CN115948263A - High-temperature high-sugar high-permeability tolerant yeast, breeding method thereof and high-concentration ethanol fermentation application - Google Patents
High-temperature high-sugar high-permeability tolerant yeast, breeding method thereof and high-concentration ethanol fermentation application Download PDFInfo
- Publication number
- CN115948263A CN115948263A CN202211603163.XA CN202211603163A CN115948263A CN 115948263 A CN115948263 A CN 115948263A CN 202211603163 A CN202211603163 A CN 202211603163A CN 115948263 A CN115948263 A CN 115948263A
- Authority
- CN
- China
- Prior art keywords
- saccharomyces cerevisiae
- fermentation
- culture
- sugar
- culture 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
- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 244
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 title claims abstract description 178
- 238000000855 fermentation Methods 0.000 title claims abstract description 131
- 230000004151 fermentation Effects 0.000 title claims abstract description 122
- 238000009395 breeding Methods 0.000 title claims abstract description 26
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims abstract description 243
- 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 abstract description 59
- 239000008103 glucose Substances 0.000 claims abstract description 59
- 230000001488 breeding effect Effects 0.000 claims abstract description 17
- 238000013537 high throughput screening Methods 0.000 claims abstract description 10
- 239000007788 liquid Substances 0.000 claims description 87
- 239000001963 growth medium Substances 0.000 claims description 79
- 230000001580 bacterial effect Effects 0.000 claims description 56
- 238000012258 culturing Methods 0.000 claims description 39
- 239000007787 solid Substances 0.000 claims description 32
- 230000004913 activation Effects 0.000 claims description 31
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 claims description 30
- 239000011248 coating agent Substances 0.000 claims description 29
- 238000000576 coating method Methods 0.000 claims description 29
- 238000009630 liquid culture Methods 0.000 claims description 25
- 238000007865 diluting Methods 0.000 claims description 23
- 238000011081 inoculation Methods 0.000 claims description 23
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 20
- 239000002609 medium Substances 0.000 claims description 20
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 claims description 18
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 claims description 16
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 12
- 239000001888 Peptone Substances 0.000 claims description 12
- 108010080698 Peptones Proteins 0.000 claims description 12
- 235000019319 peptone Nutrition 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 11
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 11
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 11
- 229940041514 candida albicans extract Drugs 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 239000012138 yeast extract Substances 0.000 claims description 11
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 10
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 claims description 10
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 10
- 229960002685 biotin Drugs 0.000 claims description 10
- 235000020958 biotin Nutrition 0.000 claims description 10
- 239000011616 biotin Substances 0.000 claims description 10
- 239000001110 calcium chloride Substances 0.000 claims description 10
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 10
- 239000004202 carbamide Substances 0.000 claims description 10
- JZCCFEFSEZPSOG-UHFFFAOYSA-L copper(II) sulfate pentahydrate Chemical compound O.O.O.O.O.[Cu+2].[O-]S([O-])(=O)=O JZCCFEFSEZPSOG-UHFFFAOYSA-L 0.000 claims description 10
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 claims description 10
- 229940044631 ferric chloride hexahydrate Drugs 0.000 claims description 10
- 229960000367 inositol Drugs 0.000 claims description 10
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 claims description 10
- NQXWGWZJXJUMQB-UHFFFAOYSA-K iron trichloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].Cl[Fe+]Cl NQXWGWZJXJUMQB-UHFFFAOYSA-K 0.000 claims description 10
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 10
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 10
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 claims description 10
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 10
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 10
- 235000013923 monosodium glutamate Nutrition 0.000 claims description 10
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 10
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 claims description 10
- 229940073490 sodium glutamate Drugs 0.000 claims description 10
- RZLVQBNCHSJZPX-UHFFFAOYSA-L zinc sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Zn+2].[O-]S([O-])(=O)=O RZLVQBNCHSJZPX-UHFFFAOYSA-L 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 9
- 229960003512 nicotinic acid Drugs 0.000 claims description 9
- 235000001968 nicotinic acid Nutrition 0.000 claims description 9
- 239000011664 nicotinic acid Substances 0.000 claims description 9
- 229960003581 pyridoxal Drugs 0.000 claims description 9
- 239000011674 pyridoxal Substances 0.000 claims description 9
- 229960003495 thiamine Drugs 0.000 claims description 9
- 239000011721 thiamine Substances 0.000 claims description 9
- 241000894006 Bacteria Species 0.000 claims description 8
- 229960004050 aminobenzoic acid Drugs 0.000 claims description 8
- 238000004321 preservation Methods 0.000 claims description 8
- 239000003761 preservation solution Substances 0.000 claims description 8
- 238000012216 screening Methods 0.000 claims description 8
- 239000003085 diluting agent Substances 0.000 claims description 7
- 238000010790 dilution Methods 0.000 claims description 7
- 239000012895 dilution Substances 0.000 claims description 7
- 239000002054 inoculum Substances 0.000 claims description 7
- 230000003068 static effect Effects 0.000 claims description 7
- 239000011148 porous material Substances 0.000 claims description 6
- 239000000243 solution Substances 0.000 claims description 5
- 230000003213 activating effect Effects 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 238000003860 storage Methods 0.000 claims description 4
- 241001052560 Thallis Species 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 230000035699 permeability Effects 0.000 claims description 3
- 108010062466 Enzyme Precursors Proteins 0.000 claims description 2
- 102000010911 Enzyme Precursors Human genes 0.000 claims description 2
- 239000000413 hydrolysate Substances 0.000 claims description 2
- 235000015097 nutrients Nutrition 0.000 claims description 2
- 231100000331 toxic Toxicity 0.000 claims description 2
- 230000002588 toxic effect Effects 0.000 claims description 2
- 238000012546 transfer Methods 0.000 claims description 2
- GHOKWGTUZJEAQD-UHFFFAOYSA-N Chick antidermatitis factor Natural products OCC(C)(C)C(O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-UHFFFAOYSA-N 0.000 claims 1
- 229930003571 Vitamin B5 Natural products 0.000 claims 1
- FAPWYRCQGJNNSJ-UBKPKTQASA-L calcium D-pantothenic acid Chemical compound [Ca+2].OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O.OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O FAPWYRCQGJNNSJ-UBKPKTQASA-L 0.000 claims 1
- 229960002079 calcium pantothenate Drugs 0.000 claims 1
- 235000009492 vitamin B5 Nutrition 0.000 claims 1
- 239000011675 vitamin B5 Substances 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 15
- 238000006243 chemical reaction Methods 0.000 abstract description 10
- 150000005846 sugar alcohols Chemical class 0.000 abstract description 10
- 229930003270 Vitamin B Natural products 0.000 description 8
- 235000019156 vitamin B Nutrition 0.000 description 8
- 239000011720 vitamin B Substances 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 4
- 210000000170 cell membrane Anatomy 0.000 description 3
- 239000000446 fuel Substances 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000002028 Biomass Substances 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 239000002803 fossil fuel Substances 0.000 description 2
- 239000005431 greenhouse gas Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 108020004463 18S ribosomal RNA Proteins 0.000 description 1
- GNFTZDOKVXKIBK-UHFFFAOYSA-N 3-(2-methoxyethoxy)benzohydrazide Chemical compound COCCOC1=CC=CC(C(=O)NN)=C1 GNFTZDOKVXKIBK-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 239000005711 Benzoic acid Substances 0.000 description 1
- FGUUSXIOTUKUDN-IBGZPJMESA-N C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 Chemical compound C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 FGUUSXIOTUKUDN-IBGZPJMESA-N 0.000 description 1
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- YTAHJIFKAKIKAV-XNMGPUDCSA-N [(1R)-3-morpholin-4-yl-1-phenylpropyl] N-[(3S)-2-oxo-5-phenyl-1,3-dihydro-1,4-benzodiazepin-3-yl]carbamate Chemical compound O=C1[C@H](N=C(C2=C(N1)C=CC=C2)C1=CC=CC=C1)NC(O[C@H](CCN1CCOCC1)C1=CC=CC=C1)=O YTAHJIFKAKIKAV-XNMGPUDCSA-N 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000002551 biofuel Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 230000005757 colony formation Effects 0.000 description 1
- 239000000306 component Substances 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 239000003502 gasoline Substances 0.000 description 1
- 230000034659 glycolysis Effects 0.000 description 1
- 230000002727 hyperosmolar Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- TVMXDCGIABBOFY-UHFFFAOYSA-N octane Chemical compound CCCCCCCC TVMXDCGIABBOFY-UHFFFAOYSA-N 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- KFLRWGSAMLBHBV-UHFFFAOYSA-M sodium;pyridine-3-carboxylate Chemical compound [Na+].[O-]C(=O)C1=CC=CN=C1 KFLRWGSAMLBHBV-UHFFFAOYSA-M 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
Images
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
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a high-temperature high-sugar high-permeability tolerant yeast, a breeding method thereof and high-concentration ethanol fermentation application, and belongs to the technical field of biology. Saccharomyces cerevisiae SG200 of the invention is preserved in China center for type culture Collection (number CCTCC M20221772) at 11 months and 11 days in 2022. The saccharomyces cerevisiae is obtained by domestication and breeding in a high-temperature high-sugar high-permeability environment and high-throughput screening, and has good anaerobic ethanol fermentation performance under the conditions of 36-40 ℃ and 200-300 g/L. The glucose consumed by fermentation at 40 ℃ is up to 220g/L, the ethanol concentration can reach 90g/L, the thallus concentration is improved by 27-72% compared with the traditional yeast strains, the ethanol production intensity in the fermentation process can reach 2.3-3.4g/L/h, and the sugar-alcohol conversion rate is maintained at 0.41-0.42g/g.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a high-temperature high-sugar high-permeability tolerant yeast, a breeding method thereof and application of high-concentration ethanol fermentation.
Background
The use of fossil fuels leads to global CO 2 The content is increased, the greenhouse effect is increased, and CO in the atmosphere is increased from 1813 to 2019 2 The content is increased from 220 to 411mg/L, and the utilization of renewable biomass energy is helpful to slow down CO 2 Among them, ethanol is considered as one of the ideal renewable alternative energy sources due to its higher octane number, good antiknock property and low pollution. Bioethanol can be classified into first-generation starch-based fuel ethanol and second-generation cellulose fuel ethanol according to the source of raw materials. Compared with gasoline, corn ethanol can reduce the greenhouse gas emission by 39-52%, and cellulose ethanol can reduce the greenhouse gas emission by 86%. In fact, countries in the world actively take new energy measures and develop relevant policies, while fossil fuel use is reduced, biomass energy industries represented by biofuel ethanol are greatly supported, global fuel ethanol demand and yield are increased sharply, 8800 ten thousand tons in 2019, and 731% of the yield is increased compared with 2000.
In fact, saccharomyces cerevisiae synthesizes ethanol by anaerobic fermentation, and after uptake of glucose by cells, glucose is converted to pyruvate by the glycolytic pathway, which is further catalytically converted to the product ethanol. However, in the process of biorefinery of cellulosic ethanol, the high temperature environment required can inhibit the growth of saccharomyces cerevisiae and hinder the cell viability, which leads to the massive aggregation and denaturation of biological macromolecules in cells, and simultaneously destroys the integrity of cell membranes, increases the membrane permeability, influences the fluidity of plasma membranes and finally leads to cell death. Meanwhile, in order to realize high-concentration ethanol fermentation, the glucose concentration is generally selected to be 200-300g/L or even higher, however, high-concentration fermentation sugar often causes a high osmotic pressure environment, promotes cell membrane osmopressure receptors of saccharomyces cerevisiae to activate a hypertonic glycerol way, and causes a strong stress effect on saccharomyces cerevisiae cells, so that the cell proliferation and activity of the saccharomyces cerevisiae are reduced, the thallus concentration and the carbon source utilization are inhibited, and further the ethanol yield, the sugar alcohol conversion rate and the production intensity are influenced. Therefore, how to improve the high-temperature high-sugar high-permeability resistance of the saccharomyces cerevisiae is particularly important for high-concentration ethanol fermentation application.
In recent years, some research progress has been reported for ethanol fermentation under high-sugar high-temperature conditions of saccharomyces cerevisiae. Song Dan (research on ethanol production by continuous fermentation of high temperature resistant Saccharomyces cerevisiae S-13 [ D ]. University of agriculture in China, 2011.) high temperature resistant Saccharomyces cerevisiae S-13 screened from laboratory as fermentation strain was fermented at 37 deg.C in a medium containing 180g/L glucose at 4% inoculum size for 48h, with ethanol yield of 86.4g/L. Zhang Jilin (protoplast fusion technology for breeding high-yield ethanol yeast [ D ]. Beijing chemical university, 2009.) commercially available Angel yeast and laboratory yeast are subjected to protoplast fusion to obtain a fusion yeast, and ethanol fermentation is carried out at 37 ℃ in a fermentation medium containing 300g/L glucose, wherein the ethanol yield can reach 137.5g/L at most. Zhang Jinwei (screening and fermentation performance research of high temperature resistant industrial saccharomyces cerevisiae [ J ]. Brewing science and technology, 2021 (06): 23-28.) A strain of environmentally screened saccharomyces cerevisiae is used as an initial strain, the Rad52 gene of the strain is knocked out, and adaptive evolution breeding is carried out on the strain to obtain a saccharomyces cerevisiae S.C.D12, the strain is subjected to ethanol fermentation by using 350g/L of liquefied mash at 37 ℃, and the final concentration of ethanol can reach 100g/L.
Disclosure of Invention
In order to effectively solve the problems of unstable and undesirable anaerobic ethanol fermentation performance caused by strong stress inhibition of high-temperature, high-sugar and high-permeability environments on saccharomyces cerevisiae cells in the high-concentration ethanol fermentation process, the invention provides a high-temperature high-sugar and high-permeability tolerant yeast, a breeding method thereof and high-concentration ethanol fermentation application. The yeast provided by the invention has the advantages that the production indexes such as thallus growth, carbon source utilization, ethanol synthesis performance, sugar alcohol conversion rate and ethanol production strength are obviously improved under the conditions of 36-40 ℃ and 200-300g/L glucose high temperature and high sugar hyperosmolarity.
The invention provides a high-temperature high-sugar high-permeability tolerant yeast, which is obtained by domesticating and breeding a Saccharomyces cerevisiae (Saccharomyces cerevisiae) SG (strain preservation number: CCTCC M20221770) in a high-temperature high-sugar high-permeability environment and screening at high flux, is preserved in a China center for type culture preservation (address: eight-way No. 299 in Wuchang district, wuhan city, hubei province, postfix 430072) at 11 months and 11 days in 2022, and is named as Saccharomyces cerevisiae (Saccharomyces cerevisiae) SG200, wherein the preservation number is as follows: CCTCC M20221772.
Furthermore, the Saccharomyces cerevisiae SG200 can have good anaerobic ethanol fermentation performance under the conditions of 36-40 ℃ and high-temperature high-sugar high-permeability of 200-300 g/L. The glucose consumed by fermentation at 40 ℃ is up to 220g/L, the ethanol concentration can reach 90g/L, the thallus concentration is improved by 27-72% compared with the traditional yeast strains EB, 4126 and 6525, the ethanol production intensity can reach 2.3-3.4g/L/h in the fermentation process, and the sugar-alcohol conversion rate is maintained at 0.41-0.42g/g.
The second aspect of the invention provides a method for breeding Saccharomyces cerevisiae SG200, which comprises a high-temperature high-sugar high-permeability environment domestication breeding and high-throughput screening method.
Further, the domestication and breeding method for the high-temperature high-sugar and high-permeability environment comprises the following steps of:
(1) Diluting Saccharomyces cerevisiae (SG) and coating on YPD solid culture medium, culturing at 36 deg.C for 12-24 hr, and preferentially selecting the first grown SG single colony;
(2) Transferring the single saccharomyces cerevisiae SG colony in the step (1) into a YPD liquid culture medium, and performing activation culture at 36 ℃ and 150rpm for 12-24h to obtain saccharomyces cerevisiae SG activation bacterial liquid;
(3) Transferring the saccharomyces cerevisiae SG activated bacterial liquid obtained in the step (2) into a fermentation culture medium according to the inoculation amount of 5-10%, continuously transferring and culturing for 20 periods at 36 ℃ and 150rpm, and maintaining each period for 24 hours to obtain saccharomyces cerevisiae bacterial liquid;
(4) Diluting and coating the saccharomyces cerevisiae bacterial liquid in the step (3) on a YPD-containing solid culture medium, culturing for 12-24h at 38 ℃, and preferentially selecting a single saccharomyces cerevisiae colony growing firstly;
(5) Transferring the single saccharomyces cerevisiae colony in the step (4) into a YPD-containing liquid culture medium, and performing activation culture at 38 ℃ and 150rpm for 12-24h to obtain a saccharomyces cerevisiae activated bacterial liquid;
(6) Transferring the saccharomyces cerevisiae activated bacterial liquid obtained in the step (5) into a fermentation culture medium according to the inoculation amount of 5-10%, continuously transferring and culturing for 30 cycles at 38 ℃ and 150rpm, and maintaining each cycle for 36 hours;
(7) Diluting and coating the saccharomyces cerevisiae bacterial liquid obtained in the step (6) on a YPD solid culture medium, culturing for 24-36h at 40 ℃, and preferentially selecting a single saccharomyces cerevisiae colony growing firstly;
(8) Transferring the single saccharomyces cerevisiae colony in the step (7) into a YPD liquid culture medium, and performing activation culture at 40 ℃ and 150rpm for 24-36h to obtain saccharomyces cerevisiae activated bacterial liquid;
(9) Transferring the saccharomyces cerevisiae activated bacterial liquid obtained in the step (8) into a fermentation culture medium containing 300g/L glucose according to the inoculation amount of 5-10%, continuously transferring and culturing for 40 cycles at 40 ℃ and 150rpm, and maintaining each cycle for 48 hours to obtain saccharomyces cerevisiae bacterial liquid;
(10) Diluting and coating the saccharomyces cerevisiae bacterial liquid in the step (9) on a YPD solid culture medium, culturing for 36-48h at 42 ℃, and preferentially selecting a saccharomyces cerevisiae single colony growing firstly;
(11) Transferring the single saccharomyces cerevisiae colony in the step (10) into a YPD liquid culture medium, and performing activation culture at 42 ℃ and 150rpm for 24-36h to obtain saccharomyces cerevisiae activated bacterial liquid;
(12) Transferring the saccharomyces cerevisiae activated bacterial liquid obtained in the step (11) into a fermentation culture medium according to the inoculation amount of 5-10%, continuously transferring and culturing for 20 periods at 42 ℃ and 150rpm, and maintaining each period for 48 hours to obtain saccharomyces cerevisiae bacterial liquid;
(13) Diluting and coating the saccharomyces cerevisiae bacterial liquid obtained in the step (12) on a YPD solid culture medium, culturing for 48-60h at 44 ℃, and preferentially selecting a saccharomyces cerevisiae single colony growing at first, namely the high-temperature high-sugar high-permeability tolerant saccharomyces cerevisiae;
(14) And (3) respectively transferring the single saccharomyces cerevisiae colonies obtained in the step (13) into YPD liquid culture media, performing activation culture at 36 ℃ and 150rpm for 12-24h, mixing with 40% glycerol solution, and performing frozen storage at-80 ℃, namely obtaining the saccharomyces cerevisiae bacterial liquid obtained by high-temperature high-sugar hyperosmotic acclimation breeding.
Still further, the high throughput screening method comprises the steps of:
(1) Respectively transferring the saccharomyces cerevisiae bacterial liquid obtained by the high-temperature high-sugar hyperosmotic domestication breeding into an YPD liquid culture medium according to the inoculum size of 1%, performing activation culture at 40 ℃ and 150rpm for 12-24h, then transferring into a fermentation culture medium according to the inoculum size of 5-10%, and performing fermentation culture at 40 ℃ and 150rpm for 24-36h to obtain a zymogen liquid;
(2) The zymocyte liquid in the step (1) is diluted in a gradient way (10) -5 ，10 -6 ,10 -7 ) Sucking 100 mu L of diluent by a liquid transfer device, uniformly coating the diluent on a solid fermentation culture medium, and culturing for 48-72h to form a single colony;
(3) A disposable 96-well plate is used, and 200 mu L of fermentation medium is added into a single well;
(4) Picking up all single colonies in the step (2), transferring to the 96-well plate in the step (3) for fermentation culture, and measuring initial OD by using a high-throughput Q-mix system 600 OD was measured again after static culture at 40 ℃ for 24 hours 600 Preliminary screening to obtain culture 24hOD 600 And initial OD 600 A Saccharomyces cerevisiae strain with a difference greater than 3.5;
(5) Activating the Saccharomyces cerevisiae strain primarily screened in the step (4), transferring the activated Saccharomyces cerevisiae strain into a hole of a 96-pore plate filled with 200 mu L of fermentation culture medium according to the inoculation amount of 10%, standing and culturing at 40 ℃ until the fermentation end point is reached, wherein no bubble is generated, the thalli are settled to the bottom of the pore plate, and re-screening to obtain the Saccharomyces cerevisiae strain with the lowest residual sugar content and the highest ethanol concentration after complete fermentation, wherein the Saccharomyces cerevisiae strain is named as Saccharomyces cerevisiae SG200.
In the application of the Saccharomyces cerevisiae SG200 high-concentration ethanol fermentation, the fermentation condition is 200-300g/L glucose anaerobic fermentation.
The third aspect of the invention provides a method for fermenting and applying high-concentration ethanol of Saccharomyces cerevisiae (Saccharomyces cerevisiae) SG200, which comprises the following steps:
(1) Diluting and coating a Saccharomyces cerevisiae (Saccharomyces cerevisiae) SG200 preservation solution on a YPD solid culture medium under an aseptic condition, and performing static culture at a constant temperature of 30-40 ℃ for 12-48h to obtain a single colony for liquid activation culture;
(2) Under the aseptic condition, inoculating the single colony in the step (1) into a YPD liquid culture medium, carrying out shake culture at 30-40 ℃ and 150-200rpm for 12-48h, and preparing an activated bacterial liquid for anaerobic ethanol fermentation culture;
(3) Under the aseptic condition, inoculating the activated bacterium liquid obtained in the step (2) into a liquid synthetic culture medium or a toxic hydrolysate culture medium according to the inoculation amount of 5% -10%, performing closed anaerobic ethanol fermentation culture at the temperature of 30-40 ℃ and the rpm of 150-200 for 24-72h.
Furthermore, the nutrient components of the fermentation medium comprise 200-300g/L glucose, 12-36g/L yeast extract, 6-18g/L peptone, 1.5-4.5g/L ammonium sulfate, 1.5-4.5g/L potassium dihydrogen phosphate, 0.5-2.5g/L urea, 0.5-1.5g/L sodium glutamate, 0.5-1.0g/L magnesium sulfate heptahydrate, 0.001-0.01g/L manganese sulfate monohydrate, 0.05-0.5g/L zinc sulfate heptahydrate, 0.001-0.01g/L copper sulfate pentahydrate, 0.01-0.1g/L calcium chloride, 0.01-0.1g/L ferric chloride hexahydrate, 0.001-0.01g/L potassium iodide, 0.05-0.15g/L inositol, 0.05-0.15g/L vitamin B5-0.15 g/L, 0.01-0.1g/L biotin, 0.01-0.01 g/L sodium nicotinate, 0.05-0.01-0.1 g/L natural amino-1 g/L HCl, and the balance of sodium sulfate, 0.05-0.01-0.1 g/L.
The invention has the beneficial effects that: the invention provides a high-temperature high-sugar hyperosmotic tolerant yeast and a breeding method thereof, and the high-temperature high-sugar hyperosmotic tolerant yeast is a Saccharomyces cerevisiae (Saccharomyces cerevisiae) SG (strain preservation number: CCTCC M20221770) which is obtained by separation in an anaerobic environment, a high-temperature environment and an inhibitor stress environment at the early stage, and is named as Saccharomyces cerevisiae SG200 through high-temperature high-sugar hyperosmotic environment domestication breeding and high-throughput screening. The saccharomyces cerevisiae SG200 provided by the invention is applied to high-concentration ethanol fermentation at the temperature of 36-40 ℃ and under the condition of 200-300g/L, the glucose consumption in the fermentation at the temperature of 40 ℃ is up to 220g/L, the ethanol concentration can reach 90g/L, the thallus concentration is improved by 27-72% compared with the traditional yeast strains, the ethanol production intensity in the fermentation process can reach 2.3-3.4g/L/h, and the sugar alcohol conversion rate can be maintained at 0.41-0.42g/g. In conclusion, the invention enriches the resources and varieties of excellent strains for producing ethanol, effectively solves the problems of unstable and undesirable anaerobic ethanol fermentation performance caused by the strong stress inhibition of high-temperature, high-sugar and high-permeability environments on saccharomyces cerevisiae cells in the fermentation process of high-concentration ethanol, and remarkably improves the production indexes such as the conversion rate of sugar alcohol for anaerobic ethanol fermentation, the production intensity of ethanol and the like.
Drawings
FIG. 1 is a schematic diagram showing the morphology and number of SG colonies of Saccharomyces cerevisiae (Saccharomyces cerevisiae) cultured at 40 ℃;
FIG. 2 is a schematic diagram showing the colony morphology and amount of Saccharomyces cerevisiae SG200 cultured at 40 ℃;
FIGS. 3 (a) to 3 (c) are schematic diagrams showing the comparison of anaerobic ethanol fermentation of Saccharomyces cerevisiae EB, 4126, 6525, SG200 with 200g/L glucose at 36 ℃;
FIGS. 4 (a) to 4 (c) are schematic diagrams showing the comparison of anaerobic ethanol fermentation of Saccharomyces cerevisiae EB, 4126, 6525, SG200 with 200g/L glucose at 40 ℃;
FIGS. 5 (a) to 5 (c) are schematic diagrams showing the anaerobic ethanol fermentation of Saccharomyces cerevisiae EB, 4126, 6525, SG200 at 40 ℃ using 300g/L glucose.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1: isolation of Saccharomyces cerevisiae SG
The method comprises the following steps of firstly collecting a bottom mud sample of a storage tank of an ethanol fermentation plant, carrying out gradient dilution coating on the collected sample, and separating to obtain a single colony, wherein the specific separation and screening operation steps are as follows:
a. collecting a bottom mud sample of a storage tank of an ethanol fermentation plant, wherein the sampling environment is anaerobic, the temperature is 36-45 ℃, the sample is rich in various inhibitors, the sampling amount is 1mL or 1g, adding the sample into a triangular shake flask containing 99mL of sterile water, and carrying out shake culture for 60-120min at the temperature of 30-45 ℃ and the speed of 150-200 rpm.
b. Gradient dilution with pipettor (10) -2 、10 -3 、10 -4 ) Coating 100 mu L of diluent on a YPD solid culture medium, coating 10 culture dishes at each dilution concentration, placing 5 culture dishes in a group in an incubator at 30-45 ℃ for anaerobic culture for 24-72h, observing the colony formation condition, selecting different single colonies, carrying out dilution and streak separation culture, and transferring to the YPD solid culture medium for streak purification and preservation.
The YPD solid medium used above had the composition (g/L): peptone 20, yeast extract 10, glucose 20, agar 15. The balance of water is added to 1000mL, the pH is natural, and the high-pressure moist heat sterilization is carried out for 15min at 121 ℃.
Wherein, anaerobic culture is carried out for 24-72h in an incubator at 30-45 ℃ on all culture dishes coated according to different dilution concentrations, and a plurality of single colonies growing out fastest under different culture conditions are respectively purified and preserved.
Example 2: identification of Saccharomyces cerevisiae SG
The strain morphology is characterized as follows: after culturing for 24-72h on YPD solid medium at the culture temperature of 30-45 ℃, the formed single colony is round, milky white, smooth and glossy in surface and regular in colony edge. According to the manual of fungus identification, a single colony strain is judged to have typical fungus morphological characteristics.
Extracting the genomic DNA of the separated and purified strain, performing 18s rDNA sequencing analysis (the sequencing result is detailed in a sequence table SEQ ID NO. 1) by a biological engineering (Shanghai) corporation, selecting Saccharomyces cerevisiae and a sequence thereof with higher homology through BLAST homologous comparison in an NCBI database, performing multi-sequence comparison analysis by using ClustalX1.81 software, and preliminarily identifying and separating the purified strain into Saccharomyces cerevisiae (Saccharomyces cerevisiae) which is preserved in a China center for type culture preservation 11/2022 and named as Saccharomyces cerevisiae (Saccharomyces cerevisiae) SG, wherein the description is referred to as Saccharomyces cerevisiae SG or Sc SG hereinafter, the preservation number is CCTCC M20221770, and the unit address is Wuhan Wuchang region eight-one-way 299 number in Hubei province.
As shown in FIG. 1, the colony morphology and number of Saccharomyces cerevisiae SG (Saccharomyces cerevisiae) were determined under the culture conditions of 40 ℃.
SEQ ID NO.1：
CCATACTCCCCCCAGACCCAAAGACTTTGATTTCTCGTAAGGTGCCGAGTGGGTCATTAAAAAAACACCACCCGATCCCTAGTCGGCATAGTTTATGGTTAAGACTACGACGGTATCTGATCATCTTCGATCCCCTAACTTTCGTTCTTGATTAATGAAAACGTCCTTGGCAAATGCTTTCGCAGTAGTTAGTCTTCAATAAATCCAAGAATTTCACCTCTGACAATTGAATACTGATGCCCCCGACCGTCCCTATTAATCATTACGATGGTCCTAGAAACCAACAAAATAGAACCAAACGGCCTATTCTATTATTCCATGCTAATATATTCGAGCAATACGCCTGCTTTGAACACTCTAATTTTTTCAAAGTAAAAGTCCTGGTTCGCCAAGAGCCACAAGGACTCAAGGTTAGCCAGAAGGAAAGGCCCCGTTGGAAATCCAGTACACGAAAAAATCGGACCGGCCAACAGGGCCCAAAGTTCAACTACGAGCTTTTTAACTGCAACAACTTTAATATACGCTATTGGAGCTGGAATTACCGCGGCTGCTGGCACCAGACTTGCCCTCCAATTGTTCCTCGGTAAGGTATTTACATTGTACTCATTCCAATTACAAGACCCGAATGGGCCCTGTATCGTTATTTATTGTCACTACCTCCCTGAATTAGGATTGGGTAATTTGCGCGCCTGCTGCCTTCCTTGGATGTGGTAGCCGTTTCTCAGGCTCCCTCTCCGGAATCGAACCCTTATTCCCCGTTACCCGTTTAAACCATGGTAGGCCACTATCCTACCATCGAAAGTTGATAGGGCAGAAATTTGAATGAACCATCGCCAGCACAAGGCCATGCGATTCGAAAAGTTATTATGAATCATCAAAGAGTCCGAAGACATTGATTTTTTATCTAATAAATACATCTCTTCCAAAGGGTCGAGATTTTAAGCATGTATTAGCTCTAGAATTACCACAGTTATACCATGTAGTAAAGGGAACTATCAAATAAACGATAACTGATTTAATGAGCCATTCGCAGTTTCACTGTAAAAATTGCTTATACTTAGAACATGCATGGCTTAATCTTTGTAGACA
Example 3: high-temperature high-sugar hyperosmotic environmental acclimation of saccharomyces cerevisiae SG
a. Diluting and coating the Saccharomyces cerevisiae (Saccharomyces cerevisiae) SG obtained in the example 2 on a YPD solid culture medium, culturing for 12-24h at 36 ℃, and preferentially picking out a single Saccharomyces cerevisiae SG colony growing firstly;
b. transferring the saccharomyces cerevisiae SG single colony in the step a into a YPD liquid culture medium containing 100g/L glucose, and performing activation culture at 36 ℃ and 150rpm for 12-24h to obtain saccharomyces cerevisiae SG activated bacterial liquid;
c. b, transferring the saccharomyces cerevisiae SG activated bacterial liquid in the step b into a fermentation culture medium containing 100g/L glucose according to the inoculation amount of 5-10%, and continuously transferring and culturing at 36 ℃ and 150rpm for 20 cycles, wherein each cycle is maintained for 24 hours;
d. c, diluting and coating the saccharomyces cerevisiae bacterial liquid in the step c on a YPD solid culture medium containing 100g/L glucose, culturing for 12-24h at 38 ℃, and preferentially selecting a saccharomyces cerevisiae single colony growing firstly;
e. transferring the single saccharomyces cerevisiae colony in the step d into a YPD liquid culture medium containing 200g/L glucose, and performing activation culture at 38 ℃ and 150rpm for 12-24h to obtain saccharomyces cerevisiae activated bacterial liquid;
f. e, transferring the saccharomyces cerevisiae activated bacterial liquid obtained in the step e into a fermentation culture medium containing 200g/L glucose according to the inoculation amount of 5-10%, continuously transferring and culturing for 30 cycles at 38 ℃ and 150rpm, and maintaining each cycle for 36 hours;
g. diluting and coating the saccharomyces cerevisiae bacterial liquid in the step f on a YPD solid culture medium containing 200g/L glucose, culturing for 24-36h at 40 ℃, and preferentially selecting a saccharomyces cerevisiae single colony growing firstly;
h. transferring the single saccharomyces cerevisiae colony in the step g into a YPD liquid culture medium containing 300g/L glucose, and performing activation culture at 40 ℃ and 150rpm for 24-36h to obtain saccharomyces cerevisiae activated bacterial liquid;
i. transferring the saccharomyces cerevisiae activated bacterial liquid obtained in the step h into a fermentation culture medium containing 300g/L glucose according to the inoculation amount of 5-10%, continuously transferring and culturing for 40 cycles at 40 ℃ and 150rpm, and maintaining each cycle for 48 hours;
j. diluting and coating the saccharomyces cerevisiae bacterial liquid in the step i on a YPD solid culture medium containing 300g/L glucose, culturing for 36-48h at 42 ℃, and preferentially selecting a saccharomyces cerevisiae single colony growing firstly;
k. transferring the single saccharomyces cerevisiae colony in the step j to a YPD liquid culture medium containing 300g/L glucose, and performing activation culture at 42 ℃ and 150rpm for 24-36h to obtain saccharomyces cerevisiae activated bacterial liquid;
l, transferring the saccharomyces cerevisiae activated bacterial liquid obtained in the step k into a fermentation culture medium containing 300g/L glucose according to the inoculation amount of 5-10%, and continuously transferring and culturing at 42 ℃ and 150rpm for 20 cycles, wherein each cycle is maintained for 48 hours;
m, diluting the saccharomyces cerevisiae bacterial liquid obtained in the step L, coating the diluted saccharomyces cerevisiae bacterial liquid on a YPD solid culture medium containing 300g/L glucose, culturing for 48-60h at 44 ℃, and preferentially selecting a saccharomyces cerevisiae single colony growing out firstly, namely the high-temperature high-sugar high-permeability tolerant saccharomyces cerevisiae;
n. transferring the single colonies obtained in step m to YPD liquid medium containing 100g/L glucose,
activating and culturing at 36 deg.C and 150rpm for 12-24 hr, mixing with 40% glycerol solution, and freezing at-80 deg.C.
The solid or YPD liquid medium and fermentation medium mainly comprise 100g/L or 200g/L or 300g/L glucose, 12g/L or 20g/L or 36g/L yeast extract, 6g/L or 12g/L peptone or 18g/L peptone, 1.5g/L or 3g/L ammonium sulfate or 4.5g/L ammonium sulfate, 1.5g/L or 3g/L potassium dihydrogen phosphate or 4.5g/L urea 1g/L, 1g/L sodium glutamate, 0.7g/L magnesium sulfate heptahydrate, 0.005g/L of manganese sulfate monohydrate, 0.4g/L of zinc sulfate heptahydrate, 0.003g/L of copper sulfate pentahydrate, 0.02g/L of calcium chloride, 0.02g/L of ferric chloride hexahydrate, 0.001g/L of potassium iodide, 0.09g/L of inositol, 0.07g/L of vitamin B, 0.3g/L of biotin, 0.04g/L of nicotinic acid, 0.01g/L of p-aminobenzoic acid, 0.02g/L of HCl-thiamine, 0.04g/L of HCl-pyridoxal, the balance of water and natural pH.
Example 4: high-throughput screening to obtain saccharomyces cerevisiae SG200
a. Respectively transferring the saccharomyces cerevisiae bacterial liquid obtained by the high-temperature high-sugar hyperosmotic domestication breeding in the embodiment 3 into a YPD liquid culture medium containing 100g/L glucose according to the inoculum concentration of 1%, performing activation culture at 40 ℃ and 150rpm for 12-24h, then transferring into a fermentation culture medium containing 200g/L glucose according to the inoculum concentration of 5-10%, and performing fermentation culture at 40 ℃ and 150rpm for 24-36h;
b. the fermentation broth from step a is diluted in a gradient (10) -5 ，10 -6 ,10 -7 ) Sucking 100 mu L of diluent by a pipette, uniformly coating the diluent on a solid fermentation culture medium containing 300g/L glucose, and culturing for 48-72h to form a single colony;
c. using a disposable 96-well plate, adding 200 mu L of fermentation medium containing 300g/L glucose into a single well;
d. picking up all single colonies in the step b, transferring the single colonies to a 96-well plate in the step c for fermentation culture, and measuring initial OD by using a high-throughput Q-mix system 600 OD was measured again after static culture at 40 ℃ for 24 hours 600 Preliminary screening to obtain culture 24hOD 600 And initial OD 600 A saccharomyces cerevisiae strain with a difference value of more than 3.5;
e. and d, activating the Saccharomyces cerevisiae strain primarily screened in the step d, transferring the Saccharomyces cerevisiae strain into a hole of a 96-pore plate filled with 200 mu L of fermentation medium (containing 300g/L of glucose) according to the inoculation amount of 10%, standing and culturing at 40 ℃ until the fermentation end point is reached, generating no bubbles, allowing the thalli to settle to the bottom of the pore plate, re-screening to obtain the Saccharomyces cerevisiae strain with the lowest residual sugar content and the highest ethanol concentration after complete fermentation, and storing the Saccharomyces cerevisiae strain in the China center for type culture collection (number CCTCC M20221772) at 11 months and 11 days in 2022.
As shown in FIG. 2, the colony morphology and number of Saccharomyces cerevisiae (Saccharomyces cerevisiae) SG200 were determined under the culture conditions of 40 ℃.
The solid or YPD liquid medium and fermentation medium components used above mainly comprise 100g/L or 200g/L or 300g/L glucose, 12g/L or 20g/L or 36g/L yeast extract, 6g/L or 12g/L or 18g/L peptone, 1.5g/L or 3g/L or 4.5g/L ammonium sulfate, 1.5g/L or 3g/L or 4.5g/L potassium dihydrogen phosphate, 1g/L urea, 1g/L sodium glutamate, 0.7g/L magnesium sulfate heptahydrate, 0.005g/L of manganese sulfate monohydrate, 0.4g/L of zinc sulfate heptahydrate, 0.003g/L of copper sulfate pentahydrate, 0.02g/L of calcium chloride, 0.02g/L of ferric chloride hexahydrate, 0.001g/L of potassium iodide, 0.09g/L of inositol, 0.07g/L of vitamin B, 0.3g/L of biotin, 0.04g/L of nicotinic acid, 0.01g/L of p-aminobenzoic acid, 0.02g/L of HCl-thiamine, 0.04g/L of HCl-pyridoxal, the balance of water and natural pH.
Example 5: comparison of anaerobic ethanol fermentation of Saccharomyces cerevisiae with 200g/L glucose at 36 deg.C
1. Saccharomyces cerevisiae SG200 ethanol fermentation
The Saccharomyces cerevisiae SG200 (hereinafter, referred to as Saccharomyces cerevisiae SG200 or Sc SG 200) obtained in example 4 was used as an experimental strain, and plate culture, activation culture and fermentation culture experiments were performed sequentially at 36 ℃, and the specific operation steps were as follows:
a. under the aseptic condition, diluting and coating the saccharomyces cerevisiae SG200 preservation solution on a YPD solid culture medium, and performing static culture at the constant temperature of 36 ℃ for 12-48h to obtain a single colony for liquid activation culture;
b. under aseptic conditions, inoculating a single saccharomyces cerevisiae SG200 colony obtained in the step a into a YPD liquid culture medium, carrying out shaking culture at 36 ℃ and 150rpm for 12-24h, and preparing activated bacterial liquid for fermentation culture;
c. and c, inoculating the saccharomyces cerevisiae SG200 activated bacterium liquid obtained in the step b into a liquid synthetic culture medium according to the inoculation amount of 10% under the aseptic condition, and performing closed anaerobic ethanol fermentation culture for 48 hours at 36 ℃ at 150 rpm.
The fermentation medium used above mainly contains 200g/L glucose, 20g/L yeast extract, 12g/L peptone, 3g/L ammonium sulfate, 3g/L potassium dihydrogen phosphate, 1g/L urea, 1g/L sodium glutamate, 0.7g/L magnesium sulfate heptahydrate, 0.005g/L manganese sulfate monohydrate, 0.4g/L zinc sulfate heptahydrate, 0.003g/L copper sulfate pentahydrate, 0.02g/L calcium chloride, 0.02g/L ferric chloride hexahydrate, 0.001g/L potassium iodide, 0.09g/L inositol, 0.07g/L vitamin B, 0.3g/L biotin, 0.04g/L nicotinic acid, 0.01g/L p-benzoic acid, 0.02g/L HCl-thiamine, 0.04g/L HCl-pyridoxal, the balance water, natural pH.
2. Saccharomyces cerevisiae EB, 4126, 6525 ethanol fermentation (control)
Saccharomyces cerevisiae EB, 4126 and 6525 (hereinafter referred to as Saccharomyces cerevisiae EB, 4126 and 6525 or Sc EB, sc 4126 and Sc 6525) are taken as control strains, are stored in a laboratory, and are respectively subjected to plate culture, activation culture and fermentation culture experiments in sequence at 36 ℃, and the specific operation steps are as follows:
a. respectively diluting and coating the preservation solutions of saccharomyces cerevisiae EB, 4126 and 6525 on a YPD solid culture medium under the aseptic condition, and standing and culturing at the constant temperature of 36 ℃ for 12-48h to obtain a single colony for liquid activation culture;
b. respectively inoculating single colonies of the saccharomyces cerevisiae EB, 4126 and 6525 obtained in the step a into a YPD liquid culture medium under the aseptic condition, carrying out shaking culture at 36 ℃ and 150rpm for 12-24h, and preparing activated bacterial liquid for fermentation culture;
c. and c, respectively inoculating the activated bacterium solutions of the saccharomyces cerevisiae EB, 4126 and 6525 in the step b into a liquid synthetic culture medium according to the inoculation amount of 10% under the aseptic condition, and performing closed anaerobic ethanol fermentation culture for 48 hours at 36 ℃ and 150 rpm.
The fermentation medium used above mainly contains 200g/L glucose, 20g/L yeast extract, 12g/L peptone, 3g/L ammonium sulfate, 3g/L potassium dihydrogen phosphate, 1g/L urea, 1g/L sodium glutamate, 0.7g/L magnesium sulfate heptahydrate, 0.005g/L manganese sulfate monohydrate, 0.4g/L zinc sulfate heptahydrate, 0.003g/L copper sulfate pentahydrate, 0.02g/L calcium chloride, 0.02g/L ferric chloride hexahydrate, 0.001g/L potassium iodide, 0.09g/L inositol, 0.07g/L vitamin B, 0.3g/L biotin, 0.04g/L nicotinic acid, 0.01g/L p-aminobenzoic acid, 0.02g/L HCl-thiamine, 0.04g/L HCl-pyridoxal, and the balance water, natural pH.
As shown in FIGS. 3 (a) to 3 (c), saccharomyces cerevisiae EB, 4126, 6525 and SG200 were compared by anaerobic ethanol fermentation at 200g/L glucose at 36 ℃. The result shows that the saccharomyces cerevisiae SG200 quickly consumes glucose, the maximum value of the thallus concentration OD600 is 7.2, and the maximum value is respectively improved by 10.8%, 28.6% and 35.8% compared with saccharomyces cerevisiae EB, 4126 and 6525. 88g/L ethanol is synthesized after saccharomyces cerevisiae SG200 is fermented for 24h, the ethanol production intensity is 3.67g/L/h, and the ethanol production intensities of saccharomyces cerevisiae EB, saccharomyces cerevisiae 4126 and saccharomyces cerevisiae 6525 at the same time are 3.35 g/L/h, 3.25 g/L/h and 3.20g/L/h respectively; the concentration of ethanol in the anaerobic fermentation of saccharomyces cerevisiae SG200 can reach 89g/L, the conversion rate of sugar alcohol is 0.45g/g, and the concentrations of saccharomyces cerevisiae EB, 4126 and 6525 are respectively 0.41g/g, 0.40g/g and 0.41g/g. Therefore, the saccharomyces cerevisiae SG200 obtained by domestication and breeding in a high-temperature high-sugar high-permeability environment and high-throughput screening has good high-concentration ethanol fermentation performance under the conditions of 40 ℃ and 200g/L glucose anaerobic fermentation.
Example 6: comparison of anaerobic ethanol fermentation of Saccharomyces cerevisiae with 200g/L glucose at 40 deg.C
1. Saccharomyces cerevisiae SG200 ethanol fermentation
The Saccharomyces cerevisiae SG200 (hereinafter, referred to as Saccharomyces cerevisiae SG200 or Sc SG 200) obtained in example 4 was used as an experimental strain, and plate culture, activation culture and fermentation culture experiments were performed sequentially at 40 ℃, and the specific operation steps were as follows:
a. under the aseptic condition, diluting and coating the saccharomyces cerevisiae SG200 preservation solution on a YPD solid culture medium, and standing and culturing at the constant temperature of 40 ℃ for 12-48h to obtain a single colony for liquid activated culture;
b. under aseptic conditions, inoculating a single saccharomyces cerevisiae SG200 colony obtained in the step a into a YPD liquid culture medium, performing shaking culture at 40 ℃ and 150rpm for 24-36h, and preparing activated bacterial liquid for fermentation culture;
c. under the aseptic condition, inoculating the saccharomyces cerevisiae SG200 activated bacterium liquid obtained in the step b into a liquid synthetic culture medium according to the inoculation amount of 10%, and carrying out closed anaerobic ethanol fermentation culture at 40 ℃ and 150rpm for 48h.
The fermentation medium used above mainly contains 200g/L glucose, 20g/L yeast extract, 12g/L peptone, 3g/L ammonium sulfate, 3g/L potassium dihydrogen phosphate, 1g/L urea, 1g/L sodium glutamate, 0.7g/L magnesium sulfate heptahydrate, 0.005g/L manganese sulfate monohydrate, 0.4g/L zinc sulfate heptahydrate, 0.003g/L copper sulfate pentahydrate, 0.02g/L calcium chloride, 0.02g/L ferric chloride hexahydrate, 0.001g/L potassium iodide, 0.09g/L inositol, 0.07g/L vitamin B, 0.3g/L biotin, 0.04g/L nicotinic acid, 0.01g/L p-aminobenzoic acid, 0.02g/L HCl-thiamine, 0.04g/L HCl-pyridoxal, and the balance water, natural pH.
2. Saccharomyces cerevisiae EB, 4126, 6525 ethanol fermentation (control)
Saccharomyces cerevisiae EB, 4126 and 6525 (hereinafter referred to as Saccharomyces cerevisiae EB, 4126 and 6525 or Sc EB, sc 4126 and Sc 6525) are taken as control strains, are stored in a laboratory, and are respectively subjected to plate culture, activation culture and fermentation culture experiments in sequence at 40 ℃, and the specific operation steps are as follows:
a. under aseptic condition, respectively diluting and coating the preservation solutions of saccharomyces cerevisiae EB, 4126 and 6525 on a YPD solid culture medium, and standing and culturing at constant temperature of 40 ℃ for 12-48h to obtain single colonies for liquid activation culture;
b. respectively inoculating single colonies of the saccharomyces cerevisiae EB, 4126 and 6525 in the step a into a YPD liquid culture medium under the aseptic condition, carrying out shaking culture at 40 ℃ and 150rpm for 24-36h, and preparing activated bacterial liquid for fermentation culture;
c. under the aseptic condition, respectively inoculating the activated bacterium liquid of the saccharomyces cerevisiae EB, 4126 and 6525 in the step b into a liquid synthetic culture medium according to the inoculation amount of 10%, and carrying out closed anaerobic ethanol fermentation culture for 48 hours at 40 ℃ and 150 rpm.
The fermentation medium used above mainly contains 200g/L glucose, 20g/L yeast extract, 12g/L peptone, 3g/L ammonium sulfate, 3g/L potassium dihydrogen phosphate, 1g/L urea, 1g/L sodium glutamate, 0.7g/L magnesium sulfate heptahydrate, 0.005g/L manganese sulfate monohydrate, 0.4g/L zinc sulfate heptahydrate, 0.003g/L copper sulfate pentahydrate, 0.02g/L calcium chloride, 0.02g/L ferric chloride hexahydrate, 0.001g/L potassium iodide, 0.09g/L inositol, 0.07g/L vitamin B, 0.3g/L biotin, 0.04g/L nicotinic acid, 0.01g/L p-aminobenzoic acid, 0.02g/L HCl-thiamine, 0.04g/L HCl-pyridoxal, and the balance water, natural pH.
As shown in FIGS. 4 (a) to 4 (c), saccharomyces cerevisiae EB, 4126, 6525 and SG200 were compared by anaerobic ethanol fermentation at 200g/L glucose at 40 ℃. The result shows that the saccharomyces cerevisiae SG200 still consumes 200g/L glucose within 24h, the maximum value of the thallus concentration OD600 is 4.6, which is respectively improved by 27.8%, 64.3% and 70.4% compared with saccharomyces cerevisiae EB, 4126 and 6525, and saccharomyces cerevisiae EB ferments 48h and consumes 200g/L glucose, while saccharomyces cerevisiae 4126 and 6525 respectively only consume 165g/L glucose. After the saccharomyces cerevisiae SG200 is fermented for 24 hours, 81.4g/L ethanol is synthesized, the ethanol production intensity is 3.39g/L/h, and the ethanol production intensity of the saccharomyces cerevisiae EB, the saccharomyces cerevisiae 4126 and the saccharomyces cerevisiae 6525 is respectively reduced to 2.50, 2.70 and 2.77g/L/h at the same time; the concentration of the anaerobic fermentation ethanol of the saccharomyces cerevisiae SG200 can reach 84.5g/L, the conversion rate of the sugar alcohol is 0.42g/g, and the contents of the saccharomyces cerevisiae EB, 4126 and 6525 are all 0.40g/g. Therefore, the saccharomyces cerevisiae SG200 obtained by domestication and breeding in a high-temperature high-sugar high-permeability environment and high-throughput screening still has good high-concentration ethanol fermentation performance under the anaerobic fermentation condition of 200g/L glucose at 40 ℃. Example 7: comparison of anaerobic ethanol fermentation of Saccharomyces cerevisiae with 300g/L glucose at 40 deg.C
1. Saccharomyces cerevisiae SG200 ethanol fermentation
The Saccharomyces cerevisiae SG200 (hereinafter, referred to as Saccharomyces cerevisiae SG200 or Sc SG 200) obtained in example 4 was used as an experimental group strain, and plate culture, activation culture and fermentation culture experiments were performed in sequence at 40 ℃, and the specific operation steps were as follows:
a. under the aseptic condition, diluting and coating the saccharomyces cerevisiae SG200 preservation solution on a YPD solid culture medium, and performing static culture at the constant temperature of 40 ℃ for 12-48h to obtain a single colony for liquid activation culture;
b. under aseptic conditions, inoculating a single saccharomyces cerevisiae SG200 colony obtained in the step a into a YPD liquid culture medium, performing shaking culture at 40 ℃ and 150rpm for 24-36h, and preparing activated bacterial liquid for fermentation culture;
c. under the aseptic condition, inoculating the saccharomyces cerevisiae SG200 activated bacterium liquid obtained in the step b into a liquid synthetic culture medium according to the inoculation amount of 10%, and carrying out closed anaerobic ethanol fermentation culture at 40 ℃ and 150rpm for 60 hours.
The fermentation medium used above mainly contains 300g/L glucose, 36g/L yeast extract, 18g/L peptone, 4.5g/L ammonium sulfate, 4.5g/L potassium dihydrogen phosphate, 1g/L urea, 1g/L sodium glutamate, 0.7g/L magnesium sulfate heptahydrate, 0.005g/L manganese sulfate monohydrate, 0.4g/L zinc sulfate heptahydrate, 0.003g/L copper sulfate pentahydrate, 0.02g/L calcium chloride, 0.02g/L ferric chloride hexahydrate, 0.001g/L potassium iodide, 0.09g/L inositol, 0.07g/L vitamin B, 0.3g/L biotin, 0.04g/L nicotinic acid, 0.01g/L p-aminobenzoic acid, 0.02g/L HCl-thiamine, 0.04g/L HCl-pyridoxal, the balance water, natural pH.
2. Saccharomyces cerevisiae EB, 4126, 6525 ethanol fermentation (control)
Saccharomyces cerevisiae EB, 4126 and 6525 (hereinafter referred to as Saccharomyces cerevisiae EB, 4126 and 6525 or Sc EB, sc 4126 and Sc 6525) are taken as control strains, are stored in a laboratory, and are respectively subjected to plate culture, activation culture and fermentation culture experiments in sequence at 40 ℃, and the specific operation steps are as follows:
a. under aseptic condition, respectively diluting and coating the preservation solutions of saccharomyces cerevisiae EB, 4126 and 6525 on a YPD solid culture medium, and standing and culturing at constant temperature of 40 ℃ for 12-48h to obtain single colonies for liquid activation culture;
b. respectively inoculating single colonies of the saccharomyces cerevisiae EB, 4126 and 6525 in the step a into a YPD liquid culture medium under the aseptic condition, carrying out shaking culture at 40 ℃ and 150rpm for 24-36h, and preparing activated bacterial liquid for fermentation culture;
c. and c, respectively inoculating the activated bacterium solutions of the saccharomyces cerevisiae EB, 4126 and 6525 in the step b into a liquid synthetic culture medium according to the inoculation amount of 10% under the aseptic condition, and performing closed anaerobic ethanol fermentation culture for 60 hours at 40 ℃ and 150 rpm.
The fermentation medium used above mainly contains 300g/L glucose, 36g/L yeast extract, 18g/L peptone, 4.5g/L ammonium sulfate, 4.5g/L potassium dihydrogen phosphate, 1g/L urea, 1g/L sodium glutamate, 0.7g/L magnesium sulfate heptahydrate, 0.005g/L manganese sulfate monohydrate, 0.4g/L zinc sulfate heptahydrate, 0.003g/L copper sulfate pentahydrate, 0.02g/L calcium chloride, 0.02g/L ferric chloride hexahydrate, 0.001g/L potassium iodide, 0.09g/L inositol, 0.07g/L vitamin B, 0.3g/L biotin, 0.04g/L nicotinic acid, 0.01g/L p-aminobenzoic acid, 0.02g/L HCl-thiamine, 0.04g/L HCl-pyridoxal, the balance water, natural pH.
As shown in FIGS. 5 (a) to 5 (c), saccharomyces cerevisiae EB, 4126, 6525 and SG200 were compared by anaerobic ethanol fermentation using 300g/L glucose at 40 ℃. The results show that 220g/L glucose can be consumed in the whole fermentation process of the saccharomyces cerevisiae SG200, the maximum value of the thallus concentration OD600 is 3.6, the maximum value is respectively improved by 71.4%, 50.0% and 56.5% compared with the maximum values of the saccharomyces cerevisiae EB, 4126 and 6525, and the maximum values of the saccharomyces cerevisiae EB, 4126 and 6525 can only consume 140-160g/L glucose in the whole fermentation process of the saccharomyces cerevisiae EB, 4126 and 6525, which shows that the inhibition effect of the high-temperature high-sugar hyperosmolar stress caused by 300g/L glucose at 40 ℃ on the saccharomyces cerevisiae EB, 4126 and 6525 is obvious. 55.8g/L ethanol is synthesized after saccharomyces cerevisiae SG200 is fermented for 24h, the ethanol production intensity is 2.33g/L/h, and the ethanol production intensity of saccharomyces cerevisiae EB, 4126 and 6525 is respectively further reduced to 0.75 g/L/h, 1.67 g/L/h and 1.30g/L/h at the same time; the concentration of ethanol in Saccharomyces cerevisiae SG200 anaerobic fermentation can reach 90g/L, the conversion rate of sugar alcohol is 0.41g/g, and the conversion rates of Saccharomyces cerevisiae EB, 4126 and 6525 sugar alcohol are 0.38, 0.38 and 0.40g/g respectively. Therefore, the saccharomyces cerevisiae SG200 obtained by domestication and breeding in a high-temperature high-sugar high-permeability environment and high-throughput screening still has good high-concentration ethanol fermentation performance under the conditions of anaerobic fermentation of 300g/L glucose at 40 ℃.
Claims (8)
1. A high-temperature high-sugar high-permeability tolerant yeast is deposited in China center for type culture Collection (CGMCC) at 11 months and 11 days of 2022, is named as Saccharomyces cerevisiae SG200, and has the deposit number of: CCTCC M20221772.
2. The method for breeding the high-temperature high-sugar high-permeability tolerant yeast according to claim 1, wherein the high-temperature high-sugar high-permeability tolerant yeast is Saccharomyces cerevisiae (SG), and the strain preservation number is as follows: CCTCC M20221770 through domestication and breeding in high-temperature high-sugar high-permeability environment and high-throughput screening.
3. The breeding method of the high-temperature high-sugar high-permeability tolerant yeast according to claim 2, wherein the domestication and breeding of the high-temperature high-sugar high-permeability environment comprises the following steps:
(1) Diluting Saccharomyces cerevisiae (SG) and coating on YPD solid culture medium, culturing at 36 deg.C for 12-24 hr, and preferentially selecting the first grown SG single colony;
(2) Transferring the saccharomyces cerevisiae SG single colony in the step (1) into a YPD liquid culture medium, and performing activation culture at 36 ℃ and 150rpm for 12-24h to obtain saccharomyces cerevisiae SG activated bacterial liquid;
(3) Transferring the saccharomyces cerevisiae SG activated bacterial liquid obtained in the step (2) into a fermentation culture medium according to the inoculation amount of 5-10%, continuously transferring and culturing for 20 periods at 36 ℃ and 150rpm, and maintaining each period for 24 hours to obtain saccharomyces cerevisiae bacterial liquid;
(4) Diluting and coating the saccharomyces cerevisiae bacterial liquid in the step (3) on a YPD-containing solid culture medium, culturing for 12-24h at 38 ℃, and preferentially selecting a single saccharomyces cerevisiae colony growing firstly;
(5) Transferring the saccharomyces cerevisiae single colony in the step (4) into a YPD-containing liquid culture medium, and performing activation culture at 38 ℃ and 150rpm for 12-24h to obtain a saccharomyces cerevisiae activated bacterial liquid;
(6) Transferring the saccharomyces cerevisiae activated bacterial liquid in the step (5) into a fermentation culture medium according to the inoculation amount of 5-10%, continuously transferring and culturing for 30 cycles at 38 ℃ and 150rpm, and maintaining each cycle for 36h;
(7) Diluting and coating the saccharomyces cerevisiae bacterial liquid in the step (6) on a YPD solid culture medium, culturing for 24-36h at 40 ℃, and preferentially selecting a saccharomyces cerevisiae single colony growing firstly;
(8) Transferring the single saccharomyces cerevisiae colony in the step (7) into a YPD liquid culture medium, and performing activation culture at 40 ℃ and 150rpm for 24-36h to obtain saccharomyces cerevisiae activated bacterial liquid;
(9) Transferring the saccharomyces cerevisiae activated bacterial liquid obtained in the step (8) into a fermentation culture medium according to the inoculation amount of 5-10%, continuously transferring and culturing for 40 periods at 40 ℃ and 150rpm, and maintaining each period for 48 hours to obtain saccharomyces cerevisiae bacterial liquid;
(10) Diluting and coating the saccharomyces cerevisiae bacterial liquid in the step (9) on a YPD solid culture medium, culturing for 36-48h at 42 ℃, and preferentially selecting a single saccharomyces cerevisiae colony growing firstly;
(11) Transferring the single saccharomyces cerevisiae colony in the step (10) into a YPD liquid culture medium, and performing activation culture at 42 ℃ and 150rpm for 24-36h to obtain saccharomyces cerevisiae activated bacterial liquid;
(12) Transferring the saccharomyces cerevisiae activated bacterial liquid obtained in the step (11) into a fermentation culture medium according to the inoculation amount of 5-10%, continuously transferring and culturing for 20 periods at 42 ℃ and 150rpm, and maintaining each period for 48 hours to obtain saccharomyces cerevisiae bacterial liquid;
(13) Diluting and coating the saccharomyces cerevisiae bacterial liquid in the step (12) on a YPD solid culture medium, culturing for 48-60h at 44 ℃, and preferentially selecting a saccharomyces cerevisiae single colony growing at first, namely the high-temperature high-sugar high-permeability tolerant saccharomyces cerevisiae;
(14) And (3) respectively transferring the single saccharomyces cerevisiae colonies obtained in the step (13) into YPD liquid culture media, performing activation culture at 36 ℃ and 150rpm for 12-24h, mixing with 40% glycerol solution, and performing frozen storage at-80 ℃, namely obtaining the saccharomyces cerevisiae bacterial liquid obtained by high-temperature high-sugar hyperosmotic acclimation breeding.
4. The method for breeding the high-temperature high-sugar high-permeability tolerant yeast according to claim 3, wherein the high-throughput screening comprises the following steps:
(15) Respectively transferring the saccharomyces cerevisiae bacterial liquid obtained by the high-temperature high-sugar hyperosmotic domestication and breeding in the step (14) into an YPD liquid culture medium according to 1% of inoculum concentration, performing activation culture at 40 ℃ and 150rpm for 12-24h, then transferring into a fermentation culture medium according to 5-10% of inoculum concentration, and performing fermentation culture at 40 ℃ and 150rpm for 24-36h to obtain a zymogen liquid;
(16) Carrying out gradient dilution on the zymocyte liquid in the step (15), wherein the dilution gradient is respectively 10 -5 、10 -6 And 10 -7 Sucking 100 mu L of diluent by a liquid transfer device, uniformly coating the diluent on a solid fermentation culture medium, and culturing for 48-72h to form a single colony;
(17) A disposable 96-well plate is used, and 200 mu L of fermentation medium is added into a single well;
(18) All single colonies in step (16) were picked, transferred to the 96-well plate in step (17) above for fermentation culture, and the initial OD was measured using a high-throughput Q-mix system 600 OD was measured again after static culture at 40 ℃ for 24 hours 600 Preliminary screening to obtain culture 24hOD 600 And initial OD 600 A saccharomyces cerevisiae strain with a difference value of more than 3.5;
(19) And (5) activating the Saccharomyces cerevisiae strain primarily screened in the step (18), transferring the activated Saccharomyces cerevisiae strain into a hole of a 96-pore plate filled with 200 mu L of fermentation medium according to the inoculation amount of 10%, standing and culturing at 40 ℃ until the fermentation end point is reached, wherein no bubble is generated, the thalli are settled to the bottom of the pore plate, and re-screening to obtain the Saccharomyces cerevisiae strain with the lowest residual sugar content and the highest ethanol concentration after complete fermentation, wherein the Saccharomyces cerevisiae strain is named as Saccharomyces cerevisiae SG200.
5. The breeding method of high temperature and high sugar hyperosmotic tolerant yeast as claimed in claim 3 or 4, wherein the nutrient components of the fermentation medium comprise 200-300g/L glucose, 12-36g/L yeast extract, 6-18g/L peptone, 1.5-4.5g/L ammonium sulfate, 1.5-4.5g/L potassium dihydrogen phosphate, 0.5-2.5g/L urea, 0.5-1.5g/L sodium glutamate, 0.5-1.0g/L magnesium sulfate heptahydrate, 0.001-0.01g/L manganese sulfate monohydrate, 0.05-0.5g/L of zinc sulfate heptahydrate, 0.001-0.01g/L of copper sulfate pentahydrate, 0.01-0.1g/L of calcium chloride, 0.01-0.1g/L of ferric chloride hexahydrate, 0.001-0.01g/L of potassium iodide, 0.05-0.15g/L of inositol, 0.05-0.15g/L of vitamin B5, 0.1-0.5g/L of biotin, 0.01-0.1g/L of nicotinic acid, 0.01-0.1g/L of p-aminobenzoic acid, 0.01-0.05g/L of HCl-thiamine, 0.01-0.1g/L of HCl-pyridoxal, and the balance of water, and the natural pH.
6. The use of high temperature, high sugar, high permeability tolerant yeast of claim 1 in high concentration ethanol fermentation.
7. The use of high temperature, high sugar, high permeability tolerant yeast for the fermentation of high concentration ethanol as claimed in claim 6, wherein the fermentation conditions are 200-300g/L glucose anaerobic fermentation.
8. The use of high temperature, high sugar, and hyperosmotic tolerant yeast for high concentration ethanol fermentation according to claim 6 or 7,
the method comprises the following steps:
(1) Diluting and coating a Saccharomyces cerevisiae (Saccharomyces cerevisiae) SG200 preservation solution on a YPD solid culture medium under an aseptic condition, and performing static culture at a constant temperature of 30-40 ℃ for 12-48h to obtain a single colony for liquid activation culture;
(2) Under the aseptic condition, inoculating the single colony in the step (1) into a YPD liquid culture medium, carrying out shake culture at 30-40 ℃ and 150-200rpm for 12-48h, and preparing an activated bacterial liquid for anaerobic ethanol fermentation culture;
(3) Under the aseptic condition, inoculating the activated bacterium liquid obtained in the step (2) into a liquid synthetic culture medium or a toxic hydrolysate culture medium according to the inoculation amount of 5-10%, performing closed anaerobic ethanol fermentation culture at the temperature of 30-40 ℃ and the rpm of 150-200 for 24-72h.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211603163.XA CN115948263A (en) | 2022-12-13 | 2022-12-13 | High-temperature high-sugar high-permeability tolerant yeast, breeding method thereof and high-concentration ethanol fermentation application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211603163.XA CN115948263A (en) | 2022-12-13 | 2022-12-13 | High-temperature high-sugar high-permeability tolerant yeast, breeding method thereof and high-concentration ethanol fermentation application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115948263A true CN115948263A (en) | 2023-04-11 |
Family
ID=87288767
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211603163.XA Pending CN115948263A (en) | 2022-12-13 | 2022-12-13 | High-temperature high-sugar high-permeability tolerant yeast, breeding method thereof and high-concentration ethanol fermentation application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115948263A (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101962620A (en) * | 2010-10-13 | 2011-02-02 | 广西大学 | Saccharomyces cerevisiae strain and application thereof |
| CN102199554A (en) * | 2011-03-14 | 2011-09-28 | 中国科学院微生物研究所 | Saccharomyces cerevisiae strain with multiple-stress resistance, and application thereof in cellulose alcohol fermentation |
| JP2013198407A (en) * | 2012-03-23 | 2013-10-03 | Saga Prefecture | Method for breeding high-temperature-resistant ethanol-productive yeast, and method for producing fermented unrefined sake using the same |
| US20180327709A1 (en) * | 2015-11-25 | 2018-11-15 | Rajendra Surana | A Method Of Producing High Amount Of Ethanol At High Temperature By Modified Yeast Strain Saccharomyces Cerevisiae |
| CN109370929A (en) * | 2018-12-05 | 2019-02-22 | 北京工商大学 | A kind of application of S. cervisiae in wine brewing |
| CN110358690A (en) * | 2019-07-24 | 2019-10-22 | 上海中溶科技有限公司 | A kind of S. cervisiae of resistance to mortifier and its selection and application |
| CN111040957A (en) * | 2019-09-30 | 2020-04-21 | 中国石油化工股份有限公司 | Ethanol-resistant and high-sugar-resistant fermentation strain and construction method thereof |
-
2022
- 2022-12-13 CN CN202211603163.XA patent/CN115948263A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101962620A (en) * | 2010-10-13 | 2011-02-02 | 广西大学 | Saccharomyces cerevisiae strain and application thereof |
| CN102199554A (en) * | 2011-03-14 | 2011-09-28 | 中国科学院微生物研究所 | Saccharomyces cerevisiae strain with multiple-stress resistance, and application thereof in cellulose alcohol fermentation |
| JP2013198407A (en) * | 2012-03-23 | 2013-10-03 | Saga Prefecture | Method for breeding high-temperature-resistant ethanol-productive yeast, and method for producing fermented unrefined sake using the same |
| US20180327709A1 (en) * | 2015-11-25 | 2018-11-15 | Rajendra Surana | A Method Of Producing High Amount Of Ethanol At High Temperature By Modified Yeast Strain Saccharomyces Cerevisiae |
| CN109370929A (en) * | 2018-12-05 | 2019-02-22 | 北京工商大学 | A kind of application of S. cervisiae in wine brewing |
| CN110358690A (en) * | 2019-07-24 | 2019-10-22 | 上海中溶科技有限公司 | A kind of S. cervisiae of resistance to mortifier and its selection and application |
| CN111040957A (en) * | 2019-09-30 | 2020-04-21 | 中国石油化工股份有限公司 | Ethanol-resistant and high-sugar-resistant fermentation strain and construction method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 董晓宇;汪江伟;袁玉莲;侯义龙;刘庆平;: "高产乙醇酿酒酵母的筛选鉴定及发酵条件优化", 大连大学学报, no. 03, 25 June 2013 (2013-06-25), pages 62 - 68 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| ES2373397T3 (en) | NON-RECOMBINANT SACCHAROMYCES STRAINS THAT GROW IN XILOSA. | |
| US8986962B2 (en) | Organism co-culture in the production of biofuels | |
| Hashem et al. | Optimization of the fermentation conditions for ethanol production by new thermotolerant yeast strains of Kluyveromyces sp | |
| CN103820346B (en) | Saccharomyces cerevisiae and application thereof in fermentation production of ethanol | |
| CN104611228A (en) | Highly oil-containing monoraphidium and culture and application thereof | |
| CN102174433B (en) | Clostridium beijerinckii with high stress resistance and application thereof | |
| US20080085536A1 (en) | Production of Cellulose in Halophilic Photosynthetic Prokaryotes (Cyanobacteria) | |
| MX2012011872A (en) | Industrial yeast capable of producing ethanol from at least one pentose. | |
| CN113913309B (en) | Alkali-resistant yeast and application thereof in producing single cell protein by utilizing biogas slurry | |
| WO2010031793A2 (en) | Thermophilic fermentative bacterium producing butanol and/or hydrogen from glycerol | |
| CN103421850A (en) | Method used for producing bioethanol with Scenedesmusabundans | |
| Atitallah et al. | Efficient bioethanol production from date palm (Phoenix dactylifera L.) sap by a newly isolated Saccharomyces cerevisiae X19G2 | |
| CN110684691A (en) | Preparation process of microbial agent based on directional screening of microorganisms | |
| CN104830705A (en) | Glucose/xylose co-metabolism saccharomyces cerevisiae strain and application thereof | |
| CN104673712A (en) | Bacterial strain for producing alcohol fuels by synchronously utilizing glucose and xylose and application of bacterial strain | |
| Talukder et al. | Fuel ethanol production using xylose assimilating and high ethanol producing thermosensitive Saccharomyces cerevisiae isolated from date palm juice in Bangladesh | |
| CN105624051A (en) | Xylose fermented yeast strain constructed based on evolution engineering and construction method of cylose fermented yeast strain | |
| CN116622612A (en) | Domestication mutation breeding method for improving formate utilization rate of saccharomyces cerevisiae | |
| CN107400646B (en) | One plant height produces Clostridium acetobutylicum and its screening and application | |
| CN109251868B (en) | Saccharomyces cerevisiae and application thereof | |
| CN107164246B (en) | High-temperature-resistant yeast and application thereof | |
| CN115948263A (en) | High-temperature high-sugar high-permeability tolerant yeast, breeding method thereof and high-concentration ethanol fermentation application | |
| CN114854795A (en) | Method for producing ethanol by fermenting raw starch with double bacteria | |
| CN1160467C (en) | Method of producing adenosylmethionine | |
| CN116218695B (en) | Stress-resistant high-yield yeast capable of efficiently utilizing hydrolysate, breeding method thereof and high-concentration ethanol fermentation application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |