CN103614418A - Method for producing fuel ethanol through synchronous saccharification and fermentation - Google Patents
Method for producing fuel ethanol through synchronous saccharification and fermentation Download PDFInfo
- Publication number
- CN103614418A CN103614418A CN201310618899.9A CN201310618899A CN103614418A CN 103614418 A CN103614418 A CN 103614418A CN 201310618899 A CN201310618899 A CN 201310618899A CN 103614418 A CN103614418 A CN 103614418A
- Authority
- CN
- China
- Prior art keywords
- fermentation
- under
- alcohol fuel
- conditions
- medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a method for producing fuel ethanol through synchronous saccharification and fermentation. The method comprises the following steps: (1) screening out thermophilic bacterial strains of cellulase from soil samples under a haystack; (2) inoculating the thermophilic bacterial strains to a basic culture medium, cultivating for 24 hours to serve as seed liquid, inoculating 10 percent of the seed liquid to a fermentation culture medium, performing shaking cultivation for 4 days by using a shaker, and concentrating to obtain an enzyme preparation; (3) placing steam exploded corn straws into hot water at the temperature of 60+/-1.0 DEG C and extracting; (4) adding the enzyme preparation obtained in the step (2) into the corn straw extracting liquid obtained in the step (3) and performing pre-enzymolysis at 60+/-1.0 DEG C for 2 hours; and (5) cooling the enzymatic hydrolysate obtained in the step (4) to 35+/-1.0 DEG C, inoculating 7 percent of Saccharomyces cerevisiae, synchronously performing enzymolysis and fermentation at the same temperature of 35+/-1.0 DEG C to obtain the fuel ethanol. By the method, the yield of fiber fuel ethanol can be increased effectively.
Description
Technical field
The invention belongs to a kind of technique of utilizing fermentation or using the method synthesising target compound of enzyme, particularly a kind of simultaneous saccharification and fermentation is produced the method for alcohol fuel.
Background technology
With vegetable fibre producing fuel ethyl alcohol by ferment, it is a focus of paying close attention to both at home and abroad at present.There are very much in recent years a lot of scholars to report the bacterial strain of High Cellulase Production, comprise: mould, bacterium and fungi.Wherein the cellulase of originated from fungus is the enzyme resource of developing at present the earliest.The research of at present both at home and abroad preparing ethanol with cellulose series biomass raw material has obtained many progress, but the too high bottleneck that remains its scale operation and apply of the production cost of fibre fuel ethanol.Its chief reason has: when monotechnics cannot be realized Mierocrystalline cellulose, hemicellulose and xylogen three component, utilize; Enzymolysis process cellulase consumption is large, and the cost of alcohol production is high.
At present, hundred million tons of the annual agriculture residues 6-7 such as agricultural crop straw that produce in China whole nation, except as feed, fertilizer, fuel etc., still hundred million tons of remaining 3-4, as waste, if for the production of ethanol, can produce 50,000,000 tons of bio-ethanols by calculating.But undeniable, along with improving constantly of state of the art, the bio-ethanol that the lignocellulose of take is raw material will bring huge economic benefit as society undoubtedly.The main component of maize straw is Mierocrystalline cellulose, hemicellulose and xylogen, is directly used in cellulase hydrolysis, and its enzymatic hydrolyzation is low, utilizes steam explosion technology well to solve this problem, to be solved but the problem of enzymolysis high cost still has.The method of the ethanol of cellulose fermentation production at present has direct fermentation, indirect fermentation method, simultaneous saccharification and fermentation method (SSF), non-isothermal simultaneous saccharification and fermentation method (NSSF), immobilized cell fermentation method etc.The most frequently used method is SSF and NSSF method at present, but these two kinds of methods also exist some shortcomings, and as inharmonious in saccharification temperature and leavening temperature in SSF method, certain density ethanol has restraining effect to enzyme.NSSF method need to increase a hydrolysis tower outside fermentation equipment, and the fluid containing yeast cell does not circulate between hydrolysis tower and fermentor tank, has increased the complicated of flow process.But no matter be any mode of action, finding efficient cellulase reduction enzymolysis cost is the key of alcohol fuel suitability for industrialized production.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, provide a kind of simultaneous saccharification and fermentation to produce the method for alcohol fuel, the method can effectively improve the yield of fibre fuel ethanol.
The scheme that the present invention solve the technical problem is: a kind of simultaneous saccharification and fermentation is produced the method for alcohol fuel, it is characterized in that: comprise the steps,
(1) get the pedotheque under haystack, spread plate substratum after dilution, temperature is to cultivate 5-7 days under 60 ± 1.0 ℃ of conditions, select the dull and stereotyped upper heat-resisting bacterial strain list bacterium colony that occurs hydrolysis transparent circle, single bacterium colony is forwarded to slant medium, under 60 ± 1.0 ℃ of conditions, cultivates the liquid nutrient medium of inoculating afterwards containing filter paper fibre for 5-7 days, shaking culture 6-8 days under 170r/min, 60 ± 1.0 ℃ of conditions, measure filter paper enzymatic hydrolyzation, filter out the bacterial strain that filter paper enzymatic hydrolyzation is high; The bacterial strain that screening obtains, after activation culture on plate culture medium, is seeded in minimum medium, under 60 ℃ ± 1.0 ℃ conditions, under 170r/min condition, after shaking culture 6-8 days, measures yield of cellulase, filters out the thermophilus strain of cellulase;
(2) thermophilus strain of step (1) gained is seeded in minimum medium, under 60 ℃ ± 1.0 ℃ conditions, under 170r/min condition, shaking culture 24 hours is as seed liquor, the seed liquor of inoculation 10% is to fermention medium, 170r/min, 60 ± 1.0 ℃ of shaking table shaking culture 4 days, obtain zymin after concentrated;
(3) the quick-fried maize straw of vapour being placed in to temperature is the hot water of 60 ± 1.0 ℃, is incubated lixiviate in 1 hour under this temperature condition;
(4) zymin of step (2) gained is added in the maize straw vat liquor of step (3) gained, under the condition of 60 ± 1.0 ℃, pre-enzymolysis is 2 hours;
(5) enzymolysis solution step (4) being obtained is cooled to 35 ± 1.0 ℃, inoculates afterwards 7% yeast saccharomyces cerevisiae, and under 35 ± 1.0 ℃ of conditions, the synchronous enzymatic hydrolysis and fermentation of equality of temperature obtains alcohol fuel.
As further technical scheme, in described step (1) and step (2), the composition of minimum medium is: CMC-Na2%; Peptone 0.5%; K2HP040.2%; MgSO4.7H2O0.05%, all the other are water.
As further technical scheme, the composition of the fermention medium in described step (2) is: CMC-Na2%, K
2hP0
40.2%, MgSO
4.7H
2o0.05%, peptone 0.1%, CaCl
20.002%, FeSO
40.0004%, MnSO
40.002%, all the other are water; The pH of fermention medium is 7.0.
As technical scheme further, the zymin in described step (2) is concentrated obtaining by the following method:
A) under normal temperature condition, the centrifugal tunning of 8000r/min, collection supernatant liquor is crude enzyme liquid;
B) utilize saturated ammonium sulphate method to concentrate crude enzyme liquid, and to adopt concentration be 10 mM/ls, the phosphoric acid buffer dialysis of pH7.0 12 hours, obtains concentrated crude enzyme liquid;
C) gel chromatography that crude enzyme liquid carries out different pore size, to obtain endoglucanase, cellobiase and glucoside zymoprotein, obtains enzyme liquid;
D) utilize ion exchange chromatography to be further purified enzyme liquid, obtain pure zymin.
As technical scheme further, in described step (4), the addition of zymin is 30U/g Mierocrystalline cellulose.
As technical scheme further, in described step (4), add 10% tensio-active agent.
As technical scheme further, by tween 80 and PEG20000, the volume ratio with 2:3 mixes described tensio-active agent.
The present invention has overcome cellulose fermentations such as utilizing at present maize straw and has produced the problem that alcohol fuel technique cellulase preparation consumption is large, production cost is high, can effectively reduce the usage quantity of cellulase preparation, improves the yield of alcohol fuel.
Embodiment
Embodiment 1
Simultaneous saccharification and fermentation is produced a method for alcohol fuel, comprises the steps,
(1) get the pedotheque under haystack, spread plate substratum after dilution, temperature is to cultivate 5-7 days under 60 ± 1.0 ℃ of conditions, select the dull and stereotyped upper heat-resisting bacterial strain list bacterium colony that occurs hydrolysis transparent circle, single bacterium colony is forwarded to slant medium, under 60 ± 1.0 ℃ of conditions, cultivate the liquid nutrient medium of inoculating afterwards containing filter paper fibre for 5-7 days, 170r/min, shaking culture 6-8 days under 60 ± 1.0 ℃ of conditions, measure filter paper enzymatic hydrolyzation, filter out the bacterial strain that filter paper enzymatic hydrolyzation is high, so-called filter paper enzymatic hydrolyzation is high selects the wherein relatively high bacterial strain of filter paper enzymatic hydrolyzation after referring to the several samples of test, the bacterial strain that screening obtains is after activation culture on plate culture medium, be seeded in minimum medium, under 60 ℃ ± 1.0 ℃ conditions, under 170r/min condition, after shaking culture 6-8 days, measure yield of cellulase, filter out the thermophilus strain of cellulase, what after measuring, yield of cellulase was relatively high is the thermophilus strain of the cellulase of needs,
(2) thermophilus strain of step (1) gained is seeded in minimum medium, under 60 ℃ ± 1.0 ℃ conditions, under 170r/min condition, shaking culture 24 hours is as seed liquor, the seed liquor of inoculation 10% is to fermention medium, 170r/min, 60 ± 1.0 ℃ of shaking table shaking culture 4 days, obtain zymin after concentrated;
(3) the quick-fried maize straw of vapour being placed in to temperature is the hot water of 60 ± 1.0 ℃, is incubated lixiviate in 1 hour under this temperature condition;
(4) zymin of step (2) gained is added in the maize straw vat liquor of step (3) gained, the addition of zymin is 30U/g Mierocrystalline cellulose, add again after 10% tensio-active agent under the condition of 60 ± 1.0 ℃ pre-enzymolysis 2 hours, by tween 80 and PEG20000, the volume ratio with 2:3 mixes tensio-active agent, can certainly use other tensio-active agent, as long as can play similar effect;
(5) enzymolysis solution step (4) being obtained is cooled to 35 ± 1.0 ℃, inoculates afterwards 7% yeast saccharomyces cerevisiae, and under 35 ± 1.0 ℃ of conditions, the synchronous enzymatic hydrolysis and fermentation of equality of temperature obtains alcohol fuel.
In described step (1) and step (2), the composition of minimum medium is: CMC-Na2%; Peptone 0.5%; K2HP040.2%; MgSO4.7H2O0.05%, all the other are water.
The composition of the fermention medium in described step (2) is: CMC-Na2%, K
2hPO
40.2%, MgSO
4.7H
2o0.05%, peptone 0.1%, CaCl
20.002%, FeSO
40.0004%, MnSO
40.002%, all the other are water; The pH of fermention medium is 7.0.
Zymin in described step (2) is concentrated obtaining by the following method:
A) under normal temperature condition, the centrifugal tunning of 8000r/min, collection supernatant liquor is crude enzyme liquid;
B) utilize saturated ammonium sulphate method to concentrate crude enzyme liquid, and to adopt concentration be 10 mM/ls, the phosphoric acid buffer dialysis of pH7.0 12 hours, obtains concentrated crude enzyme liquid;
C) gel chromatography that crude enzyme liquid carries out different pore size, to obtain endoglucanase, cellobiase and glucoside zymoprotein, obtains enzyme liquid;
D) utilize ion exchange chromatography to be further purified enzyme liquid, obtain pure zymin.
Compare with cellulase preparation common on market, reaching under identical hydrolysis result condition, the consumption of zymin has reduced more than 20%.With original pre-enzymolysis with synchronize enzymatic hydrolysis and fermentation technique and compare, use method of the present invention, the output of ethanol can improve 40% left and right.
Claims (7)
1. simultaneous saccharification and fermentation is produced a method for alcohol fuel, it is characterized in that: comprise the steps,
(1) get the pedotheque under haystack, spread plate substratum after dilution, temperature is to cultivate 5-7 days under 60 ± 1.0 ℃ of conditions, select the dull and stereotyped upper heat-resisting bacterial strain list bacterium colony that occurs hydrolysis transparent circle, single bacterium colony is forwarded to slant medium, under 60 ± 1.0 ℃ of conditions, cultivates the liquid nutrient medium of inoculating afterwards containing filter paper fibre for 5-7 days, shaking culture 6-8 days under 170r/min, 60 ± 1.0 ℃ of conditions, measure filter paper enzymatic hydrolyzation, filter out the bacterial strain that filter paper enzymatic hydrolyzation is high; The bacterial strain that screening obtains, after activation culture on plate culture medium, is seeded in minimum medium, under 60 ℃ ± 1.0 ℃ conditions, under 170r/min condition, after shaking culture 6-8 days, measures yield of cellulase, filters out the thermophilus strain of cellulase;
(2) thermophilus strain of step (1) gained is seeded in minimum medium, under 60 ℃ ± 1.0 ℃ conditions, under 170r/min condition, shaking culture 24 hours is as seed liquor, the seed liquor of inoculation 10% is to fermention medium, 170r/min, 60 ± 1.0 ℃ of shaking table shaking culture 4 days, obtain zymin after concentrated;
(3) the quick-fried maize straw of vapour being placed in to temperature is the hot water of 60 ± 1.0 ℃, is incubated lixiviate in 1 hour under this temperature condition;
(4) zymin of step (2) gained is added in the maize straw vat liquor of step (3) gained, under the condition of 60 ± 1.0 ℃, pre-enzymolysis is 2 hours;
(5) enzymolysis solution step (4) being obtained is cooled to 35 ± 1.0 ℃, inoculates afterwards 7% yeast saccharomyces cerevisiae, and under 35 ± 1.0 ℃ of conditions, the synchronous enzymatic hydrolysis and fermentation of equality of temperature obtains alcohol fuel.
2. the method that a kind of simultaneous saccharification and fermentation as claimed in claim 1 is produced alcohol fuel, is characterized in that: in described step (1) and step (2), the composition of minimum medium is: CMC-Na2%; Peptone 0.5%; K2HP040.2%; MgSO4.7H2O0.05%, all the other are water.
3. the method that a kind of simultaneous saccharification and fermentation as claimed in claim 1 or 2 is produced alcohol fuel, is characterized in that: the composition of the fermention medium in described step (2) is: CMC-Na2%, K
2hP0
40.2%, MgSO
4.7H
2o0.05%, peptone 0.1%, CaCl
20.002%, FeSO
40.0004%, MnSO
40.002%, all the other are water; The pH of fermention medium is 7.0.
4. the method that a kind of simultaneous saccharification and fermentation as claimed in claim 1 is produced alcohol fuel, is characterized in that: the zymin in described step (2) is concentrated obtaining by the following method:
A) under normal temperature condition, the centrifugal tunning of 8000r/min, collection supernatant liquor is crude enzyme liquid;
B) utilize saturated ammonium sulphate method to concentrate crude enzyme liquid, and to adopt concentration be 10 mM/ls, the phosphoric acid buffer dialysis of pH7.0 12 hours, obtains concentrated crude enzyme liquid;
C) gel chromatography that crude enzyme liquid carries out different pore size, to obtain endoglucanase, cellobiase and glucoside zymoprotein, obtains enzyme liquid;
D) utilize ion exchange chromatography to be further purified enzyme liquid, obtain pure zymin.
5. the method that a kind of simultaneous saccharification and fermentation as claimed in claim 1 is produced alcohol fuel, is characterized in that: in described step (4), the addition of zymin is 30U/g Mierocrystalline cellulose.
6. the method that a kind of simultaneous saccharification and fermentation as claimed in claim 1 is produced alcohol fuel, is characterized in that: in described step (4), add 10% tensio-active agent.
7. a kind of simultaneous saccharification and fermentation as claimed in claim 6 is produced the method for alcohol fuel, it is characterized in that: by tween 80 and PEG20000, the volume ratio with 2:3 mixes described tensio-active agent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310618899.9A CN103614418A (en) | 2013-11-27 | 2013-11-27 | Method for producing fuel ethanol through synchronous saccharification and fermentation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310618899.9A CN103614418A (en) | 2013-11-27 | 2013-11-27 | Method for producing fuel ethanol through synchronous saccharification and fermentation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103614418A true CN103614418A (en) | 2014-03-05 |
Family
ID=50165133
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310618899.9A Pending CN103614418A (en) | 2013-11-27 | 2013-11-27 | Method for producing fuel ethanol through synchronous saccharification and fermentation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103614418A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104561127A (en) * | 2014-12-30 | 2015-04-29 | 松原光禾能源有限公司 | Comprehensive utilization method of agricultural straw |
| CN105886553A (en) * | 2015-01-26 | 2016-08-24 | 中粮营养健康研究院有限公司 | Synchronous diastatic fermentation process of cellulose-containing raw materials |
| CN106011182A (en) * | 2016-08-02 | 2016-10-12 | 四川力久云智知识产权运营有限公司 | Method for preparation of fuel ethanol from waste paper |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1824782A (en) * | 2005-12-29 | 2006-08-30 | 陕西科技大学 | Method for preprocessing biomass |
| CN101868548A (en) * | 2006-05-01 | 2010-10-20 | 达特默斯大学托管会 | Lignocellulose biomass is converted into the alcoholic acid thermophilic organisms |
| CN102174594A (en) * | 2011-03-16 | 2011-09-07 | 中国科学院广州能源研究所 | Efficient enzyme hydrolysis method of lignocellulose biomass |
| CN102174410A (en) * | 2009-10-22 | 2011-09-07 | 郑州大学 | Stain for producing heat-resisting and acid-resisting cellulase with high yield and new method for fermenting cellulosic ethanol |
-
2013
- 2013-11-27 CN CN201310618899.9A patent/CN103614418A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1824782A (en) * | 2005-12-29 | 2006-08-30 | 陕西科技大学 | Method for preprocessing biomass |
| CN101868548A (en) * | 2006-05-01 | 2010-10-20 | 达特默斯大学托管会 | Lignocellulose biomass is converted into the alcoholic acid thermophilic organisms |
| CN102174410A (en) * | 2009-10-22 | 2011-09-07 | 郑州大学 | Stain for producing heat-resisting and acid-resisting cellulase with high yield and new method for fermenting cellulosic ethanol |
| CN102174594A (en) * | 2011-03-16 | 2011-09-07 | 中国科学院广州能源研究所 | Efficient enzyme hydrolysis method of lignocellulose biomass |
Non-Patent Citations (1)
| Title |
|---|
| 杨敏: "玉米秸秆制取生物乙醇关键技术的研究", 《中国优秀硕士学位论文全文数据库》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104561127A (en) * | 2014-12-30 | 2015-04-29 | 松原光禾能源有限公司 | Comprehensive utilization method of agricultural straw |
| CN105886553A (en) * | 2015-01-26 | 2016-08-24 | 中粮营养健康研究院有限公司 | Synchronous diastatic fermentation process of cellulose-containing raw materials |
| CN106011182A (en) * | 2016-08-02 | 2016-10-12 | 四川力久云智知识产权运营有限公司 | Method for preparation of fuel ethanol from waste paper |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Hu et al. | Thermotolerant Kluyveromyces marxianus and Saccharomyces cerevisiae strains representing potentials for bioethanol production from Jerusalem artichoke by consolidated bioprocessing | |
| CN102352381B (en) | Method using xylose production waste liquid to produce acetone and butanol | |
| Zhao et al. | Simultaneous saccharification and fermentation of fungal pretreated cornstalk for hydrogen production using Thermoanaerobacterium thermosaccharolyticum W16 | |
| CN102174433B (en) | Clostridium beijerinckii with high stress resistance and application thereof | |
| CN101824395B (en) | Method for culturing fermentation seed liquid by adopting solid straws as carbon source | |
| JP5711873B2 (en) | Simultaneous saccharification and fermentation of cellulosic materials | |
| CN101608192B (en) | Method for producing succinic acid employing corn cob | |
| CN103409382B (en) | A kind of strengthen the technology of lignin degradation in Phanerochaete chrysosporium solid fermentation | |
| CN102168113B (en) | Method for producing ethanol by using straw lignocellulose raw materials | |
| CN110055195A (en) | The microbial strains BF-1801 of one high-efficiency degradation cellulose | |
| CN109797121A (en) | The microbial strains LM-1801 of one plant of degraded cellulose and its application | |
| CN103614418A (en) | Method for producing fuel ethanol through synchronous saccharification and fermentation | |
| CN103614448B (en) | Method for preparing bioethanol by taking sodium alginate or algae as active ingredients | |
| CN103409383A (en) | Method used for accelerating lignin degradation in Aspergillus oryzae solid state fermentation | |
| Tang et al. | Integrated process of starch ethanol and cellulosic lactic acid for ethanol and lactic acid production | |
| Wen et al. | Optimization of liquid fermentation of microbial consortium WSD-5 followed by saccharification and acidification of wheat straw | |
| CN101942483A (en) | Method for preparing butanol by fermenting pentose with reinforced strains | |
| CN104073526B (en) | A kind of method utilizing lignocellulose raw material to produce acetone/butanol | |
| CN103103221B (en) | Method for converting cellulose into ethanol by use of mixed culture of genetically recombinant yeast | |
| CN102286572A (en) | Method for preparing fermentable sugar solution from straws | |
| CN105062928A (en) | Zymomonas mobilis resistant to high-concentration acetic acid and high-concentration furfural and application thereof | |
| CN108410746A (en) | It is a kind of can efficient-decomposition, convert cellulose saccharomyces cerevisiae and stalk fermentation method | |
| CN104450598A (en) | Cultivation method for saccharomyces cerevisiae | |
| CN101659949B (en) | Preparation method of liquid cellulase | |
| CN104263680A (en) | Thermoanaerobacter ethanolicus and method for producing ethanol by using same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20140305 |