CN103103221B - Method for converting cellulose into ethanol by use of mixed culture of genetically recombinant yeast - Google Patents
Method for converting cellulose into ethanol by use of mixed culture of genetically recombinant yeast Download PDFInfo
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 title claims abstract description 78
- 238000000034 method Methods 0.000 title claims abstract description 47
- 229920002678 cellulose Polymers 0.000 title claims abstract description 30
- 239000001913 cellulose Substances 0.000 title claims abstract description 30
- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 20
- 238000000855 fermentation Methods 0.000 claims abstract description 41
- 230000004151 fermentation Effects 0.000 claims abstract description 40
- 230000003248 secreting effect Effects 0.000 claims abstract description 25
- 108010047754 beta-Glucosidase Proteins 0.000 claims abstract description 20
- 102000006995 beta-Glucosidase Human genes 0.000 claims abstract description 20
- 108010059892 Cellulase Proteins 0.000 claims abstract description 19
- 101710112457 Exoglucanase Proteins 0.000 claims abstract description 10
- 239000007788 liquid Substances 0.000 claims description 31
- 230000008569 process Effects 0.000 claims description 28
- 238000003756 stirring Methods 0.000 claims description 26
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 24
- 239000008103 glucose Substances 0.000 claims description 24
- 240000008042 Zea mays Species 0.000 claims description 18
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 18
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 18
- 235000005822 corn Nutrition 0.000 claims description 18
- 238000002360 preparation method Methods 0.000 claims description 17
- 230000001580 bacterial effect Effects 0.000 claims description 16
- 241000609240 Ambelania acida Species 0.000 claims description 15
- 230000001954 sterilising effect Effects 0.000 claims description 14
- 229910019142 PO4 Inorganic materials 0.000 claims description 12
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 12
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 12
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 12
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 12
- 238000011081 inoculation Methods 0.000 claims description 12
- 235000021317 phosphate Nutrition 0.000 claims description 12
- 229910052700 potassium Inorganic materials 0.000 claims description 12
- 239000011591 potassium Substances 0.000 claims description 12
- 150000003013 phosphoric acid derivatives Chemical class 0.000 claims description 10
- 239000010905 bagasse Substances 0.000 claims description 9
- 239000002054 inoculum Substances 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 230000004913 activation Effects 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 2
- 239000010452 phosphate Substances 0.000 claims description 2
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 abstract description 14
- 238000004519 manufacturing process Methods 0.000 abstract description 14
- 102000004190 Enzymes Human genes 0.000 abstract description 9
- 108090000790 Enzymes Proteins 0.000 abstract description 9
- 229910052799 carbon Inorganic materials 0.000 abstract description 9
- 229940088598 enzyme Drugs 0.000 abstract description 9
- 229940106157 cellulase Drugs 0.000 abstract description 8
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 7
- 238000005516 engineering process Methods 0.000 abstract description 6
- 230000007062 hydrolysis Effects 0.000 abstract description 6
- 238000006460 hydrolysis reaction Methods 0.000 abstract description 6
- 230000014509 gene expression Effects 0.000 abstract description 4
- 239000002994 raw material Substances 0.000 abstract description 2
- 238000010353 genetic engineering Methods 0.000 abstract 2
- 230000009514 concussion Effects 0.000 description 18
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 12
- 239000000203 mixture Substances 0.000 description 12
- 235000015097 nutrients Nutrition 0.000 description 12
- 239000000306 component Substances 0.000 description 10
- 241000894006 Bacteria Species 0.000 description 6
- 239000001888 Peptone Substances 0.000 description 6
- 108010080698 Peptones Proteins 0.000 description 6
- 230000003139 buffering effect Effects 0.000 description 6
- 229940041514 candida albicans extract Drugs 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 235000011187 glycerol Nutrition 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 239000012533 medium component Substances 0.000 description 6
- 238000002156 mixing Methods 0.000 description 6
- 125000001477 organic nitrogen group Chemical group 0.000 description 6
- 235000019319 peptone Nutrition 0.000 description 6
- 238000004321 preservation Methods 0.000 description 6
- 238000005070 sampling Methods 0.000 description 6
- 239000012138 yeast extract Substances 0.000 description 6
- 230000008901 benefit Effects 0.000 description 4
- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 108010008885 Cellulose 1,4-beta-Cellobiosidase Proteins 0.000 description 2
- 229920002488 Hemicellulose Polymers 0.000 description 2
- 150000001768 cations Chemical class 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 229920001542 oligosaccharide Polymers 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000002195 synergetic effect Effects 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 102000005575 Cellulases Human genes 0.000 description 1
- 108010084185 Cellulases Proteins 0.000 description 1
- -1 EG) Proteins 0.000 description 1
- 101150062179 II gene Proteins 0.000 description 1
- 101150098499 III gene Proteins 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 230000001476 alcoholic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010364 biochemical engineering Methods 0.000 description 1
- 239000002551 biofuel Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000002478 diastatic effect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 150000004676 glycans Polymers 0.000 description 1
- 239000011121 hardwood Substances 0.000 description 1
- 150000002402 hexoses Chemical class 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000010977 jade Substances 0.000 description 1
- 239000012978 lignocellulosic material Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000020265 peanut milk Nutrition 0.000 description 1
- 150000002972 pentoses Chemical class 0.000 description 1
- 230000029553 photosynthesis Effects 0.000 description 1
- 238000010672 photosynthesis Methods 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 150000004804 polysaccharides Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
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
Abstract
The invention relates to the fields of genetic engineering and fermentation engineering, and particularly discloses a method for converting cellulose into ethanol by use of the mixed culture of genetically recombinant yeast. The method comprises the following steps of: constructing three genetically recombinant saccharomyces cerevisiae strains for secretory expression of endoglucanase EG, exoglucanase CBH and beta-glucosidase BGL respectively by use of the genetic engineering technology; performing mixed culture of the three genetically recombinant saccharomyces cerevisiae strains; and fermenting by taking cellulose raw material as a unique carbon source to efficiently and directly convert cellulose into ethanol. The method disclosed by the invention finishes enzyme production, cellulose hydrolysis and ethanol fermentation in one system, does not need additional cellulase, can directly decompose cellulose and convert into ethanol in the fermentation process, omits the saccharifying step, simplifies operation and saves cost, thereby being a green, environment-friendly and low-carbon cellulosic ethanol production technology.
Description
Technical field
The present invention relates to genetically engineered and field of fermentation engineering, more specifically, relate to a kind of gene recombination ferment that utilizes
The method that female mixed culture is ethanol by cellulose conversion.
Background technology
Lignocellulose is the abundantest renewable resources of occurring in nature content, is the main component that plant tissue forms, and accounts for the 20-45% of plant dry weight.The lignocellulose of occurring in nature is produced by photosynthesis by plant, and wherein nearly 90% not yet by the mankind, is effectively utilized, and major part is used as waste disposal, not only will expend a large amount of manpower and materials, and has caused environmental pollution and waste greatly.Lignocellulosic material three major polymers: cellulose, hemicellulose and xylogen form.If can make full use of Mierocrystalline cellulose wherein and hemicellulose, produce biofuel ethanol, both can protection of the environment, can solve international problem of energy crisis again, can produce huge economic benefit simultaneously.
Mierocrystalline cellulose is a kind of macromolecule polysaccharide structure being comprised of glucose, its enzymolysis needs the synergy of a plurality of cellulases to complete, mainly comprise three fibrid element enzymes: endoglucanase (endoglucanase, EG), exoglucanase, claim again cellobiohydrolase (cellobiohydrolase, CBH) and beta-glucosidase (β-glucosidase, BGL).EG is the β-Isosorbide-5-Nitrae-glycosidic link of staple fiber element long-chain inside randomly, forms cell-oligosaccharide; CBH divides the work with EG, can act on the crystallizing field of cellulosic molecule, and the β-Isosorbide-5-Nitrae-glycosidic link of hydrocellulose chain end, cuts cellobiose molecule successively; BGL can continue to be hydrolyzed to glucose by cell-oligosaccharide and the cellobiose of EG and CBH hydrolysis formation.The final glucose obtaining can be utilized by microorganism fermentation, as the glucose fermentation that cellulose hydrolysis can be produced with yeast saccharomyces cerevisiae etc. become ethanol.
At present the production technique of cellulosic ethanol mainly contains following several: (1) substep diastatic fermentation (separate hydrolysis and fermentation, SHF).SHF makes hydrolysis and fermentation proceed step by step.Advantage is that enzymolysis and fermentation can be carried out and not interact under optimal conditions separately, and shortcoming is that the glucose and the cellobiose that in enzymolysis process, accumulate can produce end products inhibition to cellulase, thereby affects the vigor of enzyme, simultaneously complicated operation.(2) simultaneous saccharification and fermentation (simultaneous sacchari cation and fermentation, SSF) and synchronous saccharification ferment altogether (simultaneous sacchari cation and co-fermentation, SSCF).SSF is that cellulosic enzymolysis and ethanol fermentation synchronously carry out, and the glucose that enzymolysis produces is become ethanol by microbial transformation simultaneously; SSCF is on the basis of SSF, makes its microbial strains used of fermenting (single bacterial classification or mixed strains) can utilize hexose and pentose, therefore be called common fermentation simultaneously.The end products that SSF and SSCF have removed cellulase suppresses, and has improved enzymolysis efficiency, has simplified production technique simultaneously, has shortened the production cycle, and be proved to be its effect and be better than SHF.But in SSF and SSCF technique, the condition of saccharification and fermentation adopts the method for compromise, saccharification and fermentation are not carried out under optimal conditions, and the saccharification step of SSF and SSCF adopts commercial enzyme conventionally, the cost of cellulase and enzymolysis efficiency are its hindering factors of industrial applications on a large scale.(3) integration bioprocess technology (consolidated bioprocessing, CBP).Lynd equals the concept that proposes CBP in 2005,, in the situation that additionally not adding commercial enzyme, is completed the technological process of the fermentation of cellulosic saccharification and ethanol by one or more microorganisms at a reactor.For need to adding SHF, the SSF and SSCF of commercial enzyme, CBP has reduced production cost significantly.Have report to show, the powder of hardwood of take is raw material, adopt the ethanol conversion efficiency ratio SSF of CBP high four times, and production cost is lower.Although being showing improvement or progress day by day of enzyme purification technology, the commercial enzyme price of using in SSF more obviously declines, and this new technique of CBP, because of its integrated advantage, still has unrivaled advantage.
Summary of the invention
Technical problem to be solved by this invention is, in order to overcome the above-mentioned deficiency of prior art, to provide a kind of profit
The method that is ethanol by cellulose conversion by recombinant yeast mixed culture.
Above-mentioned technical problem to be solved by this invention is achieved by the following technical programs:
Utilize the method that recombinant yeast mixed culture is ethanol by cellulose conversion, comprise following step
Rapid:
S1. the preparation of seed liquor: the first-selected strains A S2.489-PS-EG that builds respectively energy secreting, expressing endoglucanase EG is, the strains A S2.489-PS-CBH of energy secreting, expressing exoglucanase CBH and the strains A S2.489-PS-BGL of energy secreting, expressing beta-glucosidase (BGL); Then each bacterial strain of activation culture is to logarithmic phase, and makes the yeast count of each bacterial strain reach 0.8 ~ 100,000,000/mL, is mature seed liquid;
S2. prepare fermention medium sterilizing, the component that described substratum contains following weight part: 100 ~ 200 parts of bagasses, 20 ~ 40 parts of corn steep liquor (liquid state, purchased from China Resources Sai Lishida corn Industrial Co., Ltd), 10 ~ 30 parts of glucose, 5 ~ 15 parts of ammonium sulfate, 1 ~ 10 part of potassium primary phosphate;
S3. inoculation: after the mature seed liquid of AS2.489-PS-EG, AS2.489-PS-CBH and AS2.489-PS-BGL is mixed, inoculate under aseptic condition, total inoculum size is culture volume 8 ~ 15%;
S4. fermentation: the front ferment phase passes into sterile air, controlling temperature is 28 ~ 32 ° of C, stirring velocity is 150 ~ 250 revs/min, stir culture 3 ~ 5 hours, the front ferment phase finishes; Then enter main anaerobically fermenting stage ferment phase, it is 28 ~ 32 ° of C that main ferment phase keeps temperature, stops ventilating and keeps the slow stirring of 50 ~ 100 revs/min, ferments 2 ~ 4 days; In whole fermenting process, controlling pH is 3.5 ~ 4.5.
As a kind of preferred version, the inoculation described in S3 is that the mature seed liquid of AS2.489-PS-EG, AS2.489-PS-CBH and AS2.489-PS-BGL is mixed by the volume ratio of 1 ~ 5:1 ~ 5:1 ~ 5.
As a kind of further preferred version, the inoculation described in S3 is that three grades of seed liquor of AS2.489-PS-EG, AS2.489-PS-CBH and AS2.489-PS-BGL are mixed by the volume ratio of 1 ~ 3:1 ~ 3:1 ~ 3.
As a kind of most preferably scheme, the inoculation described in S3 is that three grades of seed liquor of AS2.489-PS-EG, AS2.489-PS-CBH and AS2.489-PS-BGL are mixed by the volume ratio of 1:3:2.
As a kind of preferred version, the inoculum size described in S3 is culture volume 12%.
As a kind of preferred version, the component that substratum contains following weight part: 120 ~ 180 parts of bagasses, 25 ~ 30
Part corn steep liquor, 15 ~ 20 parts of glucose, 5 ~ 10 parts of ammonium sulfate, 2 ~ 6 parts of potassium primary phosphates.
As a kind of most preferably scheme, the component that substratum contains following weight part: 150 parts of bagasses, 28 parts of jade
Rice & peanut milk, 18 parts of glucose, 7 parts of ammonium sulfate, 4 parts of potassium primary phosphates.
As a kind of preferred version, the bagasse in above-mentioned substratum (bagasse) is through mechanical disintegration to 150 ~ 250 order.
As a kind of preferred version, in S4, the front ferment phase is controlled 30 ° of C of temperature, and stirring velocity is 200 revs/min,
Stir culture 4 hours; It is 30 ° of C that the main ferment phase keeps temperature, ferments 3 days.
As a kind of preferred version, in the whole fermenting process of S4, controlling pH is 4.0.
The construction process of AS2.489-PS-EG of the present invention is referring to document: Liu Zehuan, platform is gorgeous, Tang Genyun, Quan Yancai, Wang Junmei, Xiao Wenjuan, Gong Yingxue. the clone of viride EG III gene and the integrative gene expression in industrial saccharomyces cerevisiae thereof. Zhongshan University's journal (natural science edition), 2009,48 (6): 83-88., AS2.489-PS-EG of the present invention is the bacterial classification preparing by the preparation method in described document.
The construction process of AS2.489-PS-CBH of the present invention is referring to document: Liu Zehuan, Quan Yancai, Tang Genyun, Gong Yingxue, Xiao Wenjuan, Wang Junmei. the clone of viride CBH II gene and the expression in yeast saccharomyces cerevisiae. South China Science & Engineering University's journal (natural science edition), 2009,37 (6): 91-95., AS2.489-PS-CBH of the present invention is the bacterial classification preparing by the preparation method in described document.
The construction process of AS2.489-PS-BGL of the present invention is referring to document: Liu Zehuan, Tang Genyun, Quan Yancai, Gong Yingxue, Xiao Wenjuan, Wang Junmei. clone, expression and the cellulose alcoholic fermentation thereof of viride BGLI gene in yeast saccharomyces cerevisiae. Lanzhou University's journal (natural science edition), 2009,45 (3): 61-66. is that AS2.489-PS-BGL of the present invention is the bacterial classification preparing by the preparation method in described document.
The present invention has following beneficial effect: (1) present method completes product enzyme, cellulose hydrolysis and ethanol fermentation in an individual system, do not need additionally to add cellulase, can be during the fermentation direct decomposition of cellulose be converted into ethanol, omitted saccharification step, simplified operation, providing cost savings, is the cellulosic ethanol production technique of a set of environmental protection low-carbon (LC); (2) the present invention can regulate and control the synergistic action effect of cellulase flexibly by adjusting the inoculative proportion of bacterial strain, has overcome the shortcoming that traditional technology cannot change synergistic action effect between cellulase; Bacterial strain uses therefor energy simultaneously of the present invention self cellulase-producing.
Accompanying drawing explanation
Fig. 1 is integration bioprocess technology schema of the present invention.
Fig. 2 is ethanol, the reducing sugar time-concentration curve in the embodiment of the present invention 1 fermenting process.
Fig. 3 is ethanol, the reducing sugar time-concentration curve in the embodiment of the present invention 2 fermenting processs.
Embodiment
Below in conjunction with specific embodiment, further explain the present invention, but embodiment does not limit in any form to invention.
1 three kinds of recombinant bacterial strains of embodiment are pressed the blending ratio fermented cellulose producing and ethanol situation of 1:1:1
S1. the preparation of seed liquor:
S11. build respectively the strains A S2.489-PS-EG of energy secreting, expressing endoglucanase EG, the strains A S2.489-PS-CBH of energy secreting, expressing exoglucanase CBH and the strains A S2.489-PS-BGL of energy secreting, expressing beta-glucosidase (BGL);
S12. the glycerine preservation strain of AS2.489-PS-EG, AS2.489-PS-CBH and AS2.489-PS-BGL being lined respectively to YPD(containing weight percent composition is: 2% peptone, 1% yeast extract, 2% glucose) flat board, be placed in 30 ° of C constant incubators and cultivate 2 days.Single bacterium colony on picking flat board, is inoculated in 2mL YPD liquid nutrient medium, and in 30 ° of C, 24h is cultivated in 200rpm concussion, is primary seed solution; Primary seed solution is forwarded in 20mL YPD, 30 ° of C, 12h is cultivated in 200rpm concussion, is secondary seed solution; Secondary seed solution is forwarded in 200 mL YPD liquid nutrient mediums, 30 ° of C, about 6h is cultivated in 200rpm concussion, makes yeast count reach 100,000,000, is mature seed liquid;
S2. prepare fermention medium sterilizing:
S21. the composition of fermention medium: the fermentation culture based component using is all with reagent preparation with low cost; Take corn steep liquor as organic nitrogen source, be equipped with (NH
4)
2sO
4for inorganic nitrogen-sourced, simultaneously with KH
2pO
4the pH buffering that substratum is provided, Mierocrystalline cellulose is the sole carbon source of fermenting process; Specifically consisting of of fermention medium: contain 150 grams in every liter of fermention medium through the bagasse of mechanical disintegration to 200 order left and right, 28 grams of corn steep liquors, 18 grams of glucose, 7 grams of ammonium sulfate, 4 grams of potassium primary phosphates;
S22. according to above-mentioned fermention medium component, prepare fermention medium, 121 ° of real tanks of C in fermentor tank
Sterilizing (disappearing in fact) 20 min;
S3. inoculation: after 1:1:1 mixes by volume by the mature seed liquid of AS2.489-PS-EG, AS2.489-PS-CBH and AS2.489-PS-BGL, inoculate under aseptic condition, total inoculum size is culture volume 12%;
S4. fermentation: the front ferment phase passes into sterile air, controlling temperature is 30 ° of C, stirring velocity is 200 revs/min, stir culture 4 hours, the front ferment phase finishes; Then enter main anaerobically fermenting stage ferment phase, it is 30 ° of C that main ferment phase keeps temperature, stops ventilating and keeps the slow stirring of 50 revs/min, ferments 3 days; In whole fermenting process, controlling pH is 4.
In fermenting process, every sampling in 12 hours, use ethanol production and reducing sugar residual content in Fermentation Liquor by High Performance Liquid Chromatography.The results are shown in accompanying drawing 2.Alcohol concn 60 hours main ferment phases increase gradually and reach maximum concentration 17.11g/L(account for theoretical value 50.2%), after 72 hours, start to decline.The present embodiment repeats to test 3 times.Accompanying drawing 2 is statisticses of 3 experiments.
2 three kinds of recombinant bacterial strains of embodiment are pressed the blending ratio fermented cellulose producing and ethanol situation of 1:3:2
S1. the preparation of seed liquor:
S11. build respectively the strains A S2.489-PS-EG of energy secreting, expressing endoglucanase EG, the strains A S2.489-PS-CBH of energy secreting, expressing exoglucanase CBH and the strains A S2.489-PS-BGL of energy secreting, expressing beta-glucosidase (BGL);
S12. the glycerine preservation strain of AS2.489-PS-EG, AS2.489-PS-CBH and AS2.489-PS-BGL being lined respectively to YPD(containing weight percent composition is: 2% peptone, 1% yeast extract, 2% glucose) flat board, be placed in 30 ° of C constant incubators and cultivate 2 days.Single bacterium colony on picking flat board, is inoculated in 2mL YPD liquid nutrient medium, and in 30 ° of C, 24h is cultivated in 200rpm concussion, is primary seed solution; Primary seed solution is forwarded in 20mL YPD, 30 ° of C, 12h is cultivated in 200rpm concussion, is secondary seed solution; Secondary seed solution is forwarded in 200 mL YPD liquid nutrient mediums, 30 ° of C, about 6h is cultivated in 200rpm concussion, makes yeast count reach 100,000,000, is mature seed liquid;
S2. prepare fermention medium sterilizing:
S21. the composition of fermention medium: fermentation culture based component used in the present invention is all with reagent preparation with low cost.Take corn steep liquor as organic nitrogen source, be equipped with (NH
4)
2sO
4for inorganic nitrogen-sourced, simultaneously with KH
2pO
4the pH buffering that substratum is provided, Mierocrystalline cellulose is the sole carbon source of fermenting process; Specifically consisting of of fermention medium: contain 150 grams in every liter of fermention medium through mechanical disintegration to 150 object bagasse, 28 grams of corn steep liquors, 18 grams of glucose, 7 grams of ammonium sulfate, 4 grams of potassium primary phosphates;
S22. according to above-mentioned fermention medium component, prepare fermention medium, 121 ° of real tanks of C in fermentor tank
Sterilizing (disappearing in fact) 20 min;
S3. inoculation: after 1:3:2 mixes by volume by the mature seed liquid of AS2.489-PS-EG, AS2.489-PS-CBH and AS2.489-PS-BGL, inoculate under aseptic condition, total inoculum size is culture volume 12%;
S4. fermentation: the front ferment phase passes into sterile air, controlling temperature is 30 ° of C, stirring velocity is 200 revs/min, stir culture 4 hours, the front ferment phase finishes; Then enter main anaerobically fermenting stage ferment phase, it is 30 ° of C that main ferment phase keeps temperature, stops ventilating and keeps the slow stirring of 50 revs/min, ferments 3 days; In whole fermenting process, controlling pH is 4.
In fermenting process, every sampling in 12 hours, use ethanol production and reducing sugar residual content in Fermentation Liquor by High Performance Liquid Chromatography.The results are shown in accompanying drawing 3.Alcohol concn 60 hours main ferment phases increase gradually and reach maximum concentration 20.76g/L(account for theoretical value 60.9%), cellulosic ethanol output is significantly higher than in the result of 1:1:1 ratio mixed culture.The present embodiment repeats to test 3 times.Accompanying drawing 3 is statisticses of 3 experiments.
3 three kinds of recombinant bacterial strains of embodiment are pressed the blending ratio fermented cellulose producing and ethanol situation of 2:3:4
S1. the preparation of seed liquor:
S11. build respectively the strains A S2.489-PS-EG of energy secreting, expressing endoglucanase EG, the strains A S2.489-PS-CBH of energy secreting, expressing exoglucanase CBH and the strains A S2.489-PS-BGL of energy secreting, expressing beta-glucosidase (BGL);
S12. the glycerine preservation strain of AS2.489-PS-EG, AS2.489-PS-CBH and AS2.489-PS-BGL being lined respectively to YPD(containing weight percent composition is: 2% peptone, 1% yeast extract, 2% glucose) flat board, be placed in 30 ° of C constant incubators and cultivate 2 days.Single bacterium colony on picking flat board, is inoculated in 2mL YPD liquid nutrient medium, and in 30 ° of C, 24h is cultivated in 200rpm concussion, is primary seed solution; Primary seed solution is forwarded in 20mL YPD, 30 ° of C, 12h is cultivated in 200rpm concussion, is secondary seed solution; Secondary seed solution is forwarded in 200 mL YPD liquid nutrient mediums, 30 ° of C, about 6h is cultivated in 200rpm concussion, makes yeast count reach 100,000,000, is mature seed liquid;
S2. prepare fermention medium sterilizing:
S21. the composition of fermention medium: the fermentation culture based component using is all with reagent preparation with low cost; Take corn steep liquor as organic nitrogen source, be equipped with (NH
4)
2sO
4for inorganic nitrogen-sourced, simultaneously with KH
2pO
4the pH buffering that substratum is provided, Mierocrystalline cellulose is the sole carbon source of fermenting process; Specifically consisting of of fermention medium: contain 150 grams in every liter of fermention medium through the bagasse of mechanical disintegration to 200 order left and right, 28 grams of corn steep liquors, 18 grams of glucose, 7 grams of ammonium sulfate, 4 grams of potassium primary phosphates;
S22. according to above-mentioned fermention medium component, prepare fermention medium, 121 ° of real tanks of C in fermentor tank
Sterilizing (disappearing in fact) 20 min;
S3. inoculation: after 2:3:4 mixes by volume by the mature seed liquid of AS2.489-PS-EG, AS2.489-PS-CBH and AS2.489-PS-BGL, inoculate under aseptic condition, total inoculum size is culture volume 12%;
S4. fermentation: the front ferment phase passes into sterile air, controlling temperature is 30 ° of C, stirring velocity is 200 revs/min, stir culture 4 hours, the front ferment phase finishes; Then enter main anaerobically fermenting stage ferment phase, it is 30 ° of C that main ferment phase keeps temperature, stops ventilating and keeps the slow stirring of 50 revs/min, ferments 3 days; In whole fermenting process, controlling pH is 4.
In fermenting process, every sampling in 12 hours, use ethanol production and reducing sugar residual content in Fermentation Liquor by High Performance Liquid Chromatography.The results are shown in accompanying drawing 2.Alcohol concn 60 hours main ferment phases increase gradually and reach maximum concentration 15.39g/L(account for theoretical value 45.2%).The present embodiment repeats to test 3 times.
4 three kinds of recombinant bacterial strains of embodiment are pressed the blending ratio fermented cellulose producing and ethanol situation of 3:5:2
S1. the preparation of seed liquor:
S11. build respectively the strains A S2.489-PS-EG of energy secreting, expressing endoglucanase EG, the strains A S2.489-PS-CBH of energy secreting, expressing exoglucanase CBH and the strains A S2.489-PS-BGL of energy secreting, expressing beta-glucosidase (BGL);
S12. the glycerine preservation strain of AS2.489-PS-EG, AS2.489-PS-CBH and AS2.489-PS-BGL being lined respectively to YPD(containing weight percent composition is: 2% peptone, 1% yeast extract, 2% glucose) flat board, be placed in 30 ° of C constant incubators and cultivate 2 days.Single bacterium colony on picking flat board, is inoculated in 2mL YPD liquid nutrient medium, and in 30 ° of C, 24h is cultivated in 200rpm concussion, is primary seed solution; Primary seed solution is forwarded in 20mL YPD, 30 ° of C, 12h is cultivated in 200rpm concussion, is secondary seed solution; Secondary seed solution is forwarded in 200 mL YPD liquid nutrient mediums, 30 ° of C, about 6h is cultivated in 200rpm concussion, makes yeast count reach 100,000,000, is mature seed liquid;
S2. prepare fermention medium sterilizing:
S21. the composition of fermention medium: the fermentation culture based component using is all with reagent preparation with low cost; Take corn steep liquor as organic nitrogen source, be equipped with (NH
4)
2sO
4for inorganic nitrogen-sourced, simultaneously with KH
2pO
4the pH buffering that substratum is provided, Mierocrystalline cellulose is the sole carbon source of fermenting process; Specifically consisting of of fermention medium: contain 150 grams in every liter of fermention medium through the bagasse of mechanical disintegration to 200 order left and right, 28 grams of corn steep liquors, 18 grams of glucose, 7 grams of ammonium sulfate, 4 grams of potassium primary phosphates;
S22. according to above-mentioned fermention medium component, prepare fermention medium, 121 ° of real tanks of C in fermentor tank
Sterilizing (disappearing in fact) 20 min;
S3. inoculation: after 3:5:2 mixes by volume by the mature seed liquid of AS2.489-PS-EG, AS2.489-PS-CBH and AS2.489-PS-BGL, inoculate under aseptic condition, total inoculum size is culture volume 12%;
S4. fermentation: the front ferment phase passes into sterile air, controlling temperature is 30 ° of C, stirring velocity is 200 revs/min, stir culture 4 hours, the front ferment phase finishes; Then enter main anaerobically fermenting stage ferment phase, it is 30 ° of C that main ferment phase keeps temperature, stops ventilating and keeps the slow stirring of 50 revs/min, ferments 3 days; In whole fermenting process, controlling pH is 4.
In fermenting process, every sampling in 12 hours, use ethanol production and reducing sugar residual content in Fermentation Liquor by High Performance Liquid Chromatography.The results are shown in accompanying drawing 2.Alcohol concn 60 hours main ferment phases increase gradually and reach maximum concentration 13.57g/L(account for theoretical value 39.8%).The present embodiment repeats to test 3 times.
5 three kinds of recombinant bacterial strains of embodiment are pressed the blending ratio fermented cellulose producing and ethanol situation of 4:2:5
S1. the preparation of seed liquor:
S11. build respectively the strains A S2.489-PS-EG of energy secreting, expressing endoglucanase EG, the strains A S2.489-PS-CBH of energy secreting, expressing exoglucanase CBH and the strains A S2.489-PS-BGL of energy secreting, expressing beta-glucosidase (BGL);
S12. the glycerine preservation strain of AS2.489-PS-EG, AS2.489-PS-CBH and AS2.489-PS-BGL being lined respectively to YPD(containing weight percent composition is: 2% peptone, 1% yeast extract, 2% glucose) flat board, be placed in 30 ° of C constant incubators and cultivate 2 days.Single bacterium colony on picking flat board, is inoculated in 2mL YPD liquid nutrient medium, and in 30 ° of C, 24h is cultivated in 200rpm concussion, is primary seed solution; Primary seed solution is forwarded in 20mL YPD, 30 ° of C, 12h is cultivated in 200rpm concussion, is secondary seed solution; Secondary seed solution is forwarded in 200 mL YPD liquid nutrient mediums, 30 ° of C, about 6h is cultivated in 200rpm concussion, makes yeast count reach 100,000,000, is mature seed liquid;
S2. prepare fermention medium sterilizing:
S21. the composition of fermention medium: the fermentation culture based component using is all with reagent preparation with low cost; Take corn steep liquor as organic nitrogen source, be equipped with (NH
4)
2sO
4for inorganic nitrogen-sourced, simultaneously with KH
2pO
4the pH buffering that substratum is provided, Mierocrystalline cellulose is the sole carbon source of fermenting process; Specifically consisting of of fermention medium: contain 150 grams in every liter of fermention medium through the bagasse of mechanical disintegration to 200 order left and right, 28 grams of corn steep liquors, 18 grams of glucose, 7 grams of ammonium sulfate, 4 grams of potassium primary phosphates;
S22. according to above-mentioned fermention medium component, prepare fermention medium, 121 ° of real tanks of C in fermentor tank
Sterilizing (disappearing in fact) 20 min;
S3. inoculation: after 4:2:5 mixes by volume by the mature seed liquid of AS2.489-PS-EG, AS2.489-PS-CBH and AS2.489-PS-BGL, inoculate under aseptic condition, total inoculum size is culture volume 12%;
S4. fermentation: the front ferment phase passes into sterile air, controlling temperature is 30 ° of C, stirring velocity is 200 revs/min, stir culture 4 hours, the front ferment phase finishes; Then enter main anaerobically fermenting stage ferment phase, it is 30 ° of C that main ferment phase keeps temperature, stops ventilating and keeps the slow stirring of 50 revs/min, ferments 3 days; In whole fermenting process, controlling pH is 4.
In fermenting process, every sampling in 12 hours, use ethanol production and reducing sugar residual content in Fermentation Liquor by High Performance Liquid Chromatography.The results are shown in accompanying drawing 2.Alcohol concn 60 hours main ferment phases increase gradually and reach maximum concentration 16.21g/L(account for theoretical value 47.6%).The present embodiment repeats to test 3 times.
6 three kinds of recombinant bacterial strains of embodiment are pressed the blending ratio fermented cellulose producing and ethanol situation of 5:4:3
S1. the preparation of seed liquor:
S11. build respectively the strains A S2.489-PS-EG of energy secreting, expressing endoglucanase EG, the strains A S2.489-PS-CBH of energy secreting, expressing exoglucanase CBH and the strains A S2.489-PS-BGL of energy secreting, expressing beta-glucosidase (BGL);
S12. the glycerine preservation strain of AS2.489-PS-EG, AS2.489-PS-CBH and AS2.489-PS-BGL being lined respectively to YPD(containing weight percent composition is: 2% peptone, 1% yeast extract, 2% glucose) flat board, be placed in 30 ° of C constant incubators and cultivate 2 days.Single bacterium colony on picking flat board, is inoculated in 2mL YPD liquid nutrient medium, and in 30 ° of C, 24h is cultivated in 200rpm concussion, is primary seed solution; Primary seed solution is forwarded in 20mL YPD, 30 ° of C, 12h is cultivated in 200rpm concussion, is secondary seed solution; Secondary seed solution is forwarded in 200 mL YPD liquid nutrient mediums, 30 ° of C, about 6h is cultivated in 200rpm concussion, makes yeast count reach 100,000,000, is mature seed liquid;
S2. prepare fermention medium sterilizing:
S21. the composition of fermention medium: the fermentation culture based component using is all with reagent preparation with low cost; Take corn steep liquor as organic nitrogen source, be equipped with (NH
4)
2sO
4for inorganic nitrogen-sourced, simultaneously with KH
2pO
4the pH buffering that substratum is provided, Mierocrystalline cellulose is the sole carbon source of fermenting process; Specifically consisting of of fermention medium: contain 150 grams in every liter of fermention medium through the bagasse of mechanical disintegration to 200 order left and right, 28 grams of corn steep liquors, 18 grams of glucose, 7 grams of ammonium sulfate, 4 grams of potassium primary phosphates;
S22. according to above-mentioned fermention medium component, prepare fermention medium, 121 ° of real tanks of C in fermentor tank
Sterilizing (disappearing in fact) 20 min;
S3. inoculation: after 5:4:3 mixes by volume by the mature seed liquid of AS2.489-PS-EG, AS2.489-PS-CBH and AS2.489-PS-BGL, inoculate under aseptic condition, total inoculum size is culture volume 12%;
S4. fermentation: the front ferment phase passes into sterile air, controlling temperature is 30 ° of C, stirring velocity is 200 revs/min, stir culture 4 hours, the front ferment phase finishes; Then enter main anaerobically fermenting stage ferment phase, it is 30 ° of C that main ferment phase keeps temperature, stops ventilating and keeps the slow stirring of 50 revs/min, ferments 3 days; In whole fermenting process, controlling pH is 4.
In fermenting process, every sampling in 12 hours, use ethanol production and reducing sugar residual content in Fermentation Liquor by High Performance Liquid Chromatography.The results are shown in accompanying drawing 2.Alcohol concn 60 hours main ferment phases increase gradually and reach maximum concentration 14.38g/L(account for theoretical value 42.2%).The present embodiment repeats to test 3 times.
Claims (4)
1. utilize the method that recombinant yeast mixed culture is ethanol by cellulose conversion, it is characterized in that, comprise the steps:
S1. the preparation of seed liquor: the first-selected strains A S2.489-PS-EG that builds respectively energy secreting, expressing endoglucanase EG is, the strains A S2.489-PS-BGL of the strains A S2.489-PS-CBH of energy secreting, expressing exoglucanase CBH and energy secreting, expressing beta-glucosidase BGL; Then each bacterial strain of activation culture is to logarithmic phase, and makes the yeast count of each bacterial strain reach 0.8 ~ 100,000,000/mL, is mature seed liquid;
S2. prepare fermention medium sterilizing, the component that described substratum contains following weight part: 100 ~ 200 parts of bagasses, 20 ~ 40 parts of corn steep liquors, 10 ~ 30 parts of glucose, 5 ~ 15 parts of ammonium sulfate, 1 ~ 10 part of potassium primary phosphate;
S3. inoculation: after the mature seed liquid of AS2.489-PS-EG, AS2.489-PS-CBH and AS2.489-PS-BGL is mixed, inoculate under aseptic condition, total inoculum size is culture volume 12%;
S4. fermentation: the front ferment phase passes into sterile air, controlling temperature is 30 ° of C, stirring velocity is 200 revs/min, stir culture 4 hours, the front ferment phase finishes; Then enter main anaerobically fermenting stage ferment phase, it is 30 ° of C that main ferment phase keeps temperature, stops ventilating and keeps the slow stirring of 50 ~ 100 revs/min, ferments 3 days; In whole fermenting process, controlling pH is 4.0;
Inoculation described in S3 is that the mature seed liquid of AS2.489-PS-EG, AS2.489-PS-CBH and AS2.489-PS-BGL is mixed by the volume ratio of 1:3:2.
2. method according to claim 1, is characterized in that, the component that substratum contains following weight part: 120 ~ 180 parts of bagasses, 25 ~ 30 parts of corn steep liquors, 15 ~ 20 parts of glucose, 5 ~ 10 parts of ammonium sulfate, 2 ~ 6 parts of potassium primary phosphates.
3. method according to claim 2, is characterized in that, the component that substratum contains following weight part: 150 parts of bagasses, 28 parts of corn steep liquors, 18 parts of glucose, 7 parts of ammonium sulfate, 4 parts of potassium primary phosphates.
4. according to the method described in claim 1,2 or 3, it is characterized in that, the bagasse in described substratum is through mechanical disintegration to 150 ~ 250 order.
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| WO2008064314A2 (en) * | 2006-11-22 | 2008-05-29 | The Trustees Of Dartmouth College | Recombinant yeast strains expressing tethered cellulase enzymes |
| CN101311271A (en) * | 2007-05-25 | 2008-11-26 | 青岛科技大学 | Process for producing cellulosic ethanol by recombining saccharomyces cerevisiae |
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| WO2008064314A2 (en) * | 2006-11-22 | 2008-05-29 | The Trustees Of Dartmouth College | Recombinant yeast strains expressing tethered cellulase enzymes |
| CN101311271A (en) * | 2007-05-25 | 2008-11-26 | 青岛科技大学 | Process for producing cellulosic ethanol by recombining saccharomyces cerevisiae |
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