CN101333553A - Method for producing fuel ethanol by mixed fermentation cellulosic - Google Patents
Method for producing fuel ethanol by mixed fermentation cellulosic Download PDFInfo
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- CN101333553A CN101333553A CNA2008100208428A CN200810020842A CN101333553A CN 101333553 A CN101333553 A CN 101333553A CN A2008100208428 A CNA2008100208428 A CN A2008100208428A CN 200810020842 A CN200810020842 A CN 200810020842A CN 101333553 A CN101333553 A CN 101333553A
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- fuel ethanol
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- lignocellulose
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- 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
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- 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
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Abstract
Disclosed is a method for producing fuel ethanol by mixed bacteria fermentation cellulose, which adopts lignocellulose resources to produce the fuel ethanol, and the method comprises: a. separating out Escherichia coli that can decompose cellulose from herbivore feces; implementing mutation breeding to the Escherichia coli by ultraviolet dose with a certain lethality rate, and selecting Escherichia coli mutants of EM1 and EM2 with enzyme-producing activity; and b. carrying out the mixed bacteria fermentation to the lignocellulose treated by crushing and dilute sulfuric acid for two to four days at a certain temperature by taking the EM1 or the EM2 and a Saccharomyces cerevisiae of S1 or S2 separated from sweet distillers yeast, and distilling off an ethanol solution after filtration. The method solves the process of fuel ethanol production by cellulose mixed bacteria fermentation and other related problems, so as to reduce the production cost, improve the technique for fully and reasonably applying resources in China, increase the productivity, and reduce the relying on international fossil energy.
Description
Technical field
The present invention is a kind of method that adopts the lignocellulose resource to produce alcohol fuel, belongs to the technical field that renewable energy source is made.
Background technology
Along with world industry process and modern develop rapidly, variety of issue can appear inevitably, and energy resource supply is one of wherein the most serious problem.Fossil energies such as oil, coal, Sweet natural gas are to satisfy the main resource of present stage energy demand, but the fossil energy resource is limited and non-renewable, it is predicted, the production of crude oil will drop to 5,000,000,000 barrels by 25,000,000,000 barrels of present every year to the year two thousand fifty, and coal and Sweet natural gas also can be developed with nothing left in the limited time.Therefore, increasing in the global range for the interest and the research dynamics of the new alternative energy of exploitation.Sun power, wind energy and biomass energy all are inexhaustible, nexhaustible renewable energy source resources, so be the most important thing of research and development.In recent years, the research of bioenergy and application had obtained huge progress
[1-3], wherein bio-ethanol has become the energy of a kind of alternative oil, environmental protection.But adopting starchiness cereal or molasses with traditional method is fermenting raw materials, and the alcoholic acid cost price is the twice of gasoline, and consumes a large amount of grain, competes mutually with human existence.On the other hand, there is abundant lignocellulose resource on the earth, as the fibrous residue in wheat straw, maize straw, corn cob, soybean residue, bagasse, slurrying and paper mill, sawdust, forestry waste etc.
[4], only the crop stalk in China every year is with regard to about 800,000,000 tons more than, and such material removes and is used for weaving on a small quantity, industries such as papermaking and feed processing, major part goes out of use or burns, and has both caused the wasting of resources, causes environmental pollution again
[5]If industrial and agricultural production depleted Mierocrystalline cellulose can be fermented, both can widen feed and human food prods's new source, simultaneously again can processing of waste, environment purification, protection is ecological.
Therefore, consider, exist in a large number and the research of the especially various waste cellulose resource of reproducible natural cellulose fermentative production of ethanol is extremely important with the earth from the mankind's long-term interest.Many in the world developed countries carry out the research of cellulose series biomass fermentative production of ethanol just energetically, all have every year hundreds of pieces of research papers and patent to deliver
[6-9]Most representative is the Ingram team of the upright university of Florida, USA, and they are doing a large amount of work aspect Mierocrystalline cellulose and the xylan fermentation
[10-11], made up the colibacillus engineering of fermentation xylan, gram-positive microorganism engineering bacteria, exogenous origin gene integrator chromosome engineering bacterium etc.Under optimized conditions, the bacillus coli gene engineering bacteria ferments in the every liter of fermented liquid that contains 10% glucose and 8% wood sugar and can obtain 54.4 and 41.6 gram ethanol respectively, and nearly 20 patents have successively been applied in correlative study.
The Chinese government has also produced cellulose fermentation ethanol and has introduced national development plan.As far back as in September, 2000 State Council's official approval develop the alcohol fuel pilot at home, and will become alcohol fuel to list 863 project guides in by the lignocellulose bio-transformation, now obtained some preliminary achievements.Independent development be the technology of raw material production alcohol fuel with sweet sorghum stem stalk, and carried out plantation and the producing fuel ethyl alcohol by ferment pilot of sweet sorghum on Heilungkiang and other places, formed in enterprises such as Anhui Feng Yuan and produced 600 tons test throughput per year.But generally speaking, this class research is also at the early-stage, and it is few in number directly to produce the alcoholic acid report with biomass such as genetic engineering bacterium fermented cellulose, xylans
[2,4,6,12,13], fermentation costs is also generally higher.In fossil energy worsening shortages, today that energy situation is serious day by day, the research of going into overdrive to carry out the cellulose series biomass fermentative production of ethanol is extremely important.
[1]J.Lee，Journal of Biotechnology，1997，56：1-24.
[2] Qu Yinbo, Gao Peiji. food and fermentation industries, 1993,19 (6): 62-65.
[3]N.Nagie，K.Ibsen，et al.，Applied Biochemistry and Biotechnology，1999，599-607.
[4] Zhang Jiquan, Wang Ruiming, Sun Yuying. brewing science and technology, 2003, (1): 39-41.
[5]L.G.Ljungdahl，K.E.Eriksson，Adv.Microb.Ecol.，1985，(5)：237-299.
[6] S.D.Zhu, Y.X.Wu, et al., chemistry and biotechnology, 2003,20:8-11.
[7]B.S.Dien，M.A.Cotta，et al.，Applied Microbiology and Biotechnology，2003，63：258-266.
[8]Y.Sun，J.Y.Cheng，Bioresource Technology，2002，83：1-11.
[9]M.Galbe，etal.，Applied Microbiology and Biotechnology，2002，59：618-628.
[10]B.E.Wood，Ingram，et al.，WO 2007005646A2，2007.
[11]Ingram，etal.，US 7192772，2007
[12] J.Liu, H.Z.Chen, et al., industrial microorganism, 2001,31 (2): 36-37.
[13] Feng Yujie, Li Dongmei, Chinese invention patent application, application number: 200610010183.
Summary of the invention
Technical problem: main purpose of the present invention provides the method that a kind of mixed fungus fermentation cellulose series biomass is produced alcohol fuel, solve the Mierocrystalline cellulose mixed fungus fermentation and produce the technology and the related problems of alcohol fuel, in the hope of reducing production costs, improve the technology of China to the abundant rational Application of resource, improve production capacity, reduce dependence international fossil energy.
Technical scheme: in view of this, we isolate cellulose-decomposing bacterium 14 strains from the ight soil of Red Hill forest animal garden, Nanjing herbivore and soil sample, to No. 1 bacterial strain wherein after the evaluation---intestinal bacteria (E.coli) are used after the ultraviolet mutagenesis breeding, obtained the intestinal bacteria mutant strain (EM) of high inulinase-producing activity, make rice straw that itself and yeast saccharomyces cerevisiae mixed fungus fermentation handle with simple chopping method etc., concentration of ethanol is main relevant with colibacillary inulinase-producing activity in the fermented liquid, and concentration of ethanol can reach 7.65%.
Mierocrystalline cellulose be by β-D-glucose structural unit with β-1, the straight chain polymer that the 4-glycosidic bond is formed by connecting, molecular weight can reach hundreds thousand of even up to a million, is the fine pencil attitude of infinitesimal, has very strong crystallinity, water insoluble and common organic solvents.Cellulose series biomass is organic resource the abundantest on the earth, and in timber, branch, the remaining wood fragments of wood working and wood sawdust, content of cellulose is generally 40%～60% (butt meter), and stem leaves of plants also contains a large amount of Mierocrystalline celluloses.Lignocellulose can not directly utilize for microorganism cells, need be absorbed by microorganism by the degraded of cellulase again.
With dilute sulphuric acid etc. lignocellulose is carried out pre-treatment before the fermentation, can destroy its crystalline texture, improve the associativity and the catalytic hydrolysis speed of enzyme; Cellulase, hemicellulase that microbes producing cellulase EM produces can make the fibrous material hydrolysis and saccharification be transformed into glucose; Yeast saccharomyces cerevisiae utilizes the glucose anaerobically fermenting to generate alcohol, discharges heat energy simultaneously.
This method is specially:
A. from herbivore ight soil, isolate the cellulolytic intestinal bacteria of energy; With lethality rate be 20%～90% ultraviolet dosage to above-mentioned intestinal bacteria selection by mutation, select intestinal bacteria mutant strain EM1 and EM2 with higher inulinase-producing activity;
B. with EM1 or EM2 and from koji isolating Saccharomyces Cerevisiae in S 1 or S2 at a certain temperature mixed fungus fermentation steam ethanolic soln after the filtration through pulverizing and dilute sulphuric acid was handled lignocellulose 2-4 days.
Leavening temperature is 20-40 ℃; Lignocellulose is one or more the mixing in rice straw, wheat stalk, maize straw, barley stalk, wood chip, other cellulose axis, the leaf.
Beneficial effect: along with the minimizing gradually of fossil resource, crude oil price climbs up and up, especially at the beginning of 2007 to 2008 7 months, international forward crude oil price increase more than 1 times, it is urgent day by day therefore to develop new substitute energy.On the other hand, world's cellulose family renewable resources is abundant unusually, the just limited portion of having utilized wherein, and most of cellulose resource is wasted, and also causes serious environmental to pollute simultaneously.After the technology industrialization of the present invention, can more make full use of cellulose resource; The alcohol that is produced can be used as the substitute energy of vapour, diesel oil, reduces the dependence to fossil energy; The fermented liquid residue contains a large amount of yeast, and it has higher protein content, can be used as the eutrophic animal feed.
Embodiment
A. from herbivore ight soil, isolate the cellulolytic intestinal bacteria of energy; With the ultraviolet dosage of lethality rate 20%～90% to above-mentioned intestinal bacteria selection by mutation, culture Mierocrystalline cellulose after the mutagenesis---Congo red substratum carries out screening just, the higher bacterial strain of the inulinase-producing activity that primary dcreening operation obtains sieves again with producing the enzyme substratum, filters out inulinase-producing activity high mutant strain EM1 and EM2.
B. with EM1 or EM2 and from koji isolating Saccharomyces Cerevisiae in S 1 or S2 at a certain temperature mixed fungus fermentation steam ethanolic soln after the filtration through pulverizing and dilute sulphuric acid was handled lignocellulose 2-4 days.Alcohol concn reaches as high as 7.65% in the fermented liquid.Leavening temperature is 20-40 ℃; Lignocellulose is one or more the mixing in rice straw, wheat stalk, maize straw, wheat stalk, wood chip, other cellulose axis, the leaf.
Embodiment
The first step: get 6 φ 9cm culture dish, add the E.coli bacteria suspension of 5ml suitable concn in every ware, the irradiation dose irradiation bacteria suspension of 20%～90% lethality rate is selected at the 30cm place under the 20W ultraviolet lamp.
Second step: extension rate is respectively 10
4, 10
5, 10
6, 10
7Through the bacterium liquid of mutagenesis, get 0.1ml respectively and be inoculated in the Congo red substratum of Mierocrystalline cellulose, coating evenly, place 30 ℃ to cultivate 4d after, the separation of ruling on the Congo red substratum of Mierocrystalline cellulose of the bacterial strain of transparent hydrolysis circle is arranged around selecting, obtain pure culture.With the bacterial strain dibbling behind the purifying to the Congo red substratum of Mierocrystalline cellulose 30 ℃ cultivate 3d, carry out primary dcreening operation according to the size of hydrolysis loop diameter and colony diameter ratio.
The 3rd step: according to the primary dcreening operation result, pick out hydrolysis circle and the bigger bacterial strain of colony diameter ratio, insert and produce the enzyme liquid nutrient medium, cultivate 3d, centrifugal (3500r/min for 30 ℃, 10min), last clear enzyme solution is suitably got after the dilution in the CMC solution that 1mL is added to 2mL1%, and constant temperature 20min in 50 ℃ of water-baths takes out immediately and places boiling water to keep 5min, make enzyme deactivation, obtain saccharification liquid.Get saccharification liquid 1mL, add DNS reagent 3mL, the 5min that in boiling water, develops the color, cooling back adding distil water 21mL shakes up.On spectrophotometer, measure OD value in λ 530nm place, and with the corresponding OD value comparison of starting strain, obtain good dissociant EM1 of 2 strains and EM2, its inulinase-producing activity is respectively 15.8,12.5 times of starting strain.
The 4th step: from the inclined-plane, get colibacillary good dissociant EM1 and EM2 respectively, and from koji isolating Saccharomyces Cerevisiae in S 1 and S2 bacterial strain with strong fermentation capacity, inserting liquid culture respectively activates based on 30 ℃ of cultivation 16h, with the physiological saline dilution, be adjusted to 10 then with blood counting chamber
8Cell/ml.
The 5th step: take by weighing the straw powder 10.0g of equilibrium water content, add 1.0% dilute sulphuric acid by solid-to-liquid ratio 1: 10, in 121 ℃ of insulation 2h down, the cooling back is with 30% NH
4HCO
3Regulate pH4.0～5.0, divide and be filled in the Erlenmeyer flask of 4 250ml, warm sterilization 20min postcooling under 121 ℃, in the aseptic technique case, EM1 and S1 respectively are inoculated in the head pin by the amount of 10% volume, EM1 and S2 are inserted No. two bottles, EM2 and S1, and EM2 and S2 insert respectively in No. 3 bottles, No. 4 bottles put into 30 ℃ of constant incubators 72h that ferments.Take out each filtering fermentation liquor, with filtrate distillation, and with gas Chromatographic Determination distillate component.Ethanol content is listed in the table 1 in each fermentation flask, and ethanol content reaches as high as 7.65%.
Alcoholic acid content in table 1 straw cellulose mixed fungus fermentation liquid and the distillate
| Matrass number | Insert bacterial classification | The quality of fermented liquid (g) | The quality of distillate (g) | Alcoholic acid percentage composition (%) in the distillate | Alcoholic acid quality (g) in the distillate | Alcoholic acid content (%) in the fermented liquid |
| 1 | EM1+S1 | 89.6 | 40.2 | 14.39 | 5.78 | 6.45 |
| 2 | EM1+S2 | 120.5 | 42.5 | 21.7 | 9.22 | 7.65 |
| 3 | EM2+S1 | 123.9 | 70 | 0.70 | 0.49 | 0.4 |
| 4 | EM2+S2 | 127.6 | 56.2 | 0.07 | 0.04 | 0.03 |
Claims (3)
1. the method for a producing fuel ethanol by mixed fermentation cellulosic is characterized in that this method is:
A. from herbivore ight soil, isolate the cellulolytic intestinal bacteria of energy; To above-mentioned intestinal bacteria selection by mutation, select intestinal bacteria mutant strain EM1 and EM2 with the ultraviolet dosage of certain lethality rate with high inulinase-producing activity;
B. with EM1 or EM2 and from koji isolating Saccharomyces Cerevisiae in S 1 or S2 at a certain temperature mixed fungus fermentation steam ethanolic soln after the filtration through pulverizing and dilute sulphuric acid was handled lignocellulose 2-4 days.
2. the method for producing fuel ethanol by mixed fermentation cellulosic according to claim 1 is characterized in that described certain lethality rate is 20%～90%.
3. the method for a kind of producing fuel ethanol by mixed fermentation cellulosic according to claim 1 is characterized in that lignocellulose is one or more the mixing in rice straw, wheat stalk, maize straw, barley stalk, wood chip, other cellulose axis, the leaf.
The method of 4 a kind of producing fuel ethanol by mixed fermentation cellulosic according to claim 1 is characterized in that leavening temperature is 20-40 ℃.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101845434A (en) * | 2010-04-19 | 2010-09-29 | 中国科学院青岛生物能源与过程研究所 | Method for rapidly screening thermophilic anaerobic ethanol microbe high-yield ethanol bacterial strain |
| CN101851656A (en) * | 2010-04-19 | 2010-10-06 | 中国科学院青岛生物能源与过程研究所 | Method for producing cellulosic ethanol |
-
2008
- 2008-07-29 CN CNA2008100208428A patent/CN101333553A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101845434A (en) * | 2010-04-19 | 2010-09-29 | 中国科学院青岛生物能源与过程研究所 | Method for rapidly screening thermophilic anaerobic ethanol microbe high-yield ethanol bacterial strain |
| CN101851656A (en) * | 2010-04-19 | 2010-10-06 | 中国科学院青岛生物能源与过程研究所 | Method for producing cellulosic ethanol |
| CN101851656B (en) * | 2010-04-19 | 2012-08-29 | 中国科学院青岛生物能源与过程研究所 | Method for producing cellulosic ethanol |
| CN101845434B (en) * | 2010-04-19 | 2015-01-07 | 中国科学院青岛生物能源与过程研究所 | Method for rapidly screening thermophilic anaerobic ethanol microbe high-yield ethanol bacterial strain |
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Open date: 20081231 |