% is spread cultivation in tank, control temperature 25-35 DEG C, pH4-6.5, rotating speed 180-220rpm, air flow 0.08-0.5L/L.min, carry out enlarged culturing to described microorganism concn and reach (0.1-0.5) × 10
9/ mL;
2) by step 1) in the secondary that is seeded to containing described enlarged culturing base according to the inoculum size of 5-20% of the microorganism that spreads cultivation that obtains spread cultivation in tank, control temperature 25-35 DEG C, pH4-6.5, rotating speed 40-60rpm, air flow 0.18-0.5L/L.min, carry out enlarged culturing to described microorganism concn and reach (0.1-0.5) × 10
9/ mL;
3) by step 2) in the microorganism that spreads cultivation that obtains three grades of being seeded to containing described enlarged culturing base according to the inoculum size of 5-20% spread cultivation in tank, control temperature 25-35 DEG C, pH4-6.5, rotating speed 40-60rpm, air flow 0.01-0.2L/L.min, carry out enlarged culturing to described microorganism concn and reach (0.1-0.5) × 10
9/ mL;
4) by step 3) in the level Four that is seeded to containing described enlarged culturing base according to the inoculum size of 5-20% of the microorganism that spreads cultivation that obtains spread cultivation in tank, control temperature 25-35 DEG C, pH4-6.5, air flow 0.0005-0.02L/L.min, carry out enlarged culturing to described microorganism concn and reach (0.1-0.5) × 10
9/ mL.
11. methods utilizing fermentable lignocellulosic material producing and ethanol according to any one of claim 7-10, it is characterized in that, in described enlarged culturing process at different levels, the one-level tank that spreads cultivation is that 50L spreads cultivation tank, the secondary tank that spreads cultivation is that 500L spreads cultivation tank, three grades of tanks that spread cultivation are 5 cubes of tanks that spread cultivation, and the level Four tank that spreads cultivation is 50 cubes of tanks that spread cultivation.
12. methods utilizing fermentable lignocellulosic material producing and ethanol according to any one of claim 7-11, it is characterized in that, in the process of described enlarged culturing at different levels, described enlarged culturing base independent of each other containing one or more in corn mash, molasses, glucose, sweet sorghum stalk juice or lignocellulosic material enzymolysis solution as carbon source, containing one or more in soybean cake powder, corn-dodger powder, corn steep liquor, fish meal, yeast extract paste as nitrogenous source; And optionally containing urea, inorganic salt, amino acid, trace element and/or inhibitor.
13. methods utilizing fermentable lignocellulosic material producing and ethanol according to any one of claim 1-12, it is characterized in that, also comprised before enlarged culturing is by described microorganism and described microorganism cultivated in seed culture medium 12-24h and reach (0.1-0.5) × 10 to described microorganism concn
9the step of/ml.
14. methods utilizing fermentable lignocellulosic material producing and ethanol according to any one of claim 1-13, it is characterized in that, described microorganism is selected from Saccharomyces Cerevisiae in S accharomycescerevisiae, pachysolen tannophilus Pachysolentannophilus, pichia stipitis Pichiastipitis, shehatae candida Candidashehatae, Brettanomyces Brettanomycesnaardenensis, very thin candiyeast Candidatenuis, candida tropicalis Candidatropicalis, match ditch pichia spp Pichiasegobiensis, Bacteroides polypragmatus Bacteroidespolypragmatus, chrysanthemum Erwinia Erwiniachrysanthem, plant klebsiella spp Klebsiellaplanticola, thermophilic anaerobic ethanol bacterium Thermoanaerobacterethanolicus, spherical spiral body Spirochaetacoccoidessp., plant fermentation clostridium Clostridiumphytofermentassp., Fusarium oxysporum Fusariumoxysporum, Neuraspora crassa Neurosporacrassa, fusarium avenaceum Fusariumavenaceum, zymomonas mobilis Zymomonasmobilis, intestinal bacteria Escherichiacoli and recombinant Saccharomyces cerevisiae S.cerevisiae, restructuring zymomonas mobilis Zymomonasmobilis, recombination bacillus coli Escherichiacoli.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105755071A (en) * | 2016-04-01 | 2016-07-13 | 江苏大学 | Method for preparing fermentable sugar or bioethanol by utilizing biomass and 'one-pot method' |
| CN106906150A (en) * | 2017-03-24 | 2017-06-30 | 黑龙江中丹建业生物能源有限公司 | The expansion method of the hexose co-fermentation saccharomycete of new metabolism five |
| CN108239609A (en) * | 2016-12-05 | 2018-07-03 | 斯坦陵布什大学 | Recombination yeast and its application |
| CN109161508A (en) * | 2018-09-30 | 2019-01-08 | 上海景峰制药有限公司 | A kind of microbiological culture media and the preparation method and application thereof |
| CN111849811A (en) * | 2020-07-16 | 2020-10-30 | 山佳经典(北京)科技有限公司 | Compound bacterium system for efficiently decomposing lignocellulose materials and culture method thereof |
| CN113025506A (en) * | 2019-12-24 | 2021-06-25 | 中国科学院天津工业生物技术研究所 | Recombinant filamentous fungus for producing ethanol and construction and application thereof |
| CN113881710A (en) * | 2021-10-29 | 2022-01-04 | 安徽工程大学 | Method for co-producing biological hydrogen and microalgae grease by utilizing lignocellulose fermentation |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1456673A (en) * | 2003-05-21 | 2003-11-19 | 云南大学 | Production of alcohol by fermenting by yeast tolerant to high concentrated sugar and alcohol |
| CN1952163A (en) * | 2006-11-03 | 2007-04-25 | 天津市工业微生物研究所 | Process for preparing ethanol from stem and leaf of jerusalem artichoke |
| CN1966694A (en) * | 2006-11-15 | 2007-05-23 | 山东大学 | Process for producing alcohol by co-fermentation of glucose and xylose |
| CN101434913A (en) * | 2008-12-12 | 2009-05-20 | 华东理工大学 | Wine brewing yeast strain and method for producing ethanol by efficient stalk fermentation |
| CN102181510A (en) * | 2011-03-07 | 2011-09-14 | 合肥工业大学 | Technological process for producing ethanol through steam explosion and fermentation of trunks and branches of poplar trees |
-
2014
- 2014-05-30 CN CN201410240486.6A patent/CN105200094B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1456673A (en) * | 2003-05-21 | 2003-11-19 | 云南大学 | Production of alcohol by fermenting by yeast tolerant to high concentrated sugar and alcohol |
| CN1952163A (en) * | 2006-11-03 | 2007-04-25 | 天津市工业微生物研究所 | Process for preparing ethanol from stem and leaf of jerusalem artichoke |
| CN1966694A (en) * | 2006-11-15 | 2007-05-23 | 山东大学 | Process for producing alcohol by co-fermentation of glucose and xylose |
| CN101434913A (en) * | 2008-12-12 | 2009-05-20 | 华东理工大学 | Wine brewing yeast strain and method for producing ethanol by efficient stalk fermentation |
| CN102181510A (en) * | 2011-03-07 | 2011-09-14 | 合肥工业大学 | Technological process for producing ethanol through steam explosion and fermentation of trunks and branches of poplar trees |
Non-Patent Citations (1)
| Title |
|---|
| 张晓阳，杜风光，常春，王林风主编: "《纤维素生物质水解与应用》", 31 December 2012, 郑州大学出版社 * |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105755071A (en) * | 2016-04-01 | 2016-07-13 | 江苏大学 | Method for preparing fermentable sugar or bioethanol by utilizing biomass and 'one-pot method' |
| CN108239609A (en) * | 2016-12-05 | 2018-07-03 | 斯坦陵布什大学 | Recombination yeast and its application |
| CN108239609B (en) * | 2016-12-05 | 2021-04-20 | 斯坦陵布什大学 | Recombinant yeast and application thereof |
| CN106906150A (en) * | 2017-03-24 | 2017-06-30 | 黑龙江中丹建业生物能源有限公司 | The expansion method of the hexose co-fermentation saccharomycete of new metabolism five |
| CN109161508A (en) * | 2018-09-30 | 2019-01-08 | 上海景峰制药有限公司 | A kind of microbiological culture media and the preparation method and application thereof |
| CN113025506A (en) * | 2019-12-24 | 2021-06-25 | 中国科学院天津工业生物技术研究所 | Recombinant filamentous fungus for producing ethanol and construction and application thereof |
| CN111849811A (en) * | 2020-07-16 | 2020-10-30 | 山佳经典(北京)科技有限公司 | Compound bacterium system for efficiently decomposing lignocellulose materials and culture method thereof |
| CN111849811B (en) * | 2020-07-16 | 2022-08-26 | 山佳经典(北京)科技有限公司 | Compound bacterium system for efficiently decomposing lignocellulose materials and culture method thereof |
| CN113881710A (en) * | 2021-10-29 | 2022-01-04 | 安徽工程大学 | Method for co-producing biological hydrogen and microalgae grease by utilizing lignocellulose fermentation |
| CN113881710B (en) * | 2021-10-29 | 2023-09-22 | 安徽工程大学 | Method for co-production of biological hydrogen and microalgae grease by lignocellulose fermentation |
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