CN104371938A - High-temperature-resistant saccharomyces cerevisiae and application thereof - Google Patents
High-temperature-resistant saccharomyces cerevisiae and application thereof Download PDFInfo
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- CN104371938A CN104371938A CN201410654463.XA CN201410654463A CN104371938A CN 104371938 A CN104371938 A CN 104371938A CN 201410654463 A CN201410654463 A CN 201410654463A CN 104371938 A CN104371938 A CN 104371938A
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- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
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- C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
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- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
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Abstract
The invention discloses high-temperature-resistant saccharomyces cerevisiae and an application thereof. The bacterial strain had been preserved in a depository authority, namely China center for type culture collection, specified by the state intellectual property office on July 3, 2014, with a preservation number of CCTCCNO: M2014312. High-temperature-resistant saccharomyces cerevisiae NT102002 obtained by combining ion implantation with a high-temperature technology for mutagenesis and screening is resistant to a high temperature of 40 DEG C, and can be used for synchronous diastatic fermentation with a lignocellulose raw material to prepare fuel alcohol, so that a novel feasible technical choice is provided for improving the thermal tolerance of the saccharomyces cerevisiae. Compared with the conventional mutation breeding technology, the technology adopted by the invention is simple and high in mutation rate. The obtained mutant strain is stable in character.
Description
Technical field
The present invention relates to microbial technology field, be specifically related to thermotolerant ethanol fermentation yeast and application thereof.
Background technology
In recent years; sugarcane, cassava and cellulose family agriculture and forestry organic waste material etc. are converted into alcohol fuel and have become the desirable route solving world energy sources crisis, especially utilize cellulosic waste to transform production alcohol fuel and obtain the support of countries in the world government and the common concern of vast researcher.In the process of fermentation for cellulose fuel ethanol, by glycolytic pathway, conversion of glucose is ethanol and carbonic acid gas by yeast, and ethanol conversion can reach 90 ~ 95%.But one of major issue of this route existence is: the optimum temperuture of cellulase is between 45 ~ 50 DEG C, and the suitableeest leavening temperature of yeast is generally between 28 ~ 35 DEG C, this makes the fermentation of yeast and cellulosic enzymolysis be difficult to synchronously carry out, and the suitableeest leavening temperature therefore improving ethanol yeast just becomes the key addressed this problem.
Saccharomycetic heat-resisting mechanism is complicated, is related to the change of multiple physiological process in cell and the stable of cell interior structure, wherein heat shock protein(HSP), trehalose, stress glycoprotein macromole etc. be and thalline thermotolerance relation gene the most closely.The experimental technique putting forward yeast saccharomyces cerevisiae thermotolerance at present comprises high temperature acclimation, spontaneons screening, selection by mutation, protoplast fusion breeding and genome segment S9 technology.Patent CN 102492634 A screens and obtains a strain thermotolerant yeast Ogataeasp.WXT3 from soil, can produce ethanol at 25 ~ 48 DEG C of temperature bottom fermentations; Patent CN 102041235 A screens and obtains a strain thermotolerant yeast Saccharomyces cerevisiae FE-B from rice wine yeast, and this bacterial strain can tolerate the high temperature of 36 ~ 44 DEG C; Patent CN103232948 A is by carrying out ultraviolet mutagenesis and gene rearrangement to yeast saccharomyces cerevisiae, and seed selection obtains Saccharomyces cerevisiae SY-5-11L, and under 38 DEG C of conditions, ethanol production is 13.9%(V/V); Patent CN 102154138 A utilizes gene shuffling technology screening to obtain high temperature resistant, ethanol-tolerant, acidproof high-yield ethanol yeast mutant TT31, under 35 DEG C of conditions, is that carbon source ethanol production reaches 101.9g/L with sucrose.
Because the heat-resisting mechanism of yeast is not yet clear and definite, the relation of genes involved and thermotolerance also needs to further investigate, and using gene engineering technique improves saccharomycetic temperature tolerance and also has many restrictions.Ion implantation technique, as the novel mutation source of one, with the physical chemistry complex mutation effect that it is special, has been successfully applied in the mutagenic and breeding of the improvement of many varieties of crops and microbial strains, and has achieved considerable economic benefit.Research shows, ion beam mutagenesis cause is not only the DNA Single and double strand breakage because energy deposition causes, and make genetic material molecule, atom that displacement and restructuring occur by matter, energy, electric multiplefactor effect in ion implantation process, and its mutant character has multiple and repeated feature, therefore ion implantationly a kind of complex mutation is equivalent to, more effective than single mutagenesis; (ionic species, energy, fluence, charge number etc.) can be combined in this external mutagenic processes inject the regulation and control reaching beneficial mutation and finally obtain by different parameters the mutant strain meeting breeding goal.But not yet find that application ion implantation technique changes the report of biotemperature tolerance at present.
Summary of the invention
the technical problem solved:order of the present invention is to provide a strain thermotolerant ethanol fermentation yeast and the application in lignocellulose raw material simultaneous saccharification and fermentation alcohol fuel thereof.
technical scheme:thermotolerant ethanol fermentation yeast (
saccharomyces cerevisiae) NT102002, this bacterial strain is in depositary institution's preservation that State Intellectual Property Office specifies, and preservation date is on July 3rd, 2014, depositary institution's title: China typical culture collection center, preservation address: Wuhan, China Wuhan University, deposit number: CCTCC NO:M2014312.
Described thermotolerant ethanol fermentation yeast (
saccharomyces cerevisiae) application of NT102002 in lignocellulose raw material simultaneous saccharification and fermentation alcohol fuel.
Described fermentation substratum is PDA substratum.
Described fermentation condition pH5.5-6.0, leavening temperature scope 35-40 DEG C and shaking speed 150r/min.
For a composition for lignocellulose raw material simultaneous saccharification and fermentation alcohol fuel, effective constituent be thermotolerant ethanol fermentation yeast (
saccharomyces cerevisiae) NT102002.
beneficial effect:the present invention apply ion implantation obtain in conjunction with high temperature acclimation technology mutagenesis screening thermotolerant ethanol fermentation yeast (
saccharomyces cerevisiae) high temperature resistant 40 DEG C of NT102002, can be used for lignocellulosic material simultaneous saccharification and fermentation and prepare alcohol fuel, for the temperature tolerance improving yeast saccharomyces cerevisiae provides the new feasible choice of technology; This technology is simple relative to traditional mutation breeding technologies method, and mutation rate is high, and the mutant strain proterties obtained is stablized.
Accompanying drawing explanation
Fig. 1 is yeast saccharomyces cerevisiae Survival curves figure;
Fig. 2 is yeast saccharomyces cerevisiae mutation rate figure;
Fig. 3 is 35 DEG C of NT102001 leaven line charts;
Fig. 4 is 40 DEG C of NT102002 leaven line charts;
Fig. 5 is for fermenting raw materials graphic representation with wheat bran and straw powder;
Fig. 6 is ethanol canonical plotting.
Embodiment
Following examples further illustrate content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, the amendment do the inventive method, step or condition and replacement, all belong to scope of the present invention.
If do not specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art.
the determination of embodiment 1 thermotolerant ethanol fermentation yeast mutagenic condition
Saccharomyces cerevisiae used in the present invention is in Angel Yeast Co., Ltd.
Picking yeast saccharomyces cerevisiae list bacterium colony coats PDA slant medium, under optimum temperuture 30 DEG C of conditions, be cultured to 3-4 days; In picking one ring yeast saccharomyces cerevisiae access PDA liquid nutrient medium, 30 DEG C, cultivate 24 hours under the condition of 120 revs/min.
Getting 0.1mL nutrient solution is spread evenly across on aseptic empty culture dish, to carry out N after aseptic wind to drying
+inject.The ion implanter adopted is LZD900 type multifunction ion implanter, and target chamber vacuum tightness is 10
-3pa, inject with 5s pulsed, interval 15s, Implantation Energy is 10keV, and fluence is respectively 50 × 10
14n
+/ cm
2, 100 × 10
14n
+/ cm
2, 150 × 10
14n
+/ cm
2, 200 × 10
14n
+/ cm
2, 250 × 10
14n
+/ cm
2with 300 × 10
14n
+/ cm
2.N
+after injection, take out plate, with 0.5mL sterilized water wash-out, be applied on PDA solid medium, being placed in primary dcreening operation temperature is that the incubator of 33 DEG C is cultured to and grows single bacterium colony, tally sheet colony count.According to single colony count statistics Strain survival rate, Survival curves is shown in Fig. 1, then continues to be cultured to ripe preparing against and screens.
Single bacterium colony of picking growth and maturity is connected to PDA slant medium, be placed in the incubator continuation cultivation that multiple sieve temperature is 32 DEG C, counting under each fluence can the bacterial strain quantity of continued growth breeding, calculates the mutation rate of each fluence, with the highest fluence of positive mutation rate for best fluence.Be that under the condition of 10keV, best ion fluence is 200 × 10 at Implantation Energy in the present embodiment
14n
+/ cm
2, positive mutation rate reaches 30%(and sees Fig. 2).Following examples all carry out injection mutagenesis with this fluence.
the yeast saccharomyces cerevisiae that mutagenesis screening is resistance to 35 DEG C
Saccharomyces cerevisiae used in the present invention is in Angel Yeast Co., Ltd.
Picking yeast saccharomyces cerevisiae list bacterium colony coats PDA slant medium, under optimum temperuture 30 DEG C of conditions, be cultured to 3-4 days; In picking one ring yeast saccharomyces cerevisiae access PDA liquid nutrient medium, 30 DEG C, cultivate 24 hours under the condition of 120 revs/min.
Getting 0.1mL nutrient solution is spread evenly across on aseptic empty culture dish, to carry out N after aseptic wind to drying
+inject.N
+after injection, take out plate, with 0.5mL sterilized water wash-out, be applied on PDA solid medium, being placed in temperature is that the incubator of 33 DEG C is cultured to and grows single bacterium colony, and single bacterium colony of picking growth and maturity is connected to PDA slant medium, and being placed in temperature is that the incubator of 35 DEG C continues to cultivate.The bacterial strain that can grow with this understanding is the yeast saccharomyces cerevisiae of resistance to 35 DEG C, called after yeast saccharomyces cerevisiae (
saccharomyces cerevisiae) NT102001.
Get 20g raw cellulose, 600U/g cellulase and 10mL yeast saccharomyces cerevisiae (
saccharomyces cerevisiae) NT102001 nutrient solution loads in 250 mL shaking flasks, regulate the initial ph value of fermented liquid to 5.5-6.0 with the sodium citrate buffer solution of 0.05 mol/L, add distilled water constant volume to 100mL, with 150r/min in 35 DEG C of condition bottom fermentations, the mass concentration of ethanol in fermentation ends post analysis supernatant liquor.Application yeast saccharomyces cerevisiae (
saccharomyces cerevisiae) NT102001 fermenting alcohol output comparatively yeast saccharomyces cerevisiae improve 24.03%, fermentation diagram is shown in Fig. 3.
the yeast saccharomyces cerevisiae that mutagenesis screening is resistance to 40 DEG C
The yeast saccharomyces cerevisiae that the present embodiment uses for yeast saccharomyces cerevisiae (
saccharomyces cerevisiae) NT102001.
Picking yeast saccharomyces cerevisiae (
saccharomyces cerevisiae) the mono-bacterium colony of NT102001 coats PDA slant medium, under 35 DEG C of conditions, be cultured to 3-4 days; In picking one ring yeast saccharomyces cerevisiae access PDA liquid nutrient medium, 35 DEG C, cultivate 24 hours under the condition of 120 revs/min.
Getting 0.1mL nutrient solution is spread evenly across on aseptic empty culture dish, to carry out N after aseptic wind to drying
+inject.N
+after injection, take out plate, with 0.5mL sterilized water wash-out, be applied on PDA solid medium, being placed in temperature is that the incubator of 35 DEG C is cultured to and grows single bacterium colony, single bacterium colony of picking growth and maturity is connected to PDA slant medium, and be placed in the incubator continuation cultivation that temperature is 40 DEG C, the bacterial strain that can grow with this understanding is the yeast saccharomyces cerevisiae of resistance to 40 DEG C of high temperature.Called after thermotolerant ethanol fermentation yeast (
saccharomyces cerevisiae) NT102002.
By 20g raw cellulose, 600U/g cellulase and 10mL yeast saccharomyces cerevisiae (
saccharomyces cerevisiae) NT102001 nutrient solution loads in 250 mL shaking flasks, regulate the initial ph value of fermented liquid to 5.5-6.0 with the sodium citrate buffer solution of 0.05 mol/L, add distilled water constant volume to 100mL, with 150r/min in 35 DEG C of condition bottom fermentations, the mass concentration of ethanol in fermentation ends post analysis supernatant liquor.Application yeast saccharomyces cerevisiae (
saccharomyces cerevisiae) NT102001 fermentation by saccharomyces cerevisiae ethanol production comparatively yeast saccharomyces cerevisiae improve 30.4%, fermentation diagram is shown in Fig. 4.
By 15g wheat bran, 5g straw powder, 600U/g cellulase and 10mL thermotolerant ethanol fermentation yeast (
saccharomyces cerevisiae) NT102002 nutrient solution loads in 250 mL shaking flasks, regulate the initial ph value of fermented liquid to 5.5-6.0 with the sodium citrate buffer solution of 0.05 mol/L, add distilled water constant volume to 100mL, with 150r/min in 35 DEG C of condition bottom fermentations, the mass concentration of ethanol in fermentation ends post analysis supernatant liquor.Application thermotolerant ethanol fermentation yeast (
saccharomyces cerevisiae) NT102002 fermenting alcohol output comparatively yeast saccharomyces cerevisiae improve 38.4%, fermentation diagram is shown in Fig. 5.
the cultural method of bacterial strain
The thermotolerant ethanol fermentation yeast that obtained (
saccharomyces cerevisiae) NT102002 is suitable for cultivating with PDA solid medium and preserving.Change the highest growth temperature tolerating 42 DEG C of bacterial strain, 40 DEG C of leavening temperatures can be tolerated; The maximum growth temperature of more original starting strain improves 8 DEG C, and the highest leavening temperature improves 6 DEG C; Retain stable fermentation producing and ethanol characteristic simultaneously.
the extraction of tunning and analysis
Alcohol concn detects: accurately take dehydrated alcohol 25.00 g, be placed in the volumetric flask of 1 L, dissolves also constant volume and is made into the standard reserving solution of 25 g/L, be diluted to 20g/L, 15g/L, 10g/L, 5g/L during experiment with ultrapure water with ultrapure water.With the absorption peak area of each concentration ethanol standardized solution for ordinate zou, alcohol concn is X-coordinate drawing standard curve.Typical curve as shown in Figure 6.Fermented sample alcohol concn is calculated with typical curve.Chromatographiccondition is: AT capillary chromatographic column (0.53mm × 18m), column temperature 70 DEG C, column flow rate 6.36mL/min.Fid detector, injection compartment temperature 180 DEG C, detector temperature 120 DEG C, sample size 1.0 μ L, carrier gas is nitrogen.
Claims (5)
1. thermotolerant ethanol fermentation yeast (
saccharomyces cerevisiae) NT102002, this bacterial strain is in depositary institution's preservation that State Intellectual Property Office specifies, and preservation date is on July 3rd, 2014, depositary institution's title: China typical culture collection center, deposit number: CCTCC NO:M2014312.
2. thermotolerant ethanol fermentation yeast described in claim 1 (
saccharomyces cerevisiae) application of NT102002 in lignocellulose raw material simultaneous saccharification and fermentation alcohol fuel.
3. application according to claim 2, is characterized in that described fermentation substratum is PDA substratum.
4. application according to claim 2, is characterized in that described fermentation condition pH5.5-6.0, leavening temperature scope 35-40 DEG C and shaking speed 150r/min.
5. for a composition for lignocellulose raw material simultaneous saccharification and fermentation alcohol fuel, it is characterized in that effective constituent be thermotolerant ethanol fermentation yeast (
saccharomyces cerevisiae) NT102002.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109517754A (en) * | 2018-11-20 | 2019-03-26 | 上海交通大学 | A method of high temperature bacterial strain is isolated and purified using common biochemical equipment |
| CN112852649A (en) * | 2019-11-28 | 2021-05-28 | 华东理工大学 | High-temperature-resistant saccharomyces cerevisiae strain for producing cellulosic ethanol and fermentation application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154138A (en) * | 2011-01-24 | 2011-08-17 | 天津科技大学 | Multi-resistant high-yield alcohol yeast mutant strain TT31 and screening method 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 |
-
2014
- 2014-11-17 CN CN201410654463.XA patent/CN104371938A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154138A (en) * | 2011-01-24 | 2011-08-17 | 天津科技大学 | Multi-resistant high-yield alcohol yeast mutant strain TT31 and screening method 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 |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109517754A (en) * | 2018-11-20 | 2019-03-26 | 上海交通大学 | A method of high temperature bacterial strain is isolated and purified using common biochemical equipment |
| CN112852649A (en) * | 2019-11-28 | 2021-05-28 | 华东理工大学 | High-temperature-resistant saccharomyces cerevisiae strain for producing cellulosic ethanol and fermentation application thereof |
| CN112852649B (en) * | 2019-11-28 | 2022-08-23 | 华东理工大学 | High-temperature-resistant saccharomyces cerevisiae strain for producing cellulosic ethanol and fermentation application thereof |
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